Here I present a mind-object identity theory based on a straightforward hypothesis: One's experience of an object is identical with the object itself. To defend this hypothesis, I will reconsider the notion of a physical object in terms of relative and actual properties. To address cases of misperception such as dreams and hallucinations, I will also reconsider the notion of present in relative terms. Both the object and the present are recast as object-relative. One's experience of an object is identical with the object itself. To defend this hypothesis, I will reconsider the notion of a physical object in terms of relative and actual properties, and in doing so, I will support a form of naturalism.I present a mind-object identity theory based on a straightforward hypothesis: At the outset, I'd like to stress that the proposed view is neither a form of panpsychism nor a variant of neutral monism; moreover, it does not require any emergence. The goal is showing that if nature is reconceived according to a relative notion of physical objects, consciousness will fit seamlessly into what we call the physical world.“Physicalism” is a notoriously subtle and elusive view, of which there are several variants. I agree that it is impossible to define a priori the nature of the physical Russell, , therefomind-object identity that, in contrast with the traditional mind-body identity, maintains that while consciousness is located in the physical world, it is not identical to brain processes but rather to external objects. In brief, the proposal is that, whenever a subject S experiences an object O, S's experience is nothing but O itself. O is a relative object where relative reads as in “relative velocity” rather than as in “relative to a subject.”The goal of the paper is to show that, by revisiting the nature of the physical world in terms of relative properties, it is possible to reveal a Such a proposal, of course, does not entail any form of panpsychism or idealism. In other words, if experience is identical to an object, it does not entail that objects are identical with a mental world. Instead, identity moves in an arrow from experience to the physical world. Consider the traditional mind-brain identity theory, such as Smart's version where he suggests that “sensations may be just brain processes” Smart,  p. 141. I will organize the paper as follows. First, I will outline the main idea in the case of successful standard perception, which is perhaps the most favorable case. Then I will list three key objections: subjective variability, alleged irreducible mental features, and the argument from illusion in all its variants. To address such objections, I will delve into an articulate account of the proposed building blocks of nature—i.e., the relative object—and I will articulate the role of the body as a condition of existence for the object itself. With these two key notions, I will carefully address the three objections plus a hoard of other recurring worries.Does consciousness fit into the physical world? Many philosophers and scientists believe that it doesn't unless extraordinary hypotheses—such as emergent properties, integrated information, dual aspect information, and the like—are deployed . However, they might have chosen the wrong x. In fact, as argued above, the hypothesis that x is a brain process has proven to be empirically wrong. Then, what else might x be? Remarkably, one candidate has gone unnoticed, a candidate that has been hidden in plain sight: the external object.In fact, if successful from a scientific perspective, an identity theory would be a welcomed solution because it would locate consciousness in the physical world. Unfortunately and differently from a large body of literature Kripke, , which hx is the external object. In shortWhenever standard perception takes place, along with one's body and one's brain, there is also the external object. Could the object be the thing that is identical to one's experience? Strange as it may seem, this is the case I will defend here. When it comes to cases of successful standard perception, the proposal is that S's experience of O is O itselfWhat and where is such an experience? If one is a physicalist, there must be something that is one and the same as her experience, and such a something must be physical. In addition, for identity to work, there must be something with the properties of Jacqueline's experience. Yet what? Traditional mind-brain identity theories have suggested looking for it into one's brain. Yet, nothing inside Jacqueline's brain has the properties that Jacqueline finds in her experience. Jacqueline experiences a red, round, and shiny apple, but nothing inside her brain is red, round, or shiny. How could a brain process be identical with Jacqueline's experience of the apple? Simply put, it cannot. We can thus consider another candidate, which is the only thing around that has the properties of Jacqueline's experience of the apple: the apple itself. The undoubtedly quite radical hypothesis is that Jacqueline's experience of the apple is nothing but the apple that exists relative to Jacqueline's body (more on the relative nature of the apple soon). The solution simplifies our understanding of the case. Two objects are there: her body (or her brain) and the apple. One of them is Jacqueline's experience. Only it is not her body but the apple. In this way there are no more metaphysical mysteries. What's going on, though not easy to know in detail, is purely physical. Everything is physical.Where O is the external physical object—no Berkeley's ideas are advocated. An example will help. Consider an ordinary (and favorable) case of successful standard perception: Jacqueline perceives an apple and there is an apple. From a physical perspective, there are two physical objects interacting together—the apple and Jacqueline's body. Next, of course, there is the issue of Jacqueline's conscious experience of the apple. This solution entails neither panpsychism nor idealism but it defends a reduction of experience to the properties of physical objects that exist relative to one's body. If correct, there is no need to introduce any problematic notions. In the end, there is only the physical world and its properties. The proposed solution suggests how to locate those properties that tradition has placed in a mental world and mistakenly considered to be metaphysically different.one's experience of an object is the object one experiences. Such a hypothesis is the core of the Spread Mind theory . For a traditional identity theorist, the brain is forced to do something that in nature nothing else does—namely, reaching out and becoming something else aside from itself. If Jacqueline's experience were identical with her neural activity, how could such an activity partake of the properties of the apple? Contrary to current views, there is only one thing applish enough to be Jacks' experience: the apple.The hypothesis that our experience of an object is nothing but the object as it exists relative to our body represents a huge conceptual leap. Yet, why not? After all, the suggested mind-object identity is strictly physicalist. The form of the explanation is the same of the traditional mind-brain identity theories. While they proposed to consider brain processes, I propose to consider external objects. Such a view offers unprecedented ontological parsimony. Not only does it get rid of mental properties, it also sets all subject-object relationships aside—e.g., intentionality, perspectivalness, and phenomenal character.A caveat. The theory being proposed is neither a new version of panpsychism nor a rendition of neutral monism. Rather it is a strong form of physicalism and realism. Moreover, the proposed theory does not require the addition of any phenomenal quality to the physical world. I want to be very clear on this point. The theory does not claim that the external object instantiates phenomenal properties. Rather, the external object is the experience. The physical world is assumed to be sufficient to host the thing that we have been referring to as consciousness. The reason why experience and world have been kept apart—I will argue—does not lie in their ontological incongruity, but in a series of questionable premises.The suggested conceptual shift, while theoretically momentous, does not contradict any empirical evidence. Moreover, it goes further than other attempts that have taken into consideration the dependence between one's experience and the external word such as various forms of externalism, enactivism, extended cognition, and radical embodiment .Subjective variability refers to the easily observed fact that an object appears differently depending on the subject. The same apple may appear bigger to a child or smaller to an adult. The temperature in the room may seem cold if one has a fever and warm if one has finished an intense running session. If experience is the external object and the external object is the same for everyone, why do we not all experience the same object? Is this proof that we experience subjective objects? Is this evidence that the phenomenal character of our experience is the outcome of some internal processing? I argue it is not. It is only the consequence of the relative-yet-utterly-physical nature of objects and their properties.allegedly characterized by irreducible features—such as Nagel's well-known question about what it is like to be something . Only in this way will we be able to check the properties of nature against those of our experience and determine whether they are the same.The subject-object dichotomy might have stemmed from an oversimplistic notion of the physical world that has encouraged scholars to add the level of the mental—fittingly nicknamed the “dustbin model of the mind” by Sydney Shoemaker .To overcome the abovementioned downsides, physical objects will be recast as relative objects following Galilean relativity and/or the Eleatic Principle, as is the case with relative velocity. By and large, I propose to generalize the idea of relative existence to relative to roughly mean that each property of a physical object (at least those that partake our experience) depends on other objects and is therefore intrinsically relative . By actual I mean that each property of a physical object is embedded and defined by the occurrence of an actual physical process that is located in space and time (as suggested by the Eleatic principle).The key point is to acknowledge the relative and actual nature of physical objects and of their properties. Here I take Remarkably, all physical properties we are familiar with exist only relative to other objects. In our own case, the object they are relative to is our own body and, more specifically, our sense organs and brains. Note that this fact does not entail any special metaphysical status for human bodies—i.e., a body is just yet another object.subject-dependent but object-dependent.The kind of relativity advocated here is object-object relativity. Consider the case of velocity. Ever since Galileo, it has been well-known that velocity is relative—i.e., there