A look at the face of the molten globule: Structural model of the Helicobacter pylori apoflavodoxin ensemble at acidic pH

Abstract Molten globule (MG) is the name given to a compact, non‐native conformation of proteins that has stimulated the imagination and work in the protein folding field for more than 40 years. The MG has been proposed to play a central role in the folding reaction and in important cell functions, and to be related to the onset of misfolding diseases. Due to its inherent intractability to high‐resolution studies, atomistic structural models have not yet been obtained. We present here an integrative atomistic model of the MG formed at acidic pH by the apoflavodoxin from the human pathogen Helicobacter pylori. This MG has been previously shown to exhibit the archetypical expansion, spectroscopic and thermodynamic features of a molten conformation. To obtain the model, we have analyzed the stability of wild‐type and 55 apoflavodoxin mutants to derive experimental equilibrium Φ values that have been used in biased molecular dynamics simulations to convert the native conformation into an MG ensemble. The ensemble has been refined to reproduce the experimental hydrodynamic radius and circular dichroism (CD) spectrum. The refined ensemble, deposited in PDB‐Dev, successfully explains the characteristic 1H‐nuclear magnetic resonance (NMR) and near‐UV CD spectral features of the MG as well as its solvent‐accessible surface area (SASA) change upon unfolding. This integrative model of an MG will help to understand the energetics and roles of these elusive conformations in protein folding and misfolding. Interestingly, the apoflavodoxin MG is structurally unrelated to previously described partly unfolded conformations of this protein, exemplifying that equilibrium MGs need not to reflect the properties of kinetic intermediates.

wise by the carboxyl group of Glu20 and the side chain of Val31) but also rely on non-native interactions. The probes in α1 indicate this helix maintains some native interactions with α5, β1 and β2.
The short helix 2 (α2) was probed by mutations S38A, E40A and N43A. Residue Ser38 is the Ncap of the helix, and it contacts two helical residues. The low Φ value of the S38A mutation indicates those contacts are still present, though markedly debilitated. The Φ value of the second mutation could not be calculated accurately as the energetic effect was small in the two conformations. That of the third mutation was negative, indicating non-native interactions of the mutated residue in the MG. Helix α2 appears to be formed, but highly debilitated, in the MG, and its native contacts with α3 seem to have been replaced by non-native interactions.
Helix 3 (α3), an irregular helix composed of two segments: α3a and α3b, was probed by mutations Q61A, D63A, L68A, T70A in segment α3a and D75A in segment α3b. The Q61A mutation replaces a polar buried residue whose side chain N-caps α3 by forming H-bonds with Asp63 and Trp64 nitrogen atoms, and additionally forms an H-bond with the side chain of Asp11 at the loop that binds the FMN phosphate. Substitution of Gln61 by alanine significantly stabilized both the native and the MG structure, and gave rise to a Φ value of 0.51. The contact observed in the native structure between Gln61 and Asp11 takes place due to the presence in the X-ray structure of a bound chloride atom replacing the FMN phosphate, which is obviously missing in apoflavodoxin. As previously reported, 1-3 the β1/α1 loop of apoflavodoxins is disordered in solution. Therefore, the energetic effect of this mutation is likely to report mainly on its capping interactions with α3. Thus, the Φ value of 0.51 indicates this capping is still present in the MG. Within α3, Leu68 organizes a hydrophobic core which is formed by packing against the hydrophobic methylene chain of Lys106 in α4 and against hydrophobic residues (Phe76 and Leu60) at either end of α3. The Φ value obtained for mutation L68A is 0.22, and the destabilization of the MG is significant (0.71 kcal/mol). It seems that this α3/α4 core is retained, albeit debilitated. On the other hand, the solvent exposed interactions formed by Asp63 and Asp75, at the ends of α3, with the ends of α2 are lost, as they have Φ values close to 0. Mutation T70A mainly breaks intra-helical interactions. Its negative Φ value indicates that Thr70 establishes non-native interactions in MG.
