The highly modular and stepwise folding process of such a large, complex protein explains the relatively high fidelity and correctability of its folding.The question how proteins fold is especially pointed for large multi-domain, multi-spanning membrane proteins with complex topologies. We have uncovered the sequence of events that encompass proper folding of the ABC transporter CFTR in live cells by combining kinetic radiolabeling with protease-susceptibility assays. We found that CFTR folds in two clearly distinct stages. The first, co-translational, stage involves folding of the 2 transmembrane domains TMD1 and TMD2, plus one nucleotide-binding domain, NBD1. The second stage is a simultaneous, post-translational increase in protease resistance for both TMDs and NBD2, caused by assembly of these domains onto NBD1. Our assays probe every 2–3 residues (on average) in CFTR. This in-depth analysis at amino-acid level allows detailed analysis of domain folding and importantly also the next level: assembly of the domains into native, folded CFTR. Defects and changes brought about by medicines, chaperones, or mutations also are amenable to analysis. We here show that the well-known disease-causing mutation F508del, which established cystic fibrosis as protein-folding disease, caused co-translational misfolding of NBD1 but not TMD1 nor TMD2 in stage 1, leading to absence of stage-2 folding. Corrector drugs rescued stage 2 without rescuing NBD1. Likewise, the DxD motif in NBD1 that was identified to be required for export of CFTR from the ER we found to be required already The online version contains supplementary material available at 10.1007/s00018-022-04671-x. Membrane proteins account for one-third of all of the proteins encoded by the genome of sequenced species. They mediate and integrate fundamental processes occurring on both sides of biological membranes. In humans at least 40% of membrane proteins span the membrane more than once . These sFolding of polytopic membrane proteins in vivo can be viewed as a series of sequential overlapping steps . While tWe have addressed this question using the ABC transporter CFTR (cystic fibrosis transmembrane conductance regulator) as a model protein. The ABC transporter superfamily is one of the oldest and most conserved protein superfamilies , 10. ABCCFTR contains two similar halves with each a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). The halves are connected via the intrinsically unstructured Regulatory (R) region. The cryo-EM structures and various structural models of CFTR show that the TMDs are assembled via their transmembrane helices and helical intracellular loops (ICLs) –17. The Seminal studies uncovered the orientation and integration of CFTR transmembrane helices into the membrane –20. The Despite this progress, mechanistic insight into the folding pathways of CFTR domains and especially of the later assembly events into a functional structure is only understood in general terms. This is in part because transmembrane helices are embedded in a membrane and shielded from the aqueous environment, but it is also caused by the paucity of suitable reagents to study them. Here we have developed and characterized antibodies against the TMDs of CFTR. We then used them together with antibodies against the NBDs to establish an integrated workflow of radiolabeling pulse chase, limited proteolysis, and immunoprecipitation, for temporal analysis of CFTR (domain) folding in vivo. By identifying the boundaries of domain-specific proteolytic fragments, we uncovered detailed changes in conformation during maturation of newly synthesized CFTR and describe a global folding profile of the CFTR protein. We then used the assays and information to uncover folding defects in the abundant disease-causing CFTR F508del mutant as well as in mutants that had been concluded to have a defect in export from the ER.Limited proteolysis in combination with immune-based detection of fragments is a versatile method to assay protein folding in cells , 24, 25.35S-labeled amino acids and a source of ER membranes  , 29. TheIn parallel, the lysates were subjected to limited proteolysis using Proteinase K, to probe conformation of CFTR. The more folded a protein is, the more compact it becomes, and the more protease-resistant it will be. During folding, proteins acquire increasing protease resistance, detectable as an increase in proteolytic fragment size. Immunoprecipitation of the fragments with the domain-specific antibodies then allows analysis of the conformational changes in each domain of CFTR during its folding, during the chase Fig. a, c–h 77, 31.As CFTR traveled from the ER to the Golgi complex Fig. b, the prLike the TMD1 antibodies, the TMD2 antibodies TMD2C and I4N also immunoprecipitated smaller proteolytic fragments after the pulse than after a chase. Immediately after the pulse, both antibodies detected a fragment we named T2b Fig. a, b. AftThe NBD1-specific immunoprecipitates from the same proteolytic digests contain a major band (N1a) at ~ 25 kDa immediately after the pulse, which persists throughout the chase  and on Proteinase-K consensus cleavage sites (Table S2), with electrophoretic mobility shifts of fragments digested from CFTR truncations and point mutants, and with secondary-structure predictions.upstream of the epitope of MM13-4, between residues M1 and G27. The N-terminal boundaries of T1a-d however must be downstream of L34, between epitopes seen by MM13-4 and E1-22, i.e. between residues L34 and A107. To zoom in, we generated 24 N-terminally truncated versions of CFTR from the far N-terminus (∆N2) to the first transmembrane helix (∆N76). When the deleted residues are part of a fragment, that fragment should be truncated too and shift down in the gel. Constructs were expressed in HEK293T cells and analyzed by SDS-PAGE for the impact of truncations on the patterns of proteolytic TMD1 fragments at 0-h, 1-h, or 2-h chase times  in ECL1, but only the larger T1e and T1f fragments were detected by MM13-4 (aa G27—L34)  or more N-terminal residues, but not yet after truncating 48 residues, ΔN48 Fig. b, indicaTo find the C-terminal boundary of T1c, we used the same strategy, but now with C-terminal truncations of CFTR. Introducing a stop codon at positions E384, Y385, or N386 close to the C-terminus of TMD1 (aa M394) did not change T1c mobility, whereas shorter constructs  did  and also with TMD1C (epitope aa S364–K381)  and was recognized by I4N (epitope aa Q1035–S1049)  but not 596 (epitope W1204–T1211)  is not resolved in the cryo-EM structure [37-L1480 Having characterized the antibodies and the biochemical folding process, we set out to investigate the effect of mutations in the diacidic (DxD) motif in NBD1  are the same in this cell line as in CF bronchial-epithelium CFBE cells  expressiPrevious studies have shown that CFTR folds its domains largely co-translationally; insertion of the transmembrane helices occurs during translation , 19, 57 In stage 1, the first transmembrane domain of CFTR folds co-translationally, with the N- and C-termini packing with each other and with ICL1 . NBD1 reE. coli have shown that specific ‘packing’ interactions between the N- and C-terminal transmembrane helices occur during translation, and that these are essential in forming a stable tertiary structure [The individual domains of CFTR fold to a large extent co-translationally . The foltructure . This altructure . In the tructure , TMH6 istructure , 63. Fortructure , 37, 64.Once NBD1 is translated, it folds quickly and starts co-translational domain assembly with TMD1 , 31. TheThe early folding fragment T2b starts from the coupling helix of ICL3 to the C-terminal end of TMD2, with ICL4 being protected. The four helices comprising T2b, which are TMH9-12, together with TMH1-2 of TMD1, form the domain swap-structure seen in CFTR . The N-tThe CFTR domains, especially the TMDs, must maintain a certain level of flexibility for interacting with the NBDs and opening and closing the channel, which is consistent with the linkers between all domains remaining exposed and cleavable throughout both stage 1 and stage 2 of the CFTR folding pathway.At later chase times, in stage 2 of CFTR folding, the appearance of fragments T1d–f shows protection of both ICL2 (decrease of T1a) and the N-terminus (T1d–f). Increasing protease resistance of the N-terminus is consistent with the N-terminus wrapping around the transmembrane helices of TMD1 and TMD2 . TMD1, TNBD2 and NBD1 are very similar in structure, and one would expect that they follow the same folding pathways. Yet, we here show that NBD2 in CFTR did not fold independently and required the other domains to fold. It acquired protease resistance through assembly with the TMDs in stage 2 of CFTR folding, and hence with slower kinetics than NBD1. This difference between the NBDs is underscored by three conspicuous features. First, NBD1 contains a regulatory insertion that is absent in NBD2 and which in F508del affects maturation of the mutant channel. RI deletion rescues F508del CFTR to the cell surface , 74 but A second difference is that isolated NBD2 is a much less stable domain than NBD1 and requires multiple