is no such a thing as absolute velocity. However, this physical fact does not entail that velocity is either subjective or arbitrary, but only that velocity is relative. Relative velocity is a well-defined quantity. In other words, velocity is not Distinguishing object-dependence from subject-dependence is paramount. In fact, in the philosophy of mind, relativism and relationalism have often been associated with mind-dependence and thus with subjectivity. This is not the case. The simple case of velocity suggests a different (and simpler) picture. Velocity is inherently relative, and it is relative to other objects (or their reference frames).Object relativity addresses the traditional question: “Would a tree falling in a forest with no observers make any sound?” as we would with the question “would a particle moving in an empty space with no other particle have a velocity?” In both cases, the reply is negative—not because the sound is taken to be a mental entity created by a subject, but rather because the sound is taken to be a relative physical entity coming into existence because of the causal circumstance offered by a human body, which is just yet another object. The same rationale holds for traditional relative properties such as velocity. If an object were utterly isolated, would it have any velocity? Of course not. The question would be meaningless. An entirely isolated object is neither in movement nor at rest. Likewise, without the body, the opportunity for the existence of the sound is lost. The tree will fall, and the air pressure waves will scatter, however their chance to create a sound will be lost due to the absence of a human body for the sound to occur. The sound is neither a process inside the human body nor a mental entity inside the mind. In this account, the sound is the physical cause of an effect on a human body. Without the body such an effect will not occur and thus no cause will either.whose existence is relative to a subject, is thus substituted by that of object-relativity, as something whose existence is relative to another object, which is, in our case, the human body. The latter notion, which is compatible with Galilean relativity, is enough to cope with the fundamental features we have always associated with phenomenal experience—or so I will argue later.The traditional notion of subjectivity, as something relative to the ground. The ground is the conventional reference frame. Similarly, when we say that a copper wire has a voltage of 50 Volt, we omit saying that it has a voltage of 50 Volt relative to the ground. And so forth. So, people tend to forget that these properties exist relative an implicit conventional reference frame. Of course, the adoption of an implicit conventional reference frame does not make such properties any less relative. As we will see later, this is also the reason why we so easily dismiss the causal role of our body in shaping our world. Since one's body is always present as the conventional causal reference frame of one's world, one easily forgets its key role.Why has the relative nature of the physical world—object relativity—so often been dismissed? The likely reason is that, in practice, the stipulation of a conventional reference frame allows people to omit the otherwise necessary yet verbose mention of a conventional reference frame. When we say that a car is traveling at 50 mph, we omit saying that the car is traveling at 50 mph The layman's standard view takes the world to be made of physical properties (objective and absolute) and mental properties (subjective and arbitrary). In contrast, I suggest that all properties are relative and that the former are simply conventionally chosen relative properties. The so-called objective properties are no longer the real properties the world is made of, they are only canonical properties that have been historically selected, just like the Greenwich time zone, which is no more real and objective than, say, Brisbane's time zone.actual. The objects and their physical properties are always spatiotemporally located and causally efficacious. While one could argue that there may be entities that do not produce any effect whatsoever, these entities would be, by definition, epiphenomenal and outside the scope both of our experience and of scientific observation. An entity that does not produce any effect can neither be observed nor acted upon, and, thus, it is, to all extents, causally extraneous to our world. So, even if such entities existed, they would not be the entities our world is made of. In fact, in physics all properties manifest causally themselves by means of their effects. Such effects bring the physical properties (and thus objects which are bundles of properties) into existence and fix the object in a precise spatiotemporal location. The actual nature of the object is a nomological consequence of standard physics—it follows from the upper speed limit of all physical processes. Consequently, relative properties need time to be pulled into existence and thus are spread over time spans of arbitrary length. The bottom line is that all known physical properties in our life are both relative and actual.Importantly, the object is not only relative, it is also The proposed revision is neither theoretically demanding nor scientifically challenging. It does not conflict with physics. It is consistent with both Galilean relativity and Einstein's notion of speed limit. Finally, this revision does not endorse anything like backward causation or processes snapping back in time. Nothing goes back in time. While relative objects are spatiotemporally located, because the object with its relative properties remains undefined until such properties are fixed by the conclusion of a process, the object is ontologically mature, so to speak, only after its (relative) properties get fixed by ensuing events. The flow of time moves only forward.In sum, I suggest that all the properties we are familiar with are physical properties that exist relative to other objects. Everything is object relative. Relative objects allow us to outline an utterly physicalist view of both objects and experience—the two being the same. The world is made of objects. Each object exists because its properties take place thanks to some further object. In such an object-only physical world, everything is physical, everything is an object. Each object is, at the same time, a cause and an effect. It exists because it causes effects on other objects and it allows other objects to exist by their acting upon it. In such a world, everything is identical with itself and there is no need for additional ontological levels, emergent properties, mental states, phenomenal characters, or ontologically equivocal relations. Our conscious experience is where the world is. It is that world. On such a view, the ontological separation between subjects and objects disappears—subjects are only those particular objects we are identical with, but no metaphysical gap divides them.causal reference frame relative to which all the objects in one's world take place. The body is analogous to a conventional reference frame. The body is always there, and thus it can be ignored. Yet, our body, for each of us, is paramount because it is the object relative to which everything, we are identical with, exists. If one's body is destroyed, one's world is destroyed too.What is the role of our body then? The body is just yet another object. However, in our case, it is an important object because it is the Consider the analogy of a system composed by a lake and a dam. The water flows in a river. Because of the dam, a lake fills up. Before the dam was built, no lake filled the valley. However, because of the dam, the water formed a lake. The water is not just the lake and the lake is not just water. The lake exists because of the dam. The dam was built because of the river and the possibility of creating a lake. The dam does not create the lake out of thin air, though. If no dam had been erected, no lake would exist. Crucially, the lake is not an emergent property of the dam. If there were no rain, a one-mile-high dam would not create a lake autonomously. Rather, the lake takes place because of the dam and the river, and because of several other conditions. Yet, in that valley, the dam has a key role in bringing the lake into existence. To extend the analogy, the lake is the object. The river is the incoming flow of events. The terrain is the environment. The water is the stuff the world is made of before one experiences it. The dam is one's brain and body. Where is one's experience? It is the lake. Where is the object? It, too, is the lake. The key idea is that the existence of the object is dependent on the proper causal circumstances. My conscious mind is the lake. My body is the dam. My experiences exist thanks to the body, and yet they are not the body. My experiences are nothing but the properties of the physical objects that take place relative to my body. Please note that this account brings together the relative and actual nature of external properties. Relative because they are what they are in relation to the body they interact with. Actual because they exist thanks to the actual occurrence of an effect in another body.true color of the canvas. The first case is conventionally representative of the real color of the canvas because it is more common and it is the one that corresponds to a healthy average human being. The color is relative to the physical system with which a surface interacts. So, the same canvas will be white relative to a standard trichromat; cyanish relative to a trichromat who is red-fatigued; magentish relative to a trichromat who is green-fatigued, and so forth. There is no absolute color, there are only colors relative to other objects—in this case, the visual systems of the human beings involved. Thus, color is analogous to velocity and white is analogous to the velocity relative to the ground. The alleged objective properties are nothing but conventionally chosen relative properties. The standard trichromat and the ground are conventional reference frames. “Relative” does not mean being relative to a subject (being subjective), but rather being relative to another object. Object-relativity does not imply subject-relativity nor subjectivity.The body shapes the world that surrounds us, which is a relative world. Consider a familiar property such as color. A canvas is white. By “white” we usually mean that the canvas appears white to a standard trichromat under direct sunlight relative. If we think carefully about this definition, it is quite clear that even once we have satisfied the environmental conditions (direct sunlight) the color of the canvas is still dependent on the observer. What if the observer is not a standard trichromat? What if an observer is red blind? Would the canvas still be white? Fortunately we can do a simple and easily replicable experiment whose results are very well-known since Helmholtz's pioneering work  does not depend on the object alone, but on the physical system the screen is relative to.Consider now a white patch