solubilizing mutations to produce recombinantly for example for determination of its structure . Third, not support assembly of TMD1, TMD2, and NBD2.Incorporation of the new TMD antibodies into the coupled pulse chase-limited proteolysis assay has allowed us to look at higher resolution into consequences of mutations in CFTR on conformations of the maturing protein. We here show that the most prevalent and well-studied disease-causing mutation F508del, which established cystic fibrosis as protein-folding disease, caused co-translational misfolding of NBD1 but not TMD1 nor TMD2. Our assay uncovered that folding of the first nucleotide-binding domain precedes that of the other domains and that folding of NBD1 is the Achilles' heel in the folding pathway of the entire polytopic multispanning membrane protein. Deletion of the single F508 residue causes the complete absence of stage-2 folding in that F508del NBD1 does The staging of CFTR's folding pathway in two experimentally distinguishable phases possibly opens opportunities for therapeutic interventions. Salvage of a functional channel is achievable through targeting either folding of NBD1 itself or alternatively assembly of F508del NBD1 with the other domains into a  active structure , 77. Ourupstream of export: for proper domain folding and assembly, upstream of transport. CFTR mutated in this DAD export motif phenocopied the misfolding and degradation of F508del CFTR. We also examined the mutants of the diacidic ER export signal (DAD) that accumulate in the ER and fail to be recognized by the COPII machinery. This mutation initially was thought to encode a relatively pure sorting mutant as opposed to a conformational mutant such as F508del CFTR [The assay also demonstrated that the DXD motif in NBD1 that was identified to be required for export of CFTR from the ER in fact was required already del CFTR  and thusdel CFTR , which tdel CFTR . MoreoveCollectively, we show that a repertoire of domain-specific antibodies together with a coupled pulse chase-limited proteolysis immunoprecipitation assay constitutes a powerful method to uncover folding pathways in vivo in a temporal manner, to investigate folding-function relationships of (mutant) proteins, and uncover conformational effects of modulator drugs. We have shown CFTR folding to encompass 2 discrete stages, a highly modular process of domain folding and stepwise domain assembly. On the one hand, this staging has the disadvantage that a mutation like F508del not only destroys the folding of one domain, NBD1, but also blocks entry into stage 2. On the other hand, not all domain interfaces are crucial, and the advantage is that the other 3 domains can rescue the complete protein by assembling and thereby stabilizing the defective domain. The modulator rescue of F508del CFTR domain assembly around a still defective NBD1 explains the relatively high fidelity of folding and the importance of a step-wise folding process for such complex proteins. While our paper was under revision, Fiedorczuk and Chen published the structure of F508del CFTR with bound Trikafta modulators in which they showed amongst others and consistent with our observations that the NBD1-TMD interface of pharmacologically corrected F508del CFTR is distinct from that of wild type CFTR .We provide experimental evidence from intact cells for a fundamental principle in protein folding shown before by in-vitro refolding studies: that low-contact-order interactions are formed before high-contact-order folding. The pathway of domain folding before domain assembly that CFTR follows makes sense from a point of view of fidelity of folding, order of folding, and it fits with in-vitro folding principles and models such as the hydrophobic-collapse and the nucleation-condensation models of folding. Properties of specific ABC transporters such as the unstructured regulatory region, the number and position of N-linked glycans, or the presence of intramolecular disulfide bonds all may affect folding. Yet, given the high structure conservation of the features that are dominant in CFTR folding, we anticipate the 2-stage folding process to be very similar for other ABC transporters and likely for other multi-spanning membrane proteins as well.https://hpcwebapps.cit.nih.gov/AbDesigner/) and synthesized as described [m-maleimidobenzoyl-N-hydroxysuccinimide ester  in a carrier(1):linker (50):peptide (50) molar ratio. RSA and Sulfo-MBS were dissolved separately in 500 μl of PBS at pH 8.4, mixed and incubated at room temperature for 45 min. RSA-sulfo-MBS was desalted on a PD10 column  with PBS at pH 7.2, and concentrated to 1 mL . Desalted RSA-sulfo-MBS was added to the peptides, which were dissolved in 200 μl of PBS, pH 7.2, and incubated for 2 h at room temperature [Peptides from the first extracellular loop of TMD1 (aa A107 –S118), C-terminus of TMD1 (aa S364–K381), ICL4 in TMD2 (aa Q1035–S1049), and the C-terminal part of TMD2 (aa E1172 – Q1186) were selected with the AbDesigner algorithm of NIH (escribed . The purperature . The RSACFTR single-domain constructs were generated in pBS vectors as previously described  and subc2. Cells were seeded onto polylysine-coated 6 cm dishes to reach 70% confluency and transfected using linear 40 kDa polymer polyethylenimine (PEI) as described [HEK293T cells were maintained in DMEM supplemented with 10% FBS and 2 mM GlutaMAX (growth medium) and incubated at 37 °C with 5% COescribed . After 435S Protein Labeling Mix (Perkin Elmer). Radiolabeling was stopped by adding excess, unlabeled 5 μM methionine and 5 μM cysteine. At indicated chase times, cells were washed twice with ice-cold Hanks' balanced salt solution  and solubilized in ice-cold lysis buffer  without protease inhibitors. Nuclei were removed by centrifugation for 10 min at 4 °C in a microfuge at maximum speed, and the supernatant was subjected to limited proteolysis or immunoprecipitation.HEK239T cells were used in pulse-chase assays as described , 87. Cel35S Protein Labeling Mix (Perkin Elmer), and membranes. The reaction proceeded at 30 °C for 30 min, was stopped with 1 mM cycloheximide, and membranes were pelleted through centrifugation at 10,000 × g for 3 min at 4 °C. Newly translated proteins were retrieved from the pellet fraction by dissolving the membranes in 10 μL KHM 2, 20 mM HEPES pH 7.2) containing 1% Triton X-100 for 10 min at 4 °C. Samples were either subjected to immunoprecipitation or used directly for SDS-PAGE analysis after adding 2 × Laemmli sample buffer.Target mRNA was prepared by transcribing DNA using T7 RNA polymerase according to the manufacturer’s instructions (Promega). In-vitro translations were done with rabbit reticulocyte lysate  and translated CFTR (domains) were translocated and inserted into HEK293T-derived microsomes or semi-permeable HEK293T cells as source of ER membrane as described [2 and 10 µg/mL micrococcal nuclease  were added and incubated for 12 min at room temperature. To inactivate micrococcal nuclease, 4 mM EGTA was added to chelate calcium. After a final spin down and resuspension in KHM, cells were used in the in-vitro translation-translocation assay.The preparation of semi-permeabilized cells was described before . ConflueAfter in-vitro translation, the membrane fraction containing translocated CFTR was dissolved in 10 µL 100 mM NaOAc (pH 5.5) and 1% Triton X-100. 500 U of Endoglycosidase H was added and incubated at 37 °C for 1 h. Samples were used for SDS-PAGE analysis after adding 2 × Laemmli sample buffer. PNGase F treatment was performed after immunoprecipitation. The beads were resuspended in PBS containing 0.2% SDS and heated for 5 min at 55 °C. Triton was added at a final concentration of 2% to quench the SDS, and 1 U of PNGaseF was added and incubated at 37 °C for 1.5 h. Samples were used for SDS-PAGE analysis after adding 5X sample buffer.Limited proteolysis was performed as described , 24. In Antibodies against CFTR were pre-incubated with protein-A or protein-G Sepharose beads  for 15 min at 4 °C before adding protease-treated or non-treated lysates. The lysates were added to the antibody-beads mixtures and incubated at 4 °C for either 3 h or overnight. The beads were washed twice for 15 min at room temperature. A list of immunoprecipitation conditions is provided in the supplementary materials (Table S8). Beads were resuspended in 10 μl 10 mM Tris–HCl pH 6.8 containing 1 mM EDTA, and immune complexes were eluted by adding 10 μl 2 × reducing Laemmli sample buffer  and heating for 5 min at 55 °C.Samples generated by in-vitro translation or fragments generated by limited proteolysis were resolved by 12% SDS-PAGE, whereas all other samples were resolved by 7.5 or 10% SDS-PAGE, as indicated in the legends. Gels were dried and exposed to super-resolution phosphor screens (Fuji Film) for quantifications, or to Kodak MR films for manuscript figure images. Signals from screens were visualized with a Typhoon FLA-7000 scanner  and quantified with ImageQuantTL software .The helical propensity of domain sequences was determined using secondary structure prediction algorithms via JPred v4 . InformaSupplementary file1 (PDF 4393 KB)Below is the link to the electronic supplementary material.


Gelidium sp., which is underutilised nowadays in food applications. Thus, a model system consisting of minced mackerel (Scomber scombrus) muscle and aqueous flour extract was subjected to heating treatment (50 °C) for 11 days. Resulting levels on conjugated diene (CD) and triene (CT) contents, peroxide value, thiobarbituric acid index, and fluorescent compound and free fatty acid (FFA) formation were monitored at different heating times. As a result, the presence of the aqueous extract of the alga flour led to higher levels (p < 0.05) of primary lipid oxidation compounds (CD and CT assessment) and lipid hydrolysis (FFA content). Contrarily, alga flour addition led to lower (p < 0.05) fluorescent compound formation measured in the aqueous and organic fractions resulting from the lipid extraction of the fish muscle and in the supernatant medium corresponding to the heating reaction system. All effects were found to be more important (p < 0.05) with increased alga flour concentration and heating reacting time. According to the straight relationship between the interaction compound formation and nutritional and sensory values, this study opens the way to the quality enhancement of thermally treated seafood by the addition of flour extract from Gelidium sp.This research aimed to evaluate the antioxidant properties of flour obtained from red alga  The thermal treatment of marine species constitutes an important and traditional means of seafood preservation that leads to a wide range of nutritional and delicious food ,2. HowevThe lipid fraction of marine species is now the subject of much attention as a result of its high content on ω-3 polyunsaturated fatty acids (PUFA), which have shown a positive effect in preventing several human diseases ,8. HowevFor a long time, marine algae have been considered as part of the human diet in different Asian countries. Among macroalgae, red macroalgae have shown a remarkable level of constituents exhibiting high nutritional value , and conGelidium sp., an organism commonly employed for animal feeding but poorly studied and underutilised in food applications. For it, a model system consisting of minced mackerel (Scomber scombrus) muscle and aqueous flour extract was subjected to heating treatment (50 °C) for 11 days. The resulting lipid oxidation  and hydrolysis  development were monitored at different heating times.The present research aimed to analyse, to our knowledge for the first time, the antioxidant properties of flour obtained from the red alga S. scombrus)   were acquired at Vigo harbour (North-Western Spain) and transported on ice to the laboratory. Then, fish were randomly divided into three (n = 3) groups , eviscerated, beheaded, and filleted. In each group, the white muscle of fish fillets was minced and homogenised.Fresh Atlantic mackerel . The flour exhibited the following proximate composition (%): 12.2 (moisture), 31.5 (protein), 0.2 (lipids), 14.3 (ash), and 42.8 . Additionally, the fatty acid (FA) profile was analysed according to previous research [Commercial flour obtained from research . Individg for 30 min at 4 °C. The supernatant was then recovered, and the extraction process was repeated three more times. Finally, all four supernatants were pooled together and made up to 2 L with distilled water.A mixture of alga flour (26 g) and distilled water (400 mL) was stirred for 30 s, sonicated for 30 s, and centrifuged at 3500× For the preparation of the model system, 4 g portions of minced mackerel muscle were placed in 30 mL glass tubes and mixed with 0.5, 1.5, and 4.5 mL of the above-mentioned alga flour extract, respectively. Then, 9.5, 8.5, and 5.5 mL of distilled water were added, respectively, leading to RAE-1 (low-concentrated), RAE-2 (medium-concentrated), and RAE-3 (high-concentrated) batches. The control (CTR) batch was prepared by mixing 4 g of mackerel muscle and 10 mL of distilled water.n = 3).Mixtures corresponding to all batches were firmly closed and stirred for 30 s, sonicated for 30 s, and incubated at 50 °C for 11 days. Sampling was undertaken at 0, 1, 4, 7, and 11 days. At each sampling time, samples were cooled to room temperature (18–20 °C) and centrifuged. Then, the supernatant was collected for fluorescence analysis and the solid phase  was subjected to lipid extraction for lipid damage assessment. Three independent sets were carried out that were analysed independently  mixture. Lipid quantification was carried out according to the Herbes and Allen [−1 mackerel muscle.Lipid extraction was carried out on the mackerel white muscle by using the Bligh and Dyer  method; nd Allen  method. −1, where A is the absorbance reading, and V and w denote the volume (mL) and the weight (mg) of the corresponding lipid extract, respectively.Conjugated diene (CD) formation was measured spectrophotometrically  at 233 nm  on the l−1, where A is the absorbance reading, and V and w denote the volume (mL) and the weight (mg) of the corresponding lipid extract, respectively.Conjugated triene (CT) formation was measured at 268 nm  on the l−1 lipids.The peroxide value (PV) was determined spectrophotometrically (520 nm) on the lipid extract. For it, peroxide reduction with ferric thiocyanate was carried out . Results−1 muscle.The thiobarbituric acid index (TBA-i) was assessed by employing the Vyncke  procedur−1 muscle.The free fatty acid (FFA) value was assessed on the lipid extract of the fish muscle by the Lowry and Tinsley  procedurst, where F is the fluorescence measured at each excitation/emission wavelength pair and Fst is the fluorescence intensity of a quinine sulphate solution (1 µg·mL−1 in 0.05 M H2SO4) at the corresponding wavelength pair. Fluorescent compound formation was determined as the fluorescence ratio (FR) between the two RF values: FR = RF393/463 nm/RF327/415 nm.Interaction compounds produced by the reaction of oxidised lipids and protein-type molecules were determined by fluorescence spectroscopy . The formation of fluorescent compounds was measured at 393/463 and 327/415 nm, according to previous research . For it,aqu) and organic (FRorg) phases obtained from the muscle lipid extraction [med) resulting from the heating reaction of fish muscle in the presence of alga flour extract.In the present study, the fluorescent compound formation was analysed on different substrates of the flour extract/minced muscle model system. First, it was studied on the aqueous  were developed in the present research. Analyses were carried out by applying the PASW Statistics 18 software for Windows , and a confidence interval at the 95% level (p < 0.05) was considered.Once values obtained for the different chemical parameters verified the normal distribution (Kolmogorov–Smirnov test), parametric analysis was carried out. The one-way ANOVA method was used to explore differences resulting from the effect of flour extract concentration and heating time. For this, the least-squares difference (LSD) method was employed to perform the comparison of means. As expressed above, three replicates , with all values being included in the 1.0–2.3 range (p < 0.05) with control were observed on days 7 and 11 in RAE-2 and RAE-3 batches. Consequently, the flour extract presence has led to an increased level of CD value in fish muscle, with this effect being more remarkable when increasing the extract concentration and the reacting time.An increasing trend of CD value with heating time was proved in all kinds of samples (.3 range . At the r = 0.91–0.95) (p < 0.05) values were detected in the RAE-3 batch  and in the RAE-2 batch (days 7 and 11). Therefore, a remarkable retention of this kind of lipid oxidation compound was proved by flour extract presence in the reacting medium, with this effect increasing with the extract concentration and the heating time.As for CD assessment, CT content indicated a continuous increase with heating time in all kinds of samples (91–0.95) . For thip > 0.05) were not observed between control and flour-treated samples. Therefore, no effect of the flour extract presence on this kind of lipid oxidation compound in fish muscle could be demonstrated.Peroxide formation in the present study can be considered low; thus, all values remained below a score of 1.90 . For allp < 0.05) of TBARS was detected in all batches during the 0–4-day period. After this time, a decrease of the average TBA-i was detected in all kinds of samples, except for the batch including the highest flour extract concentration. For this batch, an increasing trend in TBARS content was maintained till the end of the study. A comparison among batches revealed scarce differences; thus, the highest average values were obtained in RAE-2 batch (day 1), CTR batch (day 4), and RAE-3 batch (7–11-day period). Compared to the control, lower (p < 0.05) values were obtained on day 4 in the RAE-1 batch and higher (p < 0.05) on day 11 in the RAE-3 batch. It is concluded that the presence of the flour extract in the reacting medium did not lead to a definite trend (p > 0.05) on the level of this kind of secondary lipid oxidation compounds in fish muscle.A general formation (p < 0.05) in the lipid extract of the fish muscle (FRorg) was detected for the 0–7-day period, which was followed by a decrease of the average value at the end of the experiment (p < 0.05) of flour extract presence on the formation of these deteriorative compounds in the 4–11-day heating time. During this period, the highest average values were detected in the control batch; differences with treated-muscle batches were found to be significant (p < 0.05) on day 4 (RAE-1 batch), day 7 (RAE-2 and RAE-3 batches), and day 11 .For CTR, RAE-1, and RAE-2 batches, a progressive fluorescent