on a computer screen. From a comfortable distance, the surface of the display appears to be white. Now, if you get much closer, you will distinguish the physical structure of the display. At such a close distance you will resolve an array of the red, green, and blue light-emitting diodes. The patch will no longer appear white, but rather as a mosaic of red, green, and blue points. Is the patch white or is it a mosaic of colored lights? There is not a unique answer. The screen is both solid white All physical properties, like velocity and color, are relative to other objects. Here I cannot address all the properties we experience, yet I contend that it is possible to make an equally convincing case for all of them. In short, each physical object we experience is a bundle of relative properties that exist thanks to the interaction with our body—i.e., it is an object relative to our body. So, the canvas is not white in any absolute sense; it is white relative to a standard trichromat and cyanish relative to a red-fatigued trichromat. The existence of the world we live in is relative to our body; it is made of those objects that exist relative to our body, which is our natural causal reference frame. Like all conventional reference frames, it tends to be forgotten, since it represents the ever present condition of existence of everything our world is made of. The role of our body in shaping our world is so largely invisible unless something (often bad) happens. For example, in the case of hemineglect, the world in which one lives is dramatically reduced. The damage to a cerebral hemisphere destroys the causal circumstances that allow the existence of half of one's world. The subjects affected by such a condition behave and feel as though half of their world had disappeared. The world we live in is a sort of negative projection of our body's sensorial apparatus, which is the collection of those organs that are causally coupled with the external world. “Sensorial apparatus” is a sort of misnomer because our organs do not sense anything. Sense organs contribute to the causal circumstances that allow the existence of those objects our experiences are identical with. The layman's notion that we have an experience of the world thanks to our body corresponds to the fact that the world relative to our body comes into existence thanks to our body. Our body, though, does not sense anything.What about subjective variability? Why do different subjects experience the same object differently? If our experience was the object itself, why would it not be the same for each subject? Why do we perceive the same object in different ways depending on age, emotional status, physical conditions, and the like? How can the physical world, albeit reconceived in terms of relative properties, cope with the familiar subjective variability of experience?Positively, object-relativity explains away subjective variability. The short answer is that since the object's properties are relative to our body, each difference in our body will determine a difference in the properties of the object. If the world is a bundle of relative properties that are what they are because of their relations with other physical objects , each object will have multiple properties—e.g., multiple velocities, multiple colors, multiple shapes, and so forth. In practice, each object exists in multiple versions of itself, each relative to a given object . Relative objects endorse a much richer physical world than the layman has ever suspected. In such a world the same room is at once hot and cold. It is hot relative to my body after a run and cold relative to yours when you have a fever. The same canvas is at once both white and cyan. The same screen is both white and multicolored. The same particle is both still and moving. The same object appears different to different subjects because the same object is different relative to their different bodies.ad hoc properties. If the object is not coupled with another object, certain properties are not pulled into existence. The object has umpteen relative properties that it sustains just in case—“the case” being the circumstances offered by other objects—in this instance, our body. Analogously, a body has as many relative velocities as they are needed given other objects and reference frames.The physical object, once it is revisited in terms of relative properties, has as many relative properties as are needed in relation to as many external objects (some of which are human bodies) as are needed. This is not a problem. The object is not overburdened by per se, is part of the very fabric of relative physical reality. All physical properties are relative to a given object and thus they can embody a viewpoint. Consider velocity again. Is a velocity field not centered on and determined by a reference frame? Of course, it is, and the same can be said for all other properties. Every object defines the surrounding world in function of itself as its relative fulcrum of existence. The resulting relative world is perspectival with respect to such an object. The fact that we perceive a world perspectivally centered around our body is not the consequence of the structure of our mental space, but the consequence of a world that exists relative to our body.The relative object can also address the issue of perspectivalness. The properties that constitute each object are relative in nature, and so they will also be perspectival insofar as they are all centered around the object relative to which they take place. The notion of perspectivalness, often quoted as something that is not shared by the physical world If the world is made of relative properties, once again, a puzzled reader may wonder if physical objects have an existence independent of being an object of experience. And would this not be a form of panpsychism? Not at all! I propose that the properties of experience are nothing but physical relative properties instantiated by an object relative to another object (which in our case is the body).In sum, the richness of a relative physical world—or at least of a world where all properties are relative—explains subjectivity and perspectivalness away. There is no longer any need of a layer of multiple subjective appearances to justify the different ways in which an object manifests itself to different observers. If an object is made of relative properties, given the fact that different observers have different bodies, the same object will have distinct and different relative properties for each of them.ad hoc hypotheses introduced to back up the separation between subject and object. They cannot be found because they exist neither in the physical world nor in one's experience.What about those properties, such as intentionality and phenomenal character, that an influential philosophical tradition has attributed to the mind , while physical entities do not. Pertinently intentionality has never been found, rather it has been postulated as a magic power that would allow the mind to be about something else, a capacity not shared by physical entities. While Brentano's dualism allowed him to appeal to the alleged special powers of the mental, contemporary physicalists do not have such a freedom. In fact, the insistence on intentionality has led to the ever-popular but hopelessly misguided genre of “naturalizing intentionality”—i.e., the recurrent desperate attempts to find intentionality in a physical world that is assumed to be devoid of it—which many philosophers have criticized  intentional relation between my neural processes and the apple. I perceive the apple because, at that moment, the thing that is one and the same with my conscious experience is the apple itself. Perceiving is being. What is my experience of the apple? It is the apple. Identity is the new intentionality. Thanks to relative objects, the concept of intentionality/aboutness can finally be retired. Identity is all we need.Object-relativity allows us to set aside intentionality by embracing an identity between the mental and the physical. However, not in the sense that the mental is identical with neural processes distinct from the object one perceives, but in the more radical way that the mental is identical with the physical object itself. From this perspective there is no longer any need for the mental phenomenal character. I contend that—as with intentionality—the notion of phenomenal character was introduced to safeguard an over simplistic notion of physical objects. Yet, no one has ever experienced the alleged phenomenal character of experience. It has been another philosophical invention rather than an experiential fact.Now consider the second alleged irreducible feature of the mental: its alleged In fact, the main reason for keeping phenomenal character apart from the physical world has been that scholars have claimed that experience has a phenomenal character that does not exist in the physical world. Thus, they have claimed that, for example, my experience of red has a phenomenal character that does not exist in strawberries and cars. The main reason for such an argument was that the properties one experiences do not match with the alleged absolute objective properties of physical objects. This was only a big misunderstanding. In fact, it was not understood that properties are relative, one class of properties was selected as the real objective one. All other relative properties were downgraded to be nothing more than subjective appearances. However, I argue that, thanks to the relative nature of the object, the properties we experience can be relocated in the physical world, where we have always experienced them—namely in the external objects. So, the red is not created by neurons, the red is instantiated by the apple relative to a trichromatic visual system (which is yet another object). In this way, I argue, there is no reason to defend the existence of phenomenal character. We do not experience phenomenal properties as opposed to physical properties. In our experience, and world, there is only one red, neither phenomenal nor physics-al . No one has ever seen a phenomenal red next to a physical one. I have never experienced the phenomenal character of red. I have always experienced the red of strawberries and cars. The properties of the physical world are not different from the properties we experience day in and day out. In fact, as far as empirical evidence goes, there is only one class of properties. There is only one red.Consider colors again. Color scientists often state that colors do not exist in the physical world Galilei, . Howeverthe notion of “experience of such objects” is redundant to that of the objects themselves. Basically, the same entities are counted twice. In real life, we cannot distinguish our reference to an apple from our reference to an experience of an apple. If I want to point to my experience of the apple, I can only point to the apple I hold in my hand. It would not make any sense to point to my head, because there is nothing in my head that looks like my experience of the apple. Experience qua experience has been invented