compound formation (periment . For samaqu) resulting from the lipid extraction of muscle indicated a marked decrease (p < 0.05) in all kinds of samples on day 1 (r = 0.91–0.94). A comparison among batches revealed two different trends. Thus, on day 1, an increased FRaqu was observed by increasing the flour extract presence in the heating medium. Contrarily, at advanced heating times , lower average values were detected in RAE-2 and RAE-3 batches than in CTR and RAE-1 batches. For this reacting period, differences in the control batch were significant (p < 0.05) on days 7 and 11 for all flour-containing batches. Remarkably, the lowest average values were detected in samples corresponding to the RAE-2 batch. Therefore, an inhibitory effect of flour extract presence was implied on the formation of fluorescent compounds measured in the aqueous phase resulting from the lipid extraction of the mackerel muscle.The fluorescent compound content measured in the aqueous fraction (FRon day 1 . Contrarp < 0.05) of fluorescent compounds with heating time (r = 0.89–0.94) was detected in the aqueous medium (FRmed) of the reacting system in all batches  on the formation of this kind of compounds in the 4–11-day period. Thus, a comparison with the control led to lower (p < 0.05) values on day 4 (FE-1 and FE-3 batches) and on days 7 and 11 .A progressive formation ( batches . As for r = 0.91–0.94) (p < 0.05) for this period. Furthermore, the samples corresponding to the RAE-1 and RAE-2 batches also showed higher (p < 0.05) FFA levels than the control fish for the 7–11-day period. Therefore, a significant retention (p < 0.05) of FFA content in fish muscle was concluded by the presence of the flour extract in the heating medium, with its effect increasing with the extract concentration and the heating time. In all kinds of samples, a remarkable FFA content increase was detected at advanced times of heating reaction (91–0.94) . Thus, ap < 0.05) in the treated-fish muscle when compared with the control. Contrarily, lower levels (p < 0.05) of interaction compound formation between oxidised lipids and protein-like compounds were detected in the treated-fish samples in comparison with the CTR batch. Remarkably, such effects were shown to be more important (p < 0.05) when increasing the flour extract presence in the reacting medium and increasing the heating time. The present results have shown a strong effect of alga flour extract on the lipid oxidation development of mackerel muscle. Thus, primary lipid oxidation compounds  showed higher levels  [S. scombrus) [Salmo salar) [Previous research has reported low values of primary and secondary lipid oxidation compounds content during thermal treatment of seafood. Thus, similar peroxide and TBARS levels to those in the current studies have been obtained in canned Atlantic Chub mackerel ( colias) , canned combrus) , and cano salar) . Remarkao salar) .According to previous research ,36, suchGelidium sp. may lead to nutritional and sensory quality retention during seafood thermal processing.Previous research has shown that the formation of interaction compounds can have a remarkable effect on the nutritional and sensory values of seafood ,37. ThusThe antioxidant properties of red alga constituents have already been described, both in in vitro studies as well as in processed seafood. In such studies, different kinds of molecules have been found to be responsible for the preservative effect, according to the kind of extracting medium employed . On the basis that a water extract of red alga flour was employed in the present study, the antioxidant activity found can be explained on the basis of the presence of the hydrophilic constituents present in the red alga flour.Hypnea flagelliformis [Gracilaria verrucosa [Gracilaria gracilis [Porphyra yezoensis [Gracilaria corticata [Spyridia hypnoides, Asparagopsis taxiformis, Portieria hornemannii, and Centroceras clavulatum [Thus, and according to present results, previous in vitro studies have shown antioxidant properties of water extracts obtained from red macroalgae. Research has concerned red algae species such as liformis , Gracilaerrucosa , and Gragracilis . In all ezoensis , Gracilaorticata , and Spyavulatum . AccordiPterocladia capillacea; the HPLC analysis revealed enriched content on glucuronic acid, arabinose, and glucose (cold extract), whereas the hot extract was found to be rich in glucuronic acid and fructose. Later on, Olasehinde et al. [Gelidium pristoides. Recently, Pei et al. [Gelidium amansii; as a result, rhamnose, glucuronic acid, glucose, galactose, xylose, and L-fucose were found to be the main components.In agreement with the preservative role reported for carbohydrate compounds, previous research accounts for different attempts focused on the analysis of carbohydrate composition in different red algae. Thus, Zeid et al.  analysede et al.  observedi et al.  analysedPyropia columbina and Gracilaria chilensis as filling media during salmon  canning; the inhibition of lipid oxidation development  and the retention of astaxanthin and tocopherol contents were observed in canned salmon resulting from the alga presence in the filling medium. Later on, cooked salmon (90 ± 5 °C for 30 min) was subjected previously to an aqueous extract dipping of red algae Gigartina radula, Iridaea larga, Gigartina chamissoi, Gracilafia chilensis, Gelidium chilense, and Gigartina akottsbergii [Previous research accounts for the lipid oxidation inhibition and sensory acceptance increase of thermally treated seafood as a result of treatment with red alga extracts. Thus, Ortiz et al.  employedtsbergii . As a rep < 0.05) on FFA molecules was detected, with this being more relevant (p < 0.05) when increasing the flour extract concentration and at advanced heating times. A remarkable effect of alga flour extract on the FFA content was observed in the present study. As for the CD and CT compounds, a protective effect  and phospholipids (PL), as a result of heat catalysation ,4; conseB. bifurcata extracts in the packing system employed for Atlantic mackerel (S. scombrus) canning led to a preservative effect of FFA in canned muscle [S. colias) as a result of adding aqueous extracts of F. spiralis and U. lactuca in the filling medium employed [Previous research concerning the effect of alga extracts on FFA content during seafood thermal processing can be considered scarce and is mostly focused on canned seafood. As in the present research, the inclusion of aqueous d muscle . Similaremployed .Gelidium sp. extract on the lipid damage development of mackerel muscle. Thus, the presence of the aqueous extract of the alga flour led to higher levels (p < 0.05) of primary lipid oxidation  and FFA compounds. Contrarily, alga flour addition led to lower (p < 0.05) fluorescent compound formation, measured in the organic and aqueous fractions obtained from the muscle lipid extraction and in the supernatant medium corresponding to the heating reaction system. All effects were found to be more important (p < 0.05) when increasing the alga flour concentration and the heating reacting time. Notably, a definite trend on the peroxide and TBARS formation in fish muscle could not be concluded (p > 0.05). A protective effect on CD, CT, and FFA compounds is concluded, with this leading to the lower formation of interaction compounds between oxidised lipids and protein-like molecules. The present results have shown a strong effect of According to the straight relationship between interaction compound formation and the loss of essential amino acids, a digestibility decrease, and the development of off-odours and off-flavours, the enhancement of sensory and nutritional properties could be implied as a result of alga flour presence during seafood thermal processing. However, in order to apply the present alga flour extract to commercial thermally treated seafood, a previous optimisation of experimental conditions  is recommended to carry out.Gelidium sp. flour for the quality enhancement of thermally treated seafood. On the basis of the abundance of Gelidium sp., this procedure would agree with current global interests in food technology in the search for new strategies including preservative compounds obtained from natural sources and from sources underutilised in food applications. Further research is envisaged to optimise the extraction of preservative compounds and to analyse the molecules involved in this preservative action.It is concluded that the current study opens the way to a novel and beneficial employment of


GRB2 is an adaptor protein required for facilitating cytoplasmic signaling complexes from a wide array of binding partners. GRB2 has been reported to exist in either a monomeric or dimeric state in crystal and solution. GRB2 dimers are formed by the exchange of protein segments between domains, otherwise known as “domain-swapping”. Swapping has been described between SH2 and C-terminal SH3 domains in the full-length structure of GRB2 (SH2/C–SH3 domain-swapped dimer), as well as between α-helixes in isolated GRB2 SH2 domains (SH2/SH2 domain-swapped dimer). Interestingly, SH2/SH2 domain-swapping has not been observed within the full-length protein, nor have the functional influences of this novel oligomeric conformation been explored. We herein generated a model of full-length GRB2 dimer with an SH2/SH2 domain-swapped conformation supported by in-line SEC–MALS–SAXS analyses. This conformation is consistent with the previously reported truncated GRB2 SH2/SH2 domain-swapped dimer but different from the previously reported, full-length SH2/C-terminal SH3 (C–SH3) domain-swapped dimer. Our model is also validated by several novel full-length GRB2 mutants that favor either a monomeric or a dimeric state through mutations within the SH2 domain that abrogate or promote SH2/SH2 domain-swapping. GRB2 knockdown and re-expression of selected monomeric and dimeric mutants in a T cell lymphoma cell line led to notable defects in clustering of the adaptor protein LAT and IL-2 release in response to TCR stimulation. These results mirrored similarly-impaired IL-2 release in GRB2-deficient cells. These studies show that a novel dimeric GRB2 conformation with domain-swapping between SH2 domains and monomer/dimer transitions are critical for GRB2 to facilitate early signaling complexes in human T cells. Monomeric GRB2 is 25 kDa and consists of a central Src Homology 2 (SH2) (residues 60–152) domain flanked by two Src Homology 3 (SH3) domains   ζ chains, and Linker for activation of T cells (LAT), among others16. The terminal SH3 domains bind proline-rich regions on downstream signaling molecules such as SOS1, c-Cbl, Vav, and SLP-76, and more30. Engagement of the TCR leads to phosphorylation of multiple tyrosine residues on the adaptor protein LAT, which in turn, serve as docking sites for various SH2 domain-bearing binding partners, including GRB235. Proper signal transmission depends critically on these early phosphorylation and recruitment events; as such, disruption of these events may compromise downstream cellular output. Our lab has described a novel role for GRB2 in stabilizing large, multimeric signaling complexes at the plasma membrane of human CD4+ T cells8. Specifically, this function is mediated by the interaction between SOS1 with two GRB2 molecules and the binding of GRB2 to three possible sites on LAT. These interactions trigger LAT oligomerization into micro-clusters and facilitates the recruitment of additional signaling molecules36. The broad range of GRB2 binding partners makes it indispensable for induction of essential cellular processes ranging from metabolism to proliferation and differentiation39. Accordingly, aberrant GRB2 function and the resulting disruptions of GRB2-mediated signaling, in particular the Ras/MAPK pathway, have been implicated in malignant transformation across cell types and organ systems46. The specific importance of GRB2’s conformation and modular SH3–SH2–SH3 structure has been shown in multiple studies where modifications of GRB2 ranging from point mutations to full domain deletions lead to interesting changes in overall protein topology and function, and consequently, changes in downstream cellular output56. For these reasons, the structure and function of GRB2 and any modulation thereof have become major areas of investigation for novel interventions against human disease.Growth factor receptor-bound protein 2 (GRB2) is an ubiquitously-expressed, non-catalytic adaptor protein72. Surprisingly, only one full-length structure of GRB2 has ever been reported, which was determined by X-ray crystallography (XRC) at a resolution of 3.1 Å shortly after the protein was originally discovered71. In the crystal structure (PDB: 1GRI), GRB2 is in an unbound and globular dimeric form with an anti-parallel protomer arrangement and significant inter-molecular contacts between one protomer’s SH2 domain and the C–SH3 domain of the other protomer  and Small-Angle X-ray Scattering (SAXS)72. In contrast to the SH2/C–SH3 domain-swapped dimer crystal structure, the authors report that GRB2 in solution with its individual domains complexed with their respective ligands is in a flexible and extended monomeric form with no detectable inter-domain contacts. They also report that the domains within the full-length protein are structurally similar to their isolated domain counterparts, further suggesting that the domains do not influence each other’s conformations. The authors conclude that ligand-bound GRB2 in solution exists as a collection of conformations that is not adequately represented by the static molecule depicted by the crystal structure (PDB: 1GRI). Together, these studies indicate that the current record reflects neither the complete structural and conformational picture of full-length GRB2 nor the cellular purpose of GRB2 oligomerization.GRB2 structure has been intensively characterized, both in isolation and in association with cognate ligands  connecting the C-terminal α-helix to the rest of the SH2 domain toward an adjacent SH2 protomer, such that one SH2 domain swaps its α-helix for that of another SH2 domain. Intriguingly, this form of inter-SH2 dimerization is accompanied by either increases or decreases in SH2 binding affinity for phospho-peptides in comparison with the isolated SH2 domain. Specifically, the SH2/SH2 domain-swapped dimer was found to bind an Shc-derived ligand with a lower affinity and a CD28-derived ligand with a higher affinity when compared to the SH2 monomer69. Of note, the un-swapped segments of the SH2 domains in the SH2/SH2 domain-swapped dimer are virtually identical in structure to the isolated domain, with the exception of the hinge loop that is N-terminal to the swapped α-helix, suggesting that the loop, rather than overall domain topology of the protein, governs the interconversion of SH2 dimer and monomer. Importantly, the hinge loop mediates the SH2 domain’s ligand-binding specificity, specifically Trp12176. Though important for engaging ligands, Trp121 has been shown to be irrelevant for dimerization, pointing to a potential role for the remaining residues of the hinge loop, Val122 and Val123, in mediating domain-swapping and dimerization68. However, no studies have ever been undertaken to determine their contribution to domain-swapping, nor has SH2/SH2 domain-swapping been explored as a form of full-length GRB2 dimerization.Although the body of work on full-length GRB2 is lacking, several other studies have investigated the structures of the isolated domains with and without associated ligands , the N188/N214 mutant, and the V122/V123 hinge loop mutants. To complement our in vitro and in silico work, we identified effects of the hinge loop mutants on T cell function. As a result of this work, we report a model of GRB2 dimer with SH2/SH2 domain-swapping as an underlying mechanism for dimerization, which, when disrupted, significantly alters signal transduction and a impairs T cell cytokine output.85. We hypothesized that the strain introduced into the loop by the Pro side chains would induce opening of the loop and lower the energetic barrier to SH2/SH2 domain-swapping. Conversely, to generate a stable monomeric construct, we mutated Val123 to an Asp residue. We hypothesized that the introduction of a charged residue at the SH2/SH2 domain-swapped dimer interface would destabilize the domain-swapped dimer and favor the lower-energy monomer. In addition to the V123D and V122P/V123P constructs, we replicated the previously-reported N188D/N214D construct as a stable monomer control for our studies.In our investigation of the remaining hinge loop residues, we designed constructs intended to stabilize either a closed or extended loop orientation and consequently, abolish or induce SH2/SH2 domain-swapping. Given that Pro residues often occur in hinge loops and are important in the swapping process, we mutated Val122 and Val123 to Pro residues in order to generate a stable dimeric GRB2 construct  chromatography. Except for V122P/V123P, all other samples eluted as a single peak from Ni–NTA columns. SDS-PAGE analyses of the eluates showed homogeneous protein of ~ 27 kDa with virtually no impurities, whereas the V122P/V123P mutant also showed several distinct products of < 27 kDa  proteins in Further resolution of the samples by size-exclusion chromatography (SEC) gave two separate and relatively well-separated peaks with no evidence of aggregation or degradation for all mutants except for V122P/V123P  and SAXS. All isolated monomer and dimer subpopulations exhibited some degree of re-equilibration of monomer and dimer Fig. . Meanwhi71. In contrast to the crystal structure, fitting of the full-length SH2/SH2 domain-swapped GRB2 dimer to the WT, N188D/N214D, and V122P/V123P dimers gave χ2 values of 2.2, 16.8, and 6.7, indicating vastly improved fits  and the N- and C–SH3 domains derived from the full-length GRB2 crystal structure PDB: 1GRI)  and the slope of the higher q scattering intensities (q > 0.1 Å−1) of a Kratky plot provides insight into the degree of compactness and unfolding of a protein. A well-folded, globular structure is characterized by a symmetric peak in the mid-q region followed by a negatively-sloped tail converging on the x-axis. In contrast, an unfolded, disordered, or extended structure exhibits a peak maximum that continues to rise and plateaus at high q. In between, the curves of partially folded proteins show both folded and unfolded characteristics; for example, a peak maximum followed by longer, shallower, more linear tail suggests a more unfolded structure. All normalized, dimensionless Kratky analyses are shown in Fig. For a qualitative assessment of each protein’s globularity, we examined the scattering intensities in mid- and high-q regions through Kratky analysis. The height of the mid-q peak (q∼0.1 ÅP(r) function, by taking an indirect Fourier transform (IFT) of the scattering curve using GNOM from the ATSAS data analysis program86. The P(r) function is represented as a histogram of all inter-electron distances within the particle and is used to estimate the maximum linear dimension of the particle, Dmax. This parameter is defined as where the P(r) curve decays smoothly to zero without significant oscillations in the curve. Globular proteins exhibit a single peak that is bell-shaped. Furthermore, the