to locate the external world where it was not, namely inside our body. As a matter of fact, there are no a priori reasons to assume, as Bertrand Russell did with his version of monism , in principle nothing prevents us from considering the possibility that whenever we experience something—say when one dreams of a pink elephant—one is perceiving previous events. Previous instances of pink and elephants are the recombination that is perceived as one object. In fact, they are one object, albeit unusual. In hallucinations, one perceives a physical object that is a reshuffled combination of previously encountered objects.However, the objection still stands, when you dream of an apple today, but the apple was eaten yesterday, the apple is no longer there at the time of the dream! Thus, in such a case, it may seem that the apple cannot exist at the time of your dream. To solve this obstacle, we need to reconsider further the notion of the now (the present). There is always a greater-than-zero time span between the external object and one's brain activity in standard perception. We cannot catch flies, because they move so fast that they are always elsewhere than where we have just seen them. It is a scientific fact that standard successful perception is never synchronous with the external object. In regard to neural activity, the world we live in is always in the past and there is no fixed threshold as to by how much. When we look at the moon or the sun or the stars, the object that is identical with our experience is seconds, minutes, or years in the past, sometimes well before our body even existed. Crucially, while such objects are in the past of our body, they are in the present of our experience, which is identical to the objects rather than the body. Mistakenly, in the case of standard perception, the object we perceive is taken to be there and now, at the time and the place where our body is. Yet it is not so. The picture is a gross approximation and a physical mistake. The object we perceive is never in the same place and time where and when the corresponding brain activity occurs. It might be a few inches or thousands of miles far off. I can look at an apple on my desk or I can look at the sun or even at a faraway star. From a spatial perspective, the notion that the object is “there” can be arbitrarily stretched. “There” can mean something as far away as billions of miles.Here one might object that: “The star relative to my body is light years away, and appears so, so my experience is identical to an object several light years away. But the star is not several light years away from itself, so my experience of the star is not identical with the star itself.” Note the mistake in assuming that being away from the body means being away from the self. The objection reads “my experience is identical to an object several light years away.” Away from what? Of course, it is implicitly assumed that the object is away from me, but it is not. It is away from my body, which is the thing that I am not—unless my body is the object of my perception as is the case when I look at my hands or I focus my attention on my heartbeat.spatially, one might venture to stretch the notion of “now” temporally. In fact, every phenomenon extended in space is also extended in time. Thus, although most everyday phenomena are so close that the time delay is negligible, they are not synchronous either. In many cases, the time delay is relatively long. A familiar object like the moon is one second away from the corresponding neural activity; the sun is 8 min away; a star can be years away. And yet, they are far away from our body, not from us. They are inside our present. They are our present.Once the notion of “there” has been stretched causally present to my body rather than what takes place at the same time in which neural activity does. Thus, the star is as present as the apple, notwithstanding the different time span of the involved causal process. In memory, dreams, and hallucinations, past events are still causally present. They are as physically present as objects are in standard perception. The apple your experience is identical with it is not the apple at the time of your neural activity (there is no longer any apple), but it is the apple at the time your body met it (yesterday).As we have seen, due to nomological speed limits, everything we perceive is—to some extent—in the past. The present is not what takes place at a given time, but rather is the set of events that causes neural activity at a given time. The present is what is In short, my approach to dreams and hallucinations is straightforward—whenever S experiences O, O is present, provided that the notion of present (there and now) had been revised and stretched in causal and relative terms as suggested by contemporary physics. So dreams and hallucinations can be recast in terms of reshuffled perception of spatiotemporally spread physical objects. Therefore, they can be explained as cases of identity with spatiotemporally spread physical objects.Whenever, we experience something—be it a standard perception, a dream, or a hallucination—to the extent that what we perceive has occurred at some place and time, the object is still the cause of one's neural activity. Thus, the object may well be the thing that is identical with one's experience, no matter when and where its parts occurred.nearby or proximal objects is parochial and vague. How near should an object be to be near enough? There are not known valid thresholds. All objects and events occur previously to one's neural activity. Because of this gap, the time-lag problem kicks in. By nomological necessity, the external cause of everything that takes place in our brain is always in its past (Δt > 0). Such a past can be relatively near or very remote, as is the case with astronomical objects, but it is past nonetheless. Perception is never instantaneous. Any object is always at the beginning of a process spread over a time span and across a spatial extension. A “close enough” boundary is an empty notion. Thus, we can turn the traditional time-gap argument upside down. If stars and long-gone events are rejected as the causes of one's memories, dreams, and hallucinations, by the same token all everyday objects ought to be rejected. Such an argument, if valid, would limit one's world to the very proximal shell that surrounds one's skin and receptors, which is absurd. In fact, we do not perceive a thin layer of events enveloping our bodies. We perceive external objects wherever and whenever they are. Our experience is identical to such objects whenever and wherever they are. So, the time-gap argument may be used to show that hallucinations and dreams are forms of perception  S experiences O; and (2) O does not exist. Here I argue that the former is true, while the latter is not. Whenever S experiences O, O does exist! However, O exists where and when it likes, so to speak.For instance, take the pink building I see now, the elephant I will see tomorrow at the zoo, and a flying bird I will see next week—all together, these objects will produce an effect in my brain 1 year from now, for example the night of May 15 2019. On that night, sleep-related mechanisms will shield my body from the surroundings . As a result, those three events will cross their causal paths and produce a joint effect. They will be causally present to my brain insofar as my brain will offer them the proper circumstances for causing an effect together. Their joint effect will carve out their cause, which is bizarre-yet-physical whole composed of three aspects: pink + flying + elephant = a pink flying elephant. I will then dream of a pink flying elephant, and yet the pink flying elephant will be physical, like my son's face looking at me right now. Emilio's face is composed of two eyes, a nose, and a mouth. These are three separate objects that, because of my body in front of him and thanks to my fusiform gyrus, produce a joint effect and are brought into existence. Likewise, the pink flying elephant is made of three separate objects that, because of my body in that future bed, will produce a joint effect and will thus be brought into existence as a composite object. In standard perception objects are very often composed of parts taking place roughly at the same time. An exception is represented by music where we perceive tunes made of notes occurring at different times. In dreams and hallucinations, we are identical with objects made of parts scattered in space and time.To recap, during wakefulness, a series of mechanisms guarantee that one's world is made of only the relative proximal objects. This is useful and efficient. Normally, one is coupled with the most pressing parts of one's environment. In various circumstances, though, one's world is made of combinations of objects belonging to the whole causal story of one's body. Such combinations are just as real and physical as the more proximal objects. While they may be less useful, such combinations are as real as the former. Thus, hallucination is perception of causally reshuffled actual objects. Hallucination is perception. Perception is experience. Experience is identity.One might wonder whether the claim that hallucination and perception are the same is not too radical. In fact, the prevailing stance in neuroscience is the same, namely that perception is like hallucination. Most neuroscientists and philosophers of mind—e.g., Koch, Tononi, Seth, Eagleman, Revonsuo, Zeki—have claimed that perception is a form of reliable hallucination. They have claimed that the world we see is a mental representation created by the brain. Thus, it would be unfair to challenge the presented proposal on this issue and not to do the same to such views. Crucially, these neuroscientists claim that perception is like hallucination, while the Spread Mind claims that hallucination is like perception.As to the relevance for psychology, I'd like to add that, in this account, hallucination differs from perception on a practical level while being the same on an ontological level. In traditional accounts, hallucinations are mental representations triggered by internal causes and perceptions are mental representations caused by external objects. According to the spread mind, hallucinations are external objects causally connected to our body through relatively unusual pathways, while perceptions are external objects causally connected through familiar pathways. Hallucinations are akin to seeing objects in a kaleidoscope or in a reflection. One sees real objects, only that, because of reflections, one sees them in the wrong place. Yet, one still sees actual objects. Seeing an object in a mirror is not ontologically different from seeing something in standard conditions. In both cases there are light rays connecting the object with our retina.The good news is that this model is amenable of empirical verification and thus it can be falsified. If one ever hallucinated something whose elementary constituents were not part of our physical relative world, the presented model would fail. So far, though, the empirical evidence points in the opposite direction—everything we experience is made of parts that have been part of the world we have lived in and so it is still causally present relative to our bodies.per se—a point already well-taken by Daniel Dennett  that experiences, we are the objects that exist relative to our bodies. The body does not feel. The body is the physical condition for the existence of the things our experience (us) is identical with. Once we embrace this conceptual shift, many objections will lose their apparent intuitive strength.A first group of objections is raised around the concerns that our experiences might possibly be far away in space and time from our bodies. A different way to illustrate the same concern is to point out that conscious experience cannot be stretched at huge distances as the proposed theory implies. Some authors would be more comfortable if the experiential present was stretched only over a local portion of space and time. They might find it difficult to accept that the present can extend out to a star that may have ceased to exist by the time light from it hits the perceiver's retina. I have already addressed this issue. Once again, such objections are the outcome of assuming that, whatever we are, our conscious mind is roughly located where our body is. Yet, this is precisely the matter of contention. In fact, my proposal denies explicitly the colocalization of body and experience. Our experience is not where our body is. Our bodies are the condition for the existence of the things that are identical with our experiences, no matter where and at what time they are located. Bodies are not the place where our experience is.Many readers move from the commonsensical notion that the physical basis of consciousness is located in a relatively small spatiotemporal area centered around the brain, roughly a few centimeters large and a few hundreds of milliseconds long. Yet, such a commonsensical notion is based on two implicit unwarranted assumptions: (1) that consciousness is located inside the head , (2) that the physical processes leading to conscious experience must be comparable in size and time to the processes taking place in a human body . Both assumptions are unsupported by evidence. In fact, why should it be so? Is there any physical law that dictates that physical phenomena have only a limited spatial length and time span? No. Is there any proof that we are inside our brain? No. The notion of a “local portion of space and time” can appear familiar and reassuring, but one may still ask “local to what?” Implicitly, the notion of being local implies to be close to the supposed locus of one's mind. But this makes sense only if it is assumed beforehand that such a locus is inside the brain. Conceptually, if consciousness is relocated where the object is—because the experience is identical with such an object—there will be no more distance between “us” and the “external object.” Our experience, which is us, will occur where the object is—the two being identical. So, there is no reason to deny placing experience far away from the body. There will be no distance whatsoever between such objects and us. We are “there” where the object is, rather than “here” where the body is. So, if my experience is a relative physical property instantiated by the star relative to my body, my experience will not be light years away from me, it will be light years away from my body—the far away stars and my experience being the same. Clearly, for the layman “here” does not refer to the location where my experience is, but to the location where my body is.As to whether many years and many light years should be less acceptable than a few cm and fractions of seconds, as it is routinely accepted in neuroscience, one can only point out that the difference is a parochial notion unsupported by any physical evidence. Between the far away star and our eyes there is a photon. Between the apple on the table and our eyes there is a photon too. What is the difference? The length of the photon? Surely nobody will seriously argue that short photons are fine and long ones are not! Likewise, the idea that there is a timeframe in which current experiences happen is parochial. The timeframe in which an effect takes place in our body  is not the timeframe of the cause that is identical with one's experience. Neural processes take place in a time-limited timeframe, but such a timeframe refers only to the last part of a longer physical process, whose onset (and not its end) is the physical entity identical to our experience. we're the cause, not the effect.Another related group of objections is centered about the notion of agency, a huge topic I can hope only to mention here. One might worry that if our experiences were nothing but the (more or less far away) objects, we would be acted by the objects themselves. The short answer is no: we are not acted by the objects, we are identical to them. As aforementioned, we are the cause rather than the effects. A longer answer would require one to dig deeper into the causal structure between external relative objects and bodies. So, for example, while my experience of the star may cause me to sing, because of the proposed identity, the star itself is the cause of my singing. One may be puzzled by this statement since one may assume that causes require proximity, and thus the photons that hit my retina cause the experience that cause the singing. Yet, why should only proximal causes be the relevant ones? In our everyday life, we do not apply the notion of causal proximity. Otherwise perfectly reasonable statements such as “The asteroid that hits the earth 75 million years ago caused the development of mammals” or “Smoking from 1950 to 1990 caused my aunt to die of emphysema in 2004” would be rendered meaningless. Reasoning in terms of causal proximity would have nefarious consequences for internalism too—our actions would be acted by the very last neural activity in the chain, which is equally absurd.However, it might be objected that the problem of identifying experience with the object itself is that we want to be the case that our experiences cause things, e.g., Jack salivates upon seeing a juicy apple. The reply is that Jack's body salivates, not Jack. Jack is the cause that makes Jack's body salivate. Jack's experience is the apple that Jack's body brings into existence. What's the cause of Jack's body salivating? The apple. Thus, strange as it may seem, it is the apple that acts through Jack's body, which is consistent with the claim that objects are one's experience. Since the apple is Jack, everything adds up. It's Jack that make Jack's body salivate. So the identity between objects and experiences is consistent with experiences being the cause of one's actions.O exists relative to S's body. Here, experience is revisited in terms of physical existence, which is what should be expected. In passim, traditional mind-brain identity theories aimed to do so. So, the proposal is that the physical thing one calls “my experience of the apple” is “the apple that exists relative to one's body.” There is no longer the need to postulate a subject—be it the brain, the subject, or an immaterial self—who experiences the world. There are only lumps of objects that exist relative to bodies, or relative to other objects. Yet, bodies do not experience. Brains do not feel. Eyes do not see. In fact, nothing experiences, feels, or sees. Things exist though. Things exist relative to bodies, brains, and eyes. Ontology is phenomenology as it should always have been in a physical world. The existence of such relative physical objects is identical to what has been called the experience of the same objects. Experience is relative existence.Of course, I do not mean that such relative objects (or objects with relative properties) have experiences. I do not mean that, since the experience of the apple is the apple that exists relative to my body, then the apple experiences. That would be both crazy and question begging. Objects do not have experiences, rather our experiences are identical to the properties of the external object relative to our body. The proposed account maintains there is no such a thing as “S experiencing O.” The proposal is that “S experiencing O” is a way to refer to the fact that One may object that it is not any easier to claim that there is an identity between object properties and experience than between brain processes and experience. Yet, this impression may result from assuming that the properties of the object are of absolute nature rather than being relative properties carved out by the causal relation with one's body.While the former do not coincide with our experience, the latter are just what our experience is. One may worry that the surface of the apple is not like my experience of the apple, yet the surface is not a relative property of the apple. The surface is the alleged objective feature of the apple, which is independent of the visual system with which it interacts. The surface as it takes place relative to the visual system of a trichromat is a different entity/property and it is just the kind of thing we find in our experience. Or so I claim.As to the latter part, I want to stress once again the difference between physical properties as something that is absolute and physical properties as something that is relative to one's body. While the former does not coincide with our experience, I claim that the latter is just what our experience is. I mean, the surface is not the relative property of the apple. The surface is the alleged objective feature of the apple, which is independent of the visual system with which it interacts. The surface as it takes place relative to the visual system of a trichromat is a different entity/property and it is just the kind of thing we find in our experience. Or so I claim.Likewise, we do not need to smear the relative object over the whole causal process, because relative objects do not have to reach the body as the magic gate to one's consciousness. The idea that the external object must reach the body is—once again—based on the idea that we are located where our body is. On the contrary, the body is the fulcrum of a causal chain, whose cause is external to it. So, while photons are not red, and neither are apples+photons+eyes+brain, there must be something that is red, otherwise what will we see? The red I see there is the relative property that the apple instantiates relative to my body. The red is the cause of a neural process that provides the condition for the existence of that specific property—no matter how long and complex the causal chain is. If the red thing is Mars, so much the better.Likewise, one might disagree that an object does not instantiate a given property until such a property becomes the target of one's experience. Not exactly. Rather, an object does not have a given property until the conditions for the existence of such property are met. For instance, would an isolate particle have any velocity if there were no other particles around? Of course not. Would it make any sense to ask what the velocity of an particle