more spherical the particle, the more symmetric the P(r) function will be. Otherwise, non-spherical, elongated, or irregular proteins have an extended curve with a longer tail and may exhibit multiple peaks if modular. All of the dimeric samples were significantly more extended in comparison with their monomeric counterparts. The Dmax values of the WT dimer and monomer were 140 and 90 Å, respectively  function for the WT dimer in comparison to the WT monomer suggests that the dimer is far more extended and flexible than the monomer  in comparison with that of the monomer (90 Å) (Table P(r) function and Dmax values comparing the N188D/N214D dimer (150 Å) with the N188D/N214D monomer (90 Å)  function may both be used to assess the overall size of the protein through estimation of the radius of gyration, Rg. Rg generally serves a measure of the way in which a protein’s mass is distributed about the protein’s center-of-mass. Importantly, Rg calculated from the P(r) function has the advantage of being derived from all available data points on the scattering curve, rather than the low-q region as in the Guinier approximation88. Agreement between the Guinier-derived Rg and P(r) function-derived Rg values reinforced that good quality data were collected (Table P(r) function Rg that was larger than the Guinier Rg. As mentioned previously, this difference may be due to increased flexibility as well as aggregation. Overall, these findings indicate minor differences when comparing the monomeric counterparts to each other and the dimeric counterparts to each other, but support earlier observations that dimeric conformations of GRB2 are overall more extended than monomeric conformations.Guinier analysis and the ed Table . One exc89. A total of 20 independent iterations were averaged to generate a consensus reconstruction. The resulting three-dimensional envelope exhibited a lobular, elongated shape with dimensions grossly similar to our proposed SH2/SH2 domain-swapper dimer model. When directly superposed onto our model, we observed the reconstruction and the model to be consistent, with each of the model’s domains mostly situated inside each of the three lobes of the extended envelope  function, which also indicated an elongated structure  conform to the GRB2 SH2 motif pYxN and are directly bound by GRB2, whereas the N-terminal pY132 is bound by PLC-γ1. Early studies have shown multi-point binding between GRB2 and phosphorylated LAT, such that a fully-phosphorylated LAT could simultaneously interact with three GRB2 moleculesIn our studies, we determined that both the phosphorylation of LAT and the clustering of phospho-LAT at the T cell plasma membrane was reduced in cells expressing either of the V123D and V122P/V123P GRB2 mutants, in comparison with WT-expressing cells. The lack of LAT phosphorylation in cells expressing V123D or V122P/V123P GRB2 is interesting. This suggests that the monomer–dimer transitions regulate LAT phosphorylation, but the mechanism underlying this phenomenon is unclear. It is possible that GRB2 conformers either promote LAT phosphorylation or block dephosphorylation of LAT by phosphatases, but further studies are needed identify the mechanism. The most dramatic reduction of phospho-LAT clustering was observed in GRB2-deficient cells, suggesting that the presence of each GRB2 conformer alone was enough to induce at least some LAT clustering. This would also suggest that both forms are needed for optimal LAT clustering, although the details remain unclear. These findings raise several possibilities as to the mechanism underlying the inter-relatedness of GRB2 oligomerization through domain-swapping and T cell signaling. It would be interesting to determine whether interaction between LAT and either GRB2 dimer or monomer variably influences the recruitment of additional GRB2 molecules and potentially other binding partners to the remaining phospho-tyrosine sites; it could be that initial binding of a GRB2 dimer to LAT allows for a speedier engagement of a third GRB2 molecule and PLC-γ1. Another possibility is that co-localization of two GRB2 molecules to LAT itself induces dimerization. Further studies are needed to fully elucidate the sequence and impact of these interactions.98. Recently, Gads, another member of the GRB2 family of adaptor, was found to bind LAT in a cooperative manner as a dimer. Gads exhibits a similar structure as GRB2, with a central SH2 domain and flanking SH3 domains, except that it contains an additional glutamine- and proline-rich spacer between the SH2 and C-terminal SH3 domains. Gads canonically binds Slp-76 rather than SOS1, but both proteins interact with tyrosine-phosphorylated LAT through their SH2 domains104. The three LAT phospho-tyrosine sites  have been shown to bind GRB2 or Gads with comparable affinity, and like GRB2, two of the three sites are required for stable Gads recruitment35. However, unlike GRB2, where any of the two sites are sufficient, Gads requires two specific sites, 171 and 191, for stable binding. Similar to GRB2, Gads has been found to dimerize in an SH2-dependent manner, where the SH2-mediated dimer was further stabilized by the additional domains found in full-length Gads96. Interestingly, the Gads dimer interface was found to be distinct from the phospho-tyrosine-binding pocket such that the binding site remained accessible. Competitive binding experiments revealed preferential binding of the Gads dimer to bivalently-phosphorylated LAT peptide, even in the presence of excess, competing amounts of monovalently-phosphorylated LAT96. Furthermore, dimer abrogation through inactivation of the dimerization interface decreased this preferential bivalent binding of Gads to LAT and impaired the ability of Gads to discriminate between monovalently- and bivalently-phosphorylated LAT. Together, these findings imply that like the Gads SH2/SH2 domain-swapped dimer, a GRB2 SH2/SH2 domain-swapped dimer may not only be capable of simultaneously engaging two LAT phospho-tyrosine sites, but that it may prefer to do so. In a cellular setting, inactivation of the dimerization interface disrupted the antigen receptor-induced recruitment of Gads to phospho-LAT and impaired TCR responsiveness, suggesting profound biological consequences for the elimination of SH2-mediated Gads dimerization. Applying these findings to GRB2, it would be interesting to determine whether GRB2, like Gads, shares this ability and preference for engaging bivalently-phosphorylated LAT in the SH2-mediated dimeric form or whether the binding of SH2-mediated GRB2 dimer to phospho-LAT modulates the function of the SH3 domains and/or the availability of the SH3 binding sites. An additional question revolves around whether competition between GRB2 dimer and Gads dimer binding to these shared sites on phospho-LAT influences the overall structure and/or functional output of the LAT signalosome. It is intriguing to consider the possibility that downstream signaling outcomes can be fine-tuned at the level of interactions between phospho-LAT and signaling proteins including GRB2 dimer or monomer.The particular mechanism of SH2-mediated dimerization involving α-helix swapping has been observed in studies of other SH2 domain-bearing signaling proteins that impact T cell signaling105. SH2-mediated dimerization may represent a new mechanism through which signal transduction pathways are formed, controlled, and sensitized to discriminated between weak signals and authentic environmental triggers. Further studies will be needed to delineate the full biological function of SH2 domain-swapped GRB2 and the details underlying the mechanisms by which GRB2 oligomerization modulates T cell processes. It will also be interesting to determine whether SH2 domain-swapping may serve as a branchpoint through which larger GRB2 networks may form. Future directions for this work will revolve around complementing these findings with methods that provide increased resolution, such as crystallography, cryoEM, and/or NMR. Additionally, hydrogen–deuterium exchange mass spectrometry (HDX) would allow for empirical determination of flexible regions in the SH2 domain-swapped dimer.Our studies open new lines of investigation into the oligomeric states and consequent functional integrity of many other SH2 domain-swapped signaling molecules2 in complete RPMI media  (Gibco). Cells were maintained at concentration of 0.1–1 × 106 cells/mL. HEK 293 T cells were cultured under the same conditions in complete DMEM media  (Gibco). Cells were maintained at < 80% confluency. For experiments, cells were stimulated at a density of 0.5 × 106 cells/mL with 5 μg/mL anti-CD3 antibody (BioLegend) immobilized in 6-well or 12-well tissue culture plates for 18–24 h prior to analysis. Unless otherwise stated, cells were lysed in hot 1X Laemmli buffer (BioRad), heated at 95 °C for 5 min, sonicated to reduce viscosity, and then stored at − 20 °C prior to electrophoresis or analyzed immediately.HUT 78 T lymphocytes were cultured at 37 °C and 5% CO106. Six silent mutations were introduced to the add-back GRB2 sequences to generate GRB2 messenger resistant to shRNA-mediated degradation. GRB2 derivatives bearing the mutations discussed were constructed by site-directed mutagenesis. The V123D putative monomer mutation was introduced by PCR using the following primers: 5′-GAAGTACTTCCTCTGGGTGGATAAGTTCAATTC-3′ and 5′-AGCTCATTCAAAGAATTGAACTTATCCACCCAGAG-3′. For the V122P/V123P putative dimer mutant, the primers were the following: 5′-CGGGAAGTACTTCCTCTGGCCGCCGAAGTTCAATT-3′ and 5′-GCTCATTCAAAGAATTGAACTTCGGCGGCCAGAGGAAG-3′. The N188D mutation was introduced by overlap extension PCR using the following primer pairs: 5′-CTTCAAGGTGCTCCGAGATGGAG-3′ and 5′-GGGTCTGAGTCATCCATGACATGGATAAAATCTC-3′; 5′-GTCATGGATGACTCAGACCCCAACTGGTG-3′ and 5′-GGGCGACCGGACTCTAGAG-3′. The N214D mutation was introduced using the following primers: 5′-GGACATAGAACAGGTGCCACAGCAG-3′ and 5′-AGTCGCGGCCGCTTAGACGTTCCGGTCCACGGGGGTGAC-3′. Primers were purchased from Integrated DNA Technologies . Expression of shRNA was under control of the U6 promoter, whereas add-back GRB2 expression was under control of the EF-1α promoter. Transduced cells were kept in selection with 1 μg/mL puromycin (Santa Cruz). For bacterial expression, the sequence encoding full-length, wild-type human GRB2 was cloned into the pET-28a(+) vector  and E. coli BL21 (DE3) cells transformed with the vectors as previously described36. The same primers as listed above were used to generated Histidine-tagged variants of GRB2 for recombinant expression.For mammalian expression, HUT 78 T lymphocytes were transduced with pLK4 lentiviruses containing GRB2 shRNAs alone or with sequences encoding add-back wild-type or mutant GRB2 as previously described600 of 0.6–0.8. Cultures were then cooled to 20 °C, induced with 500 uM IPTG, and allowed to grow for 18–24 h. Cells were then pelleted and frozen at − 80 °C prior to protein isolation. Pellets were thawed in a hot waterbath for 2–5 min and resuspended in cold lysis buffer  supplemented with EDTA-free protease inhibitor cocktail (Roche) at 60 mL per 1 L of culture. Resuspensions were subjected to 4 rounds of sonication (30 s on/30 s off) and centrifuged at 18,000 rpm at 4 °C for 1 h. Supernatants were clarified with 0.45 um filters and applied to a 5 mL Ni–NTA agarose column. All subsequent steps were performed at room temperature. Protein was eluted over a linear gradient of 25 mM to 250 mM imidazole. Fractions containing GRB2 were pooled and concentrated using Amicon Ultra-15 centrifugal filter units (MWCO 10 kDa) (Millipore). Particular care is required for sample preparation from this point on, given the increasingly unfavorable conditions  and thus, increasing potential for precipitation, as has been reported70. The protein was further purified by SEC and the concentrate injected onto a 120 mL Superdex 75 10/300 preparative-grade gel filtration column (GE) equilibrated with 20 mM Tris pH 8.0, 150 mM NaCl, 100 mM L-arginine, and 1 mM DTT on a Bio-Rad BioLogic DuoFlow chromatography system. The apparent molecular weight of the protein was approximated using a calibration curve generated with known standards. Fractions containing the protein were again pooled and concentrated using Amicon filters in preparation for dialysis. The protein was dialyzed at room temperature at 500× the sample volume into buffers of gradually decreasing L-arginine concentration: 25 mM, 10 mM, and 0 mM. Protein aliquots in 30% glycerol were flash-frozen and stored at − 80 °C, or used immediately in experiments. Aliquots were reserved at various points throughout the isolation process for SDS–PAGE to determine protein purity and integrity. Samples were diluted with hot 4× Laemmli buffer, heated at 95 °C for 5 min, and then stored at − 20 °C prior to electrophoresis or analyzed immediately. Samples were then loaded onto pre-cast 4–20% Mini-PROTEAN TGX Stain-free polyacrylamide gels (Bio-Rad) and run at 120 V for up to 1 h prior to visualization of resolved bands using the Bio-Rad Gel Doc EZ Imager.Bacterial cells transformed with human His-tagged GRB2 were grown on an LB plate supplemented with 50 μg/mL kanamycin that was inverted and incubated at 37 °C overnight. A starter culture of 5 mL of Superbroth (SB) medium with antibiotic was inoculated with a single clone and incubated at 37 °C with shaking at 250 rpm for 6–8 h, or until cloudy. The starter culture was then inoculated into 1 L of medium and cells grown at 37 °C with shaking for 3–4 h to an OD6 cells were seeded into one 15 cm tissue culture dish in cDMEM and incubated overnight. At 1–2 h prior to transfection, the media was replaced with fresh cDMEM. Lentiviral DNA was introduced into 293 T cells using the calcium phosphate transfection method. Per transfection, plasmids were combined at a ratio of 6:4:3  with CaCl2 at a final concentration of 125 mM and HEPES-buffered saline at a final concentration of 50 mM HEPES pH 7.05, 140 mM NaCl, and 1.5 mM Na2HPO4). DNA was allowed to precipitate for 30 min at room temperature, then added drop-wise to 293 T cells. After 12–18 h, the transfection media was replaced with fresh cDMEM media combined with unsupplemented DMEM at a ratio of 20/80 for a final FBS concentration of 2%. Viral supernatant was harvested at 24 h intervals over the following 3 days, then spun down and filtered through 0.45-um Durapore Millex (Millipore) filters. Virus was diluted in a solution of 10% (w/v) PEG-8000 and 300 mM NaCl, rotated overnight at 4 °C, and spun down at 3000 g for 1 h at 4 °C. The viral pellet was held overnight at 4 °C in 1× PBS at 1/100th the original volume of the viral supernatant and spun down to pellet serum protein and other particulates. Concentrated virus was stored at -80 °C or used immediately to transduction. HUT 78 T lymphocytes at 0.5 × 106 cells/mL were allowed to grow for 2–3 days in the presence of concentrated virus and hexadimethrine bromide (Polybrene) transfection reagent (Sigma Aldrich) at a concentration of 8 μg/mL before removal of the virus and initiation of puromycin selection. Cells were allowed to expand in the presence of increasing puromycin concentration, from 0.25 μg/mL to a final selection concentration of 1 μg/mL. Whole cell lysates were probed for loss of GRB2 expression and expression of GRB2 mutants after 2–3 passages in puromycin.To generate pseudo-typed lentiviral particles, shRNA-encoding pLK4 vectors were transfected into 293 T cells along with pCL-Eco and Pax2 packaging vectors and VSV-G envelope vector. At 18–24 h prior to transfection, 7.5 × 105 cells/mL. Cells were stimulated by adding 0.5 mL of cell suspension to 24-well plates coated with anti-CD3 for 24 h. IL2 protein concentrations in culture supernatants were measured using standard TMB ELISA utilizing a spectrophotometric plate reader with a reading absorbance at 450 nm.Cells were washed in complete RPMI 1640, and then resuspended at 2–5 × 10Following SDS-PAGE, resolved proteins were transferred onto a PVDF membrane (Millipore) and then blocked for 1 h at room temperature in a 1:1 solution of SEA Block buffer (Thermo Scientific) in 1X PBS. Membranes were incubated for 1 h at room temperature or overnight at 4 °C with primary antibodies followed by two washes in 0.05% Tween-20 in 1X PBS. Secondary anti-mouse or anti-rabbit DyLight 680- or 800-conjugated antibodies were applied for 1 h at room temperature followed by 2 washes. Blots were then visualized using the Licor Odyssey Infrared detector. Densitometric analysis of protein bands was performed using Odyssey’s v3.0 software and normalized to GAPDH. The followed primary antibodies were used: GRB2 ; LAT pY226 ; and GAPDH (Meridian Bioscience).SEC–MALS–SAXS data sets were collected using the 18-ID-D BioCAT Beamline at the Advanced Proton Source (APS) at Argonne National Laboratory . Samples were centrifuged for 5 min at 13,000 rpm to remove any potential aggregates prior to column loading. Samples containing 4–9 mg/mL of GRB2 WT or mutants in 250 μL were injected onto a 24 mL Superdex 75 Increase 10/300 analytical-grade column (GE) equilibrated with 20 mM Tris pH 8.0, 150 mM NaCl, and 1 mM DTT at a flow rate of 0.5 mL/minute on an Agilent 1300 chromatography system. Column eluant was analyzed in line by the UV absorbance detector of the Agilent 1300 chromatography system, then subsequently directed into the DAWN Heleos-II light scattering (LS) and OptiLab T-rEX refractive index detectors in series. Finally, the elution trajectory directed samples into a 1.0 mm ID quartz capillary SAXS sample cell. Scattering data were collected every 1 s using a 0.5 s exposure and detected with a Pilatus 3 1 M pixel detector (DECTRIS) with a 12 keV (1.033 Å wavelength) X-ray beam covering a q-range of 0.0045 < q < 0.35 Å − 1 . Accurate protein molecular weights from MALS data were determined using the ASTRA software (Wyatt Technology).107. An average of 30 frames prior to an eluted peak was used for buffer subtraction. Protein peaks were also run through evolving factor analysis (EFA) to deconvolute peaks into the individual scattering components where applicable. The forward scattering intensity I(0) and radius of gyration (Rg) were calculated from the Guinier fit. The normalized Kratky plot, pair distance distribution plot, or P(r), and Porod volume (VP) were calculated using the program GNOM embedded in the BioXTAS RAW software86. The calculation of theoretical scattering curves for the crystal structure PDB 1GRI was performed using the program CRYSOL, part of the ATSAS software package (version 3.1.0)87. The initial all-atom model of the full-length SH2/SH2 domain-swapped dimer was generated with PyMOL version 2.5.2 using PDB structures 1GRI (full-length GRB2 dimer) and 6ICH (SH2 domain-only dimer)108. Each chain of the 1GRI dimer was superimposed over one of the SH2 domains comprising the 6ICH dimer. Intermodular linkers were built, N-terminal His-tags added, and missing amino acids inserted using YASARA109. Reconstruction of the electron density was calculated from SAXS data using the program DENSS version 1.6 embedded in the BioXTAS RAW software89. The final representative model was colorized and annotated using PyMOL and Chimera version 1.16110.SAXS data reduction, buffer subtraction, and further analysis were performed using BioXTAS RAW version 2.1.1Supplementary Information.