is before another particle enters the scene? Of course not. There was no velocity in particle A until particle B was included. The same holds true for any other property. Consider color. Imagine a red ruby on a planet many light years away from earth, on which there are neither humans nor alien trichromat observers. What could we say about the color of the ruby? Does it have any color waiting to be seen? Of course not. Color exists only relative to the right physical system, which is missing on that planet. At some point, a trichromat is born on earth and points his telescope toward this distant planet and observes the ruby. At that moment, for the first time, the color property of the ruby takes place actually and relatively to the body of the trichromat at the other end of the telescope. The ruby has existed light years away and possibly even before the trichromat observer was born. Was it colorless? Yes, just like an isolated particle was velocity-less until considered relative to another particle. When the trichromat is born and focuses his telescope on the planet with the ruby, color takes place. When and where exactly? On the far away planet at the time when the light started its journey, no matter how many years in the past. Red comes into existence when it takes place relative to a proper external object.One further objection might stem from a confusion between thinking and experiencing. The spread mind is a hypothesis about the nature of our experience—i.e., consciousness or phenomenal experience, it does not address the issue of thinking and cognition. In fact, while thinking may be accompanied by some experience, it is not like having an experience of what thoughts are about. A congenitally blind subject can think about colors without any experience of colors . One may think of Atlantis only to find out that the lost sunk island was only a literary invention. One may think of the concept of a dinosaur existing millions of years ago, but that does not entail any experience of the dinosaur as it was millions of years ago. One can see a picture of a dinosaur or the skeleton of a dinosaur exhibited in a museum. They are all events taking place in one's environment. In brief, thinking about something and experiencing something are two separate issues. The Spread Mind addresses the latter issue.Yet, one may still worry that experience may be requested to move from one place in the universe to another at impossible speed. To clarify, what does it happen when one looks up to the sky and is seeing a star located in a distant part of our galaxy (say location A) and then one moves the eyes just slightly and is seeing another star right (location B). But while the starts are right next to each other in one's optic, the stars themselves are light years apart. Does this imply that, as one's experience moves from one star to another, experience has covered several light years in an instant? Of course not. Experience does not move from A to B. Rather experience is made of a star at location A at time t1, and then it is made of another start at location B at time t2. There is no substance corresponding to one's experience moving from point A to point B. The two experiences are bound by having two effects at close temporal and spatial distance in one's brain. What matters, from a physical perspective, is the causal processes going from the start to one's brain and from one brain event to the next. These causal processes do not contradict any physical laws. The case is similar to the difference between causal processes (which are constrained to light speed) and pseudoprocesses (which are not constrained by light speed) as they have been described by Hans Reichenbach . So, it is possible that, say, a faraway star and a very modest light source at a shorter distance will instantiate the same relative property and thus they will be the basis for  the same experience. They will be the same experience because they will be nothing but the same relative property. Identity entails constitution as well as supervenience. Experience is not caused by object, it is identical to them.A Materialist Theory of the Mind, by asking the question: “What is a man?” His prompt reply was that a man “is a certain sort of material object” (Armstrong, In 1968, David Armstrong began his seminal book, This paper endorses Armstrong's premise that consciousness is physical, but it proceeds in a radically different direction. Here I have defended an identity theory—only that rather than being a mind-brain identity it is a mind-object identity theory. The material object that is a man is no longer the human body but rather the external object that exists thanks to the causal circumstances offered by one's body.The presented mind-object identity moves from a revision in the way we understand physical objects which is consistent with both empirical evidence and contemporary physics. Conscious experience is not extraneous and incongruous to the physical world. I do not try to naturalize consciousness; rather I propose to revisit the notion of material object in such a way that experience can be found inside the physical world.My main claim is that what has been traditionally called “subjective, in the sense of being relative to a subject” can be replaced with “relative, in the sense of being relative to another object.” In the case of human beings, our body provides the required object relative to which the world instantiates the properties we are familiar with.Previous naturalistic frameworks could not explain conscious experience which was assumed to be extraneous and to some extent alien to the natural order, thus spawning a progeny of chasms: subject vs. object, mental vs. physical, mind vs. nature. While such ontological chasms may be flattering for narcissist subjects keen to be set apart from the world Freud, , they arhidden in plain sight.In conclusion, when I perceive a red apple, what, where and when is my experience of the apple? What is the least expensive ontological candidate? My answer is that my experience is identical with the external physical object that exists relative to my body. The relative object is an ideal candidate for identity with one's experience. Nothing is ontologically closer to our experience of the world than the objects the world is made of. Once we set aside the ancient prejudices that our minds are roughly where our bodies are, it is quite straightforward to see that consciousness is—and has always been—there, all around our body, The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer SB and handling editor declared their shared affiliation at the time of review.


Pin1 is a peptidyl-prolyl isomerase responsible for isomerizing phosphorylated S/T-P motifs. Pin1 has two domains that each have a distinct ligand binding site, but only its PPIase domain has catalytic activity. Vast evidence supports interdomain allostery of Pin1, with binding of a ligand to its regulatory WW domain impacting activity in the PPIase domain. Many diverse studies have made mutations in Pin1 in order to elucidate interactions that are responsible for ligand binding, isomerase activity, and interdomain allostery. Here, we summarize these mutations and their impact on Pin1′s structure and function. The N-t(PPIase) . Connect(PPIase) ,5. The p(PPIase) ,7,8. An (PPIase) ,10. LigaPin1 is a very promiscuous mitotic regulator, and regulates phosphoproteins through changing the phosphorylation/dephosphorylation state, as well as protein stability through either enhancing or protecting against ubiquitin-mediated proteasomal degradation . Pin1 isIt is clearly apparent that Pin1 is capable of binding and isomerizing many different substrates, all with the commonality of a pS/T-P motif . DespitePin1 has been very extensively studied for the last 20 years, with most structural work aimed at understanding the dynamics and allostery of this two-domain protein. Various studies have been performed looking at Pin1 binding to many different ligands. In this review, we focus specifically on a summary of which mutants in Pin1 impact catalytic activity and ligand binding affinity and to what extent. As such, this review highlights mutations made in biochemical and structural studies. We hope this review aids in computational studies and methodological developments in order to characterize an allosteric system on a per residue basis.The association of variants with specific diseases is outside the scope of this review. However, the interested reader is referred to an excellent review on single nucleotide polymorphisms and mutations of Pin1 in cancers . Many otDK of the two events must be different by orders of magnitude, as if they are too similar they are indistinguishable. Even if two binding events were measured using either of these two methods, these methods alone would not be enough to disentangle which binding affinity corresponds to each binding site. In all the studies that have reported binding affinities of Pin1 using fluorescence anisotropy or ITC, only one binding affinity was reported for the whole protein, likely because the binding affinities of each domain are in the same order of magnitude, resulting in an effective DK. The smaller the DK value, the larger the binding affinity between the two molecules.cis-trans and trans-cis cross and the respective diagonal peaks in a NOESY experiment using many different mixing times [1H chemical shifts of ligand are assigned without enzyme present. The 1H chemical shifts are often distinct for trans and cis species due to slow exchange, typically with peak intensity ratios around 10:1 trans to cis. During a NOESY experiment, usually used to measure through-space interactions, each hydrogen is frequency labeled by its respective chemical shift and after a certain mixing time (time given for isomerization to occur), the hydrogens are frequency labeled again. If the proton isomerized to a different chemical shift during that time, there will be a cross peak between the two distinct frequencies. For example, if a ligand is in the trans conformation during the first frequency detection and then undergoes isomerization during the mixing time, the cis conformation is detected at the second frequency labeling event. Isomerization of a ligand can occur without enzyme present, but this is often a slow process (seconds-minutes) that is unlikely to occur during a NOESY mixing time (milliseconds-seconds). For pCDC25c and FFpSPR ligands, no cross peaks are detected without enzyme present. Once Pin1 is added, cross-peak intensities can be measured at various mixing times to extract rates of exchange. A perk of this experiment is that both c→t and t→c rates can be measured, and the sum of which is the exchange rate (kCT + kTC = kEXSY). While many studies report both kCT and kTC, some studies only report kEXSY. Therefore, in the bulk of this report we look at kEXSY values for ease of comparison, but kCT and kTC values are reported in the kEXSY value, the higher is the activity of the enzyme.To quantify Pin1 isomerase activity, most mutants were studied using NMR exchange spectroscopy (EXSY) by fitting the ng times . Before kcat/KM has been performed through a chymotrypsin-coupled chromophoric assay of p-nitroaniline (pNA) [trans conformer. Therefore, the substrate used to study Pin1 activity is suc-AEPF-pNA, with glutamate acting as a phosphomimetic. A similar assay can be done with trypsin when peptides contain Pro-Arg-pNA motifs, like with peptide WFYpSPR-pNA [kcat/KM, the higher is the activity of the enzyme.Another method to measure activity by determining ne (pNA) . ChymotrpSPR-pNA . SupplemBoth domains have been studied in isolation, where they have different binding affinities and activities than the full-length (FL) protein A. It sho−1s−1, it increases to 20,160 mM−1s−1 for WFYpSPR-pNA , only WT Pin1 WW had any measurable affinity using isothermal titration calorimetry with a DK of 35 ± 25 μM. Of the other variants that were measured, none showed any measurable binding and heat transfer via ITC. The authors conclude that the WT WW domain is tuned for phospho-peptide binding at the expense of increased folding time and stability [A study was performed to investigate the structure-folding relationship of the WW domain specifically looking at the WW binding loop (residues 17–20) as it is the rate limiting step of folding for this domain . In thistability .Full-length mutant W34A was investigated in other studies with ligand pCDC25c, and was found to have reduced activity compared to WT Pin1 . W34 appOverall, it can be concluded that structural integrity of the WW β-sheet is well tolerated for most mutations except for W11, Y24, N26 and P37. Mutating and/or truncating the WW binding loop (residues 17–20) and mutating S32 and W34 severely impacts substrate binding.Unlike the mutations studied in the WW domain involving full-length Pin1, mutations evaluated in the PPIase domain were from a mixture of studies with either the isolated PPIase domain or full-length Pin1. For the analysis of each mutation, it is noted whether this mutation was studied in the isolated or full-length construct. The C-terminal PPIase domain is residues 50–163, and contains a β-sheet core with surface-exposed α-helices A. The PPUnigenic evolution using random mutagenesis in yeast showed three hypomutable regions in the PPIase domain: R56-P70, A107-G120, and F139-I158 . In addiCysteine 113 has been noted as a key residue for the activity of Pin1, yet a C113D mutation still maintains some isomerase activity in the isolated PPIase domain . CysteinWhile it is clear that residue C113 participates directly in catalysis, the role of residues H59 and H157 in the active site is less obvious. The original hypothesis was that these histidines stabilize the transition state of the ligand . InitialThe tumor suppressor death-associated protein kinase 1 (DAPK1) has been shown to phosphorylate S71. This phosphorylation event reduces the catalytic activity of Pin1  as residThe combination of mutation studies show that residue C113 is important for catalysis, residues H59 and H157 are vital for the structure of the catalytic site, and K63, R68, and R69 are responsible for phospho-substrate binding.The two domains of Pin1 are connected via a flexible linker (residues 40–49), and there is an interdomain interface where the two domains are transiently associated. The interdomain interface is comprised of residues 28–31 of the WW domain, and 137–142 of the PPIase domain. It has been suggested that this interface is responsible for the allosteric connection between the two ligand binding sites .The most well characterized mutant in the ID interface is I28A. Mutating this residue causes chemical shift perturbations in the ID interface of the PPIase domains (residues 137–141), and decreases ID contact between the two domains ,8. This kEXSY = 74.8 s−1) than 40ΔNSSSG (kEXSY = 66 s−1), but this discrepancy was not addressed [Due to the higher activities of the isolated PPIase domain and the I28A mutant compared to WT Pin1, there was a hypothesis that interdomain contact negatively regulates catalysis since I28A causes a further extended state and the isolated PPIase domain has no interdomain contact ,8. This ddressed .in vivo study compared the activity of WT, S138E (phospho-mimetic), and S138A (phospho-deficient) Pin1 and showed that S138E had 4 times greater catalytic activity than WT Pin1 in a cellular assay, and the S138A mutant had 2-fold decrease in activity [E. coli and was extremely unstable at room temperature, therefore it was unsuitable for NMR studies. These challenges were not present for S138A-PPIase mutant [Another mutation investigated on the isolated PPIase domain was S138A, which is proposed to mimic the structural impact of the WW domain ID contact. S138 is located in the ID interface of the PPIase domain. Pin1 is phosphorylated at residue S138 by mixed-lineage kinase 3 (MLK3) which results in an increase in catalytic activity of Pin1. An activity . The S13e mutant . The S13e mutant  N-HSQC, e mutant . This woThe I28A and S138A mutations disrupt the interdomain allostery, which results in higher isomerase activity. The linker length between the two domains does not appear to have a clear correlation to the isomerase activity.cis or trans state by replacing the peptidyl-prolyl linkage by alkene isosteres [cis- and trans-locked inhibitors, the WW is specific for the trans [cis- and trans-locked inhibitors, suggesting that the binding modes for the two states are different. As the PPIase domain has a preference to bind cis-locked FFpSPR (lower DK), when trans-FFpSPR is first titrated in, which binds to both domains, subsequent addition of cis-FFpSPR displaces the trans state from the PPIase domain, and the cis binding signatures become apparent. The PPIase has about 10 times higher affinity for the cis-inhibitor than the trans-inhibitor (7 μM versus 66 μM), and this could be a contributing factor to why the exchange rate between cis-trans (kCT) is typically 10 times higher than trans-cis (kTC), with WT Pin1 usually having values reported about kCT = 40 s−1 and kTC = 4 s−1 [An in-depth study of non-isomerizable ligands was investigated on full-length Pin1, as well as its isolated domains. Ligand FFpSPR has a very similar sequence to Pintide, WFpSPR, but has increased solubility. Chemists were able to “lock” the proline in either a sosteres . These nhe trans . In addi = 4 s−1 . Even upDK ~ 6–10 μM), FFpSPR has a much greater affinity in the FL WW (DK ~ 43 μM) compared to the isolated WW (DK ~ 246 μM). This suggests that that PPIase domain has a positive impact on the WW domain’s ability to bind FFpSPR, while the PPIase domain doesn’t have an effect when the WW binds to pCDC25c. This is potentially due to the fact that FFpSPR stabilizes the compact state (increased interdomain contact) and this contact allosterically changes the WW binding site to enhance binding. Since the extended state (decreased interdomain contact) is preferred for pCDC25c, there isn’t a mechanism for the PPIase to strengthen the affinity of the WW domain. While the isomerase activity of FL Pin1 and the isolated PPIase domain is somewhat variable depending on the study, we can confidently conclude that isomerization of pCDC25c is higher in the isolated PPIase than FL Pin1 (kEXSY range of ~37–58 s−1 versus ~26–44 s−1). On the other hand, the kEXSY of FFpSPR is between 44–97 s−1 for FL Pin1 while only a single measurement of 91 s−1 for the isolated PPIase. Therefore, no confident conclusions can be made in regard to FFpSPR effect on activity, but at first glance it appears it doesn’t have much of a change on activity compared to pCDC25c. Ligand pCDC25c causes a stabilized extended state with less interdomain contact, and the isolated PPIase doesn’t have any interdomain contact (since the WW is absent). A primary hypothesis is that the WW domain is a negative regulator of the PPIase activity in the compact state, and the extended state has higher isomerase activity  [There is a plethora of evidence suggesting that ligands pCDC25c (sequence EQPLpTPVTDL) and FFpSPR have different allosteric pathways in Pin1, with the former stabilizing the extended state and the latter stabilizing the compact state . InteresThe changes in affinity and activity of Pin1 upon mutations are mapped on the Pin1 structure in Conclusively, this review summarized mutations previously studied in Pin1 with their impacts on affinity and activity, and we hope other researchers can use this to evaluate and develop models to describe allostery. A suitable model for allostery would have to be able to explain the extensively mapped out effects of the mutations. This overview could also serve as an aid to make further mutations. Some future projects could look at the double mutants I28A and A140I, as these two residues have been predicted as a co-evolving pair and could give further insights into interdomain allostery . Previou


MuRF1 (TRIM63) is a RING-type E3 ubiquitin ligase with a predicted tripartite TRIM fold. TRIM proteins rely upon the correct placement of an N-terminal RING domain, with respect to C-terminal, specific substrate-binding domains. The TRIM domain organization is orchestrated by a central helical domain that forms an antiparallel coiled-coil motif and mediates the dimerization of the fold. MuRF1 has a reduced TRIM composition characterized by a lack of specific substrate binding domains, but contains in its helical domain a conserved sequence motif termed COS-box that has been speculated to fold independently into an α-hairpin. These characteristics had led to question whether MuRF1 adopts a canonical TRIM fold. Using a combination of electron paramagnetic resonance, on spin-labeled protein, and disulfide crosslinking, we show that TRIM63 follows the structural conservation of the TRIM dimerization domain, observed in other proteins. We also show that the COS-box motif folds back onto the dimerization coiled-coil motif, predictably forming a four-helical bundle at the center of the protein and emulating the architecture of canonical TRIMs.  •The dimerization domain of MuRF1 (TRIM63) is an antiparallel coiled-coil.•The COS-box motif folds back onto the center of the dimerization coiled-coil motif.