The evolution of microstructure and texture was studied using a scanning electron microscope and electron backscattered diffraction. The results indicated that cementite spheroidization occurred and greatly reduced at 750 °C due to a phase transformation. Dynamic recrystallization led to a transition from {112}<110> texture to {111}<112> texture. Below 800 °C, the intensity and variation of texture with deformation temperature is more significant than that above 800 °C. The contents of the {111}<110> texture and {111}<112> texture were equivalent above 800 °C, resulting in the better uniformity of γ-fiber texture. Nucleation of <110>//ND-oriented grains increased, leading to the strengthening of <110>//ND texture. Microstructure analysis revealed that the uniform and refined grains can be obtained after deformation at 800 °C and 850 °C. The texture variation reflected the fact that 800 °C was the critical value for temperature sensitivity of warm deformation. At a large strain rate, the lowest dislocation density appeared after deformation at 800 °C. Therefore, 800 °C is a suitable temperature for the warm forming application, where the investigated material is easy to deform and evolves into a uniform and refined microstructure.Warm compression tests were carried out on low carbon and low alloy steel at temperatures of 600–850 °C and stain rates of 0.01–10 s In industrial conditions, more than 80% of energy is consumed in heating and the rest in rolling . Taking Grain refinement occurred in both medium carbon and low carbon alloy steels during warm deformation ,6,7. UltGrain refinement was also directly related to the spheroidization of cementite during warm deformation ,16. WarmIt is well recognized that texture is one of the key factors affecting the properties of BCC metals and alloys , and texAt present, there have been a large number of studies on warm deformation of carbon steel. In most previous studies, steel sheets were investigated to obtain blank material with better plasticity for further cold processing , such asIn this study of warm deformation, low carbon and low alloy steel (20CrMoA) with high quenching ability, good machinability, cold strain plasticity, and which is widely used in shafts and gears , was inv−1 with the deformation ratio of 70% as shown in The chemical composition of the studied low carbon and low alloy structural steel was 0.2%C, 0.24%Si, 0.52%Mn, 0.92%Cr, 0.16%Mo (in wt%). The initial microstructure of the low carbon steel was lamellar with alternate formations of ferrite and pearlite. The cylindrical specimens, with a diameter of 8 mm and a height of 12 mm sampled from a homogenized bar, were compressed on a Gleeble-3500 thermal simulator. In this experiment, the deformation temperature was 600, 650, 700, 750, 800 and 850 °C. Each specimen was heated to the specified deformation temperature at a rate of 10 °C/s and held for 3 min under isothermal conditions for heat balance. The specimen was compressed along the axial direction (ND) at strain rates of 0.01, 0.1, 1, and 10 sThe EBSD map of initial microstructure was shown in −1, the microstructure of steel comprises pearlite and ferrite, even though the deformation time is longer  are shown in The fractions of recrystallized grains, substructure, and deformed grains are shown in There is no significant difference in grain size under different deformation conditions, with an average grain size of around 3.1 μm. −1, the proportion of recrystallized grain after deformation at 850 °C is only 3.73%, which is much less than 32.08% at 800 °C and 5.56% at 650 °C with the strain rate of 0.1 s−1. This phenomenon is mainly attributed to phase transformation. Complete austenitizing occurs during deformation at 850 °C due to the fact that the deformation temperature is higher than the AC3 temperature. Consequently, the recrystallized grains at 850 °C are all austenite. However, 850 °C is lower than the complete recrystallization temperature of austenite grains, so only a small amount of dynamic recrystallization occurs in the microstructure.−1, which is much higher than 14.77% at 800 °C and 1 s−1, but the area fraction of substructure is apparently low. There are three main reasons for this phenomenon [The volume fraction of recrystallized grain is 32.08% at 800 °C and 10 senomenon ,39: (i) −1, even at a lower percentage of recrystallized grains, indicating that the substructures were not composed of subgrains with LAGBs. DRV excessively consumed the distortion energy at a higher temperature [The distributions of misorientation angle and the percentages of HABs reveal the recrystallization behavior shown in perature ,37, so t−1 is larger than that at 10 s−1.−1, indicating that the microstructure with a more uniform grain distribution can be obtained at 800 °C or 850 °C.To analyze the dispersion degree of grain distribution, the standard deviation of grain size under each deformation condition was calculated as shown in 2 = 45° section of the Euler space [er space . Figure er space  revealeder space ,43.−1 and disappeared at 700 °C and 0.1 s−1, indicating that {112}<110> was completely consumed during the recrystallization process, even if the recrystallization proportion was very low. With the increasing temperature or decreasing strain rate, the intensity of <111>//ND texture and {001}<110> (R-Cube) decreased. Meanwhile, the intensity of {001}<110> (Cube) increased under conditions favorable for recrystallization, which indicated that the Cube is recrystallized texture.As shown in −1, the intensity of {111}<110> and {001}<100> at 750 °C was higher than that at 700 °C and 800 °C. It indicated that a small amount of austenite has a significant effect on {111}<110> and {001}<100> in the initial stage of phase transformation.At the fixed strain rate of 1 s−1. It is inferred that there was a critical temperature range between 750 °C and 800 °C, where the texture intensity decreased rapidly with the phase transformation due to rising temperature, and the deformation behavior was more sensitive to the temperature below 800 °C. The steel was in the end stage of phase transformation and austenite was dominant at 800 °C where texture type and intensity variation were similar to austenite, thus the texture intensity was higher than that at 850 °C. It is worth noting that above 800 °C the nucleation of <110>//ND oriented grains increased, leading to the strengthening of <110>//ND texture. In addition, when the deformation temperature rose from 750 °C to 800 °C, the reduction of texture intensity was much larger than that at the same temperature interval (from 800 °C to 850 °C) and a larger strain rate of 10 s−1 and above 800 °C, the texture of λ-fiber has better uniformity.−1 and 10 s−1 are shown in −1, the dislocation density decreases with the increase of deformation temperature. The sample after deformation at 800 °C has the lowest dislocation density and most homogenous strain distribution. At a fixed strain rate of 10 s−1, the dislocation density in the sample deformed at 850 °C looks more uniform, but apparently higher than the others. The sample deformed at 800 °C has a lower dislocation density than the sample with 750 °C, which will show relative stable properties in subsequent processing.Kernel Average Misorientation (KAM) distribution can reflect the degree of deformation. The blue region possesses the dislocation with the lowest density, while the red region possesses the dislocation with the highest density ,46. The (1)The spheroidization of cementite is related to deformation temperature. At low temperature, the broken fragments are dominant, while the cementite spheroidizes into small particles and short rods at 700 °C. In the dual-phase region, the cementite is dissolved due to the austenitizing, and the spheroidizing particles decrease rapidly. After deformation at 800 °C, equiaxed grains with the size of 3 μm were observed, which is caused by the dynamic recrystallization of ferrite.(2)In the ferrite region, the texture mainly consists of <111>//ND, <001>//ND fibers. With the increasing temperature or/and the decreasing strain rate, the intensity of {001}<100> (Cube) texture decreases and the {112}<110> texture transforms to {111}<112> texture.(3)800 °C is the critical value for temperature sensitivity of warm deformation. At temperatures below 800 °C, the texture mainly consisted of strong γ-fiber and λ-fiber texture, and the variation of texture strength with deformation temperature is significant. Above 800 °C, the γ-fiber texture shows relatively uniform distribution. The <110>//ND oriented grains appears, leading to the strengthening of <110>//ND texture.(4)Equiaxed grains with smaller average grain size and uniform distribution can be obtained at 800 °C. Under a large strain rate, the fraction of KAM values indicating lower dislocation at 800 °C is much higher than at other deformation temperatures. For this reason, 800 °C is a suitable temperature for warm forming applications.