•Proteins in the classes CI and CII of the TRIM family exhibit a canonical TRIM fold. The TRIM protein family is the largest family of RING E3 ubiquitin ligases C signifies C-terminal subgroup) according to their domain composition TRIM proteins are defined by their domain composition. Invariably, they share a tripartite fold consisting of a RING domain (R), one or two RING-like B-box domains (B), and a helical domain (HD) that forms a coiled-coil motif 4TN3; The ubiquitin ligase activity of TRIM proteins is reliant upon the placement of the RING domain with respect to the substrate-binding domain. There are currently no crystal structures of full-length TRIM proteins, but several structures of TRIM components are available that reveal the organization of the TRIM fold (reviewed in Ref. Muscle-specific RING Finger proteins (MuRFs): MuRF1, MuRF2 and MuRF3 that regulate the trophicity of striated muscle tissue. Because of its patho-physiological significance, MuRF1 is the best-studied member of the family. MuRF1 is strongly upregulated by atrophic stimuli, and it has been associated with the muscle atrophy that ensues upon immobilization, denervation, nutritional deprivation, aging or chronic disease 3Q1D) showed the domain to form dimers. A distinct feature of MuRFs is that the C-terminal fraction of their HD domain (spanning predicted helices H2–H3) contains a conserved sequence motif, termed the COS -box ab initio structure prediction for the COS-box of MuRF1 TRIM proteins in class C-II are unusual in that they lack variable C-terminal domains The measurement of distances between nitroxide groups (introduced in proteins using site-specific labels) by pulsed electron paramagnetic resonance (EPR) is an established technique for the sampling of protein folds. This method often makes use of the pulsed electron–electron double resonance (PELDOR) protocol, also called double electron–electron resonance (DEER) In this study, we employed disulfide cross-linking and the PELDOR experiment to investigate the quaternary structure of the MuRF1-HD dimer and the fold of its COS-box. Using site-directed mutagenesis, native cysteine residues were removed from the MuRF1-HD construct and new cysteine groups introduced at desired positions for labeling with MTSSL. Positions were chosen in this study guided by known TRIM structures taken from The Protein Data Bank MuRF1 is composed of a RING domain in N-terminal position, a MuRF-specific helical motif, a B-box type II (B), an HD that includes the COS-box sequence motif, and a disordered C-terminal acidic tail (AT) . The HD in silico. Matching the simulated subpopulations of spin labels to the features of EPR distance distributions provides an insight into the nature of their various peaks and permits identifying possible outlier features. In the absence of an atomic structure, the modeling of spin labels can be performed using homology models with caution.To obtain a more detailed characterization of the MuRF1-HD fold, we sampled intermolecular distances across protomers in the dimer using EPR and applying the PELDOR protocol. The PELDOR experiment measures the dipolar coupling between the unpaired electrons in spin-labels introduced in proteins. The data can be interpreted as a distance and a distance distribution, if the data are of sufficient quality. The application of the data provided by PELDOR to the interpretation of the underlying protein structure is often complicated by the conformation and dynamics of the flexible spin labels. The dynamics of the latter is not necessarily homogeneous. Spin labels can form spatial subpopulations, possibly due to steric hindrance effects or unspecific interactions with the protein surface, which yield split distance distributions of complex appearance. In such cases, it is the modal distance and the shape of the distance distribution that are informative, with an experimental distance error factor not being directly deductible from the distributions themselves. When a reliable atomic structure of the protein under study exists, a complex distance distribution can be studied through the modeling and molecular dynamics simulation of spin labels introduced into the structure In order to study MuRF1-HD using EPR, nine residues in the cysteine-null MuRF1-HD segment were exchanged for cysteines using site-directed mutagenesis: E192C, E200C, K212C, E222C, R230C, E243C, K297C, E313C and R320C . The resNext, we analyzed the MuRF1 inter-label distances recorded by EPR by investigating their compatibility with the fold of other TRIMs characterized structurally to date. For this, homology models of MuRF1-HD were calculated with Modeller In most of the previously published TRIM protein structures A, the poTo further validate the conformation of the COS-box of MuRF1, we performed a triangulation experiment by determining distances using a double label strategy with labels placed at E192R1 and R320R1 simultaneously. Because measurement in systems containing more than two spin labels can be problematic The TRIM fold appears to be an anisometric, rod-like fold, where functional domains are organized onto a central antiparallel coiled-coil of approximately 18 nm length. Only a few examples of partial structures from this family are known to date, the first structures from the central TRIM rod domain being reported in 2014 Here, we have studied the HD dimerization domain of MuRF1 (TRIM63), a small TRIM protein of class C-II that lacks C-terminal specific domains but contains a conserved sequence motif, the COS-box. Earlier data on MuRF1 6-tag and a SUMO domain N-terminal to the inserted gene. A cysteine-null MuRF1-HD sample was generated by exchanging native cysteine residues (C173 and C293) into serines using QuikChange® (Agilent). For cross-linking and EPR analyses, cysteines were introduced into the cysteine-nullMuRF1-HD at positions F163, V184, E192, E200, S202, K212, E222, L228, R230, Q235, E243, L253, S256, A263, L276, K297, E313 and R320, using the same mutagenesis protocol. All expression plasmids were confirmed by sequencing.The HD of MuRF1, MuRF1-HD , was cloned into a modified pET28a vector (Novagen) that adds a HisEscherichia coli BL21 (DE3) (Agilent). Cells were cultivated in Luria-Bertani medium supplemented with 50 μg/mL kanamycin at 37 °C to an OD600 = 0.8. Protein expression was induced using 0.2 mM IPTG, and cultures were grown further for 3.5 h at 37 °C. Cells were harvested by centrifugation and lysed by sonication in 50 mM Tris–HCl (pH 8.0), 500 mM NaCl, 5% (v/v) glycerol and 5 mM β-mercaptoethanol in the presence of an EDTA-free protease inhibitor cocktail (Roche). The lysate was clarified by centrifugation and subsequent filtration. The purification of all proteins from supernatants followed Ni2+-chelating affinity chromatography. The His6-SUMO tag was cleaved with His-Ulp1 protease in overnight dialysis  at 5–7 °C and the proteolyzed mixture subjected to subtractive Ni2+-NTA chromatography.All proteins were expressed in Double-cysteine mutant proteins were reduced by dilution to 20 μM in reducing buffer  and then dialyzed overnight at 4 °C into the same buffer containing no reducing agent. Aliquots were then mixed with the same volume of 2 × SDS-PAGE sample buffer containing either 0 (nonreducing) or 1 M β-mercaptoethanol (reducing), incubated for 5 min at 99 °C in a dry bath and immediately analyzed by SDS-PAGE with Coomassie-blue staining.Cysteine residues were reduced by incubation in 5 mM DTT for 2 h at room temperature. DTT was removed with size exclusion chromatography using a 10/300 Superose S12 column  in labeling buffer [20 mM Hepes (pH 6.8), 100 mM NaCl). MuRF1 constructs were then labeled by incubation with a 10-fold molar excess of MTSSL for 2 h at room temperature. Excess label was removed by dialysis in 20 mM Hepes (pH 8.0) and 100 mM NaCl at 5–7 °C overnight.2O, using centrifugal ultrafiltration. Buffer exchanged samples were concentrated to a final concentration of 100 to 200 μM MuRF1 dimer in a volume of 50 μL and mixed in a 1:1 (v/v) ratio with D8-glycerol. For PELDOR measurements, 75 μL of each sample was transferred to a quartz tube and flash frozen to form a glass. MuRF1 labeling efficiency was assessed by Continuous Wave (CW) EPR using an amino-TEMPO standard as reference  operating in the 1.5–300 K temperature range. Pulses were amplified using a pulsed traveling wave tube amplifier with a nominal power output of 150 W. The four-pulse, dead-time free PELDOR sequence was used, with the pump pulse frequency positioned at the maxima of the nitroxide spectrum. The frequency of the observer pulses was incremented by 80 MHz relative to the pump position. The observer sequence used a 32-ns π-pulse; the pump π-pulse was typically 16 ns. The experiment repetition time was 4 ms, and the number of shots at each time point was 50. The number of time points and the number of scans used were varied for each sample, but sufficient data were collected to obtain an adequate signal-to-noise ratio. Data were analyzed using the DeerAnalysis 2013 software package P(r).PELDOR experiments were performed using a Bruker ELEXSYS E580 spectrometer operating at Q-band with a cylindrical resonator ER 5106QT-2w and a Bruker 400-U second microwave source unit. All measurements were taken at a temperature of 50 K with an over coupled resonator giving a P(r), were calculated from the resulting MuRF1 homology models using MTSSL-Wizard Pairwise sequence alignments were performed using the EMBOSS Needle server d residues are boxed in red. For each TRIM protein, helical sequences have been colored , according to features observed in the respective PDB structures for TRIM25, TRIM69, TRIM20 and TRIM5α. For TRIM63, helices have been colored according to secondary structure prediction from Jpred (http://www.compbio.dundee.ac.uk/jpred).Alignment of TRIM-HD sequences. Heptad positions  The raw PELDOR data with background correction function. (B) Background corrected data with fit to the distance distribution, and (C) the distance distribution with background validation, for construct labeled at residues 192 and 320. The lower and upper error bounds (two times standard deviation) are displayed as gray error bars. (D) The Pake pattern derived from the background corrected data with fit. (E) The L-curve used to determine optimum fit characteristics. (F) A comparison between the experimentally determined distance distribution (black) and the distributions calculated from the model based on PDB 4LTB, with spin labels added using MtsslWizard Fig. S2PELDOR data. Tikhonov regularization of the PELDOR data. Column 1 shows the Pake patterns, and column 2 shows the L-curves for the data shown in Fig. S3PELDOR data from label positions E243R1 and K297R1. The PELDOR data derived from these positions gave data that are insufficient to derive an accurate distance but are indicative of label distances being in excess of 8 nm.Fig. S4Results of the background verification using DeerAnalysis on sample E192R1. The lower and upper error bounds (two times standard deviation) are displayed as gray error bars. Mean distance, 76 Å; Std, 2.4 Å.Fig. S5CW spectra for samples used in PELDOR distance estimations. CW spectra were taken during preparation and labeling of samples to estimate labeling efficiency. Comparison to a sample of free R1 label was used with double integration to estimate the concentration of spin label which, in concert with an estimation of protein concentration taken from UV measurement and predicted absorption coefficient, was used to estimate the efficiency of labeling. Labeling efficiency was estimated to be 90% or greater for all samples, except that for R320 which appeared to be lower judging from the PELDOR oscillation depth. Some samples contained small amounts of free label, which would distort the calculation, but at the levels observed, had little effect on the PELDOR experiment. PELDOR experiments resulted in oscillation depths of 0.3 and 0.15 giving sufficient signal to determine distances, which are otherwise unaffected by labeling efficiency.Fig. S6The following are the supplementary materials related to this article.