Helix 4 (α4) was probed by three mutations. Mutation I100A is an aliphatic deletion that removes contacts with the short strand β6c at the characteristic loop of long-chain flavodoxins that splits the fifth strand of the main β-sheet. Although its Φ value is small (0.12), the destabilization of the MG is significant (0.47 kcal/mol), which suggests that α4 still packs onto this long loop. The hydroxyl deletion Y104F breaks a H-bond linking α4 to a short strand (β6b) also belonging to the long loop. Its negative Φ value indicates that the native H-bond is likely replaced by non-native interactions. The E105A mutation probes intra-helical contacts, i.e. an H-bond with Lys108 and van der Waals contacts with Phe101, and the Φ value obtained is not reliable.
Helix 5 (α5) was probed by 8 mutations. Three of them were different replacements of Thr148 (to Ser, Ala or Val) that led to one unreliable Φ value and two Φ values close to zero, respectively. D149A and V156A probed interactions with α1 and their Φ values were close to zero. The three remaining mutations R151A, I152A, V159A probed internal α5 interactions. Two of them also resulted in Φ values close to zero, and R151A yielded a negative Φ value deriving from a large stabilizing effect in the MG. This indicates the interactions formed by this residue in the MG are non-native. The analysis of the mutations introduced in α5 suggests this helix has lost in the MG internal interactions and packing interactions with α1.
Mutations in the Loops. The loop connecting strand β1 and helix α1 has been probed by mutations T10A, D11A and S12A. This loop interacts with the phosphate group of FMN in the holoprotein and it appears to be unstructured in the homologous apoflavodoxin from Anabaena PCC7119. 1,3 As expected, the three mutations have negligible effects on the stability of the native structure. However, the stabilization of the MG by removal of the Asp11 carboxylate (D11A) suggests this residue establishes non-native interactions in the MG.
The loop connecting α2 and β3 has been probed by F45A. The mutated residue is buried and contributes to packing helix α2 onto the β-sheet by making contacts with strands β1 and β2. Unfortunately, the energetic effect of the mutation on the native state is small and the associated Φ value is unreliable.
The loop connecting α3 and β3 is probed by mutation L60A, affecting a buried residue. Leu60 intercalates between the beginning of helix α3 and the end of α4 and contacts strand β3. The closeto-zero Φ value of L60A indicates the packing of this leucine residue between helices α3 and α4 is absent in the MG.
The loop connecting α4 and β4 has been probed by mutations D88A, Q89A, S93A, T95A, T95S and E98A. This loop is one of the FMN binding loops interacting with the isoalloxazine ring of the FMN redox cofactor in Hp holoflavodoxin. In Hp apoFld this loop packs against the other isoalloxazine binding loop (loop β3/α3) and against the extra loop characteristic of long-chain flavodoxins. 4,5 The Φ values obtained for the indicated mutations are either non-reliable (T95S), close to zero (Q89A, T95A, E98A) or negative (D88A, S93A). Therefore, the interactions formed by this loop in native flavodoxin are generally broken and, in some cases, replaced by non-native interactions in the MG. Finally, the loop connecting α5 and β5b has been probed by mutations E141A and Q144A.
Glu141 mainly interacts with other residues of the loop, while Gln144 organizes a H-bond network connecting the N-terminus of α5 with the long loop splitting strand β5 and with the β4/α4 isoalloxazine binding loop. The close-to-0 Φ value of E141A indicates the loop is disorganized, while the negative Φ value of Q144A (with its significant effect on the stability of the MG) suggests this residue forms nonnative interactions in MG.
Schematic Structure of MG. With the above structural interpretation of the thirty-two canonical and thirteen non-canonical but reliable Φ values, a schematic structure of the MG can be proposed (Figure 1d,e of the main text). Flavodoxins are three-layered α/β proteins with a central β-sheet of five strands with a 21345 order. The β-sheet layer is essentially retained in the MG albeit with a progressive loss of native interactions, or their substitution by non-native ones in the strands located in the C-terminal half of the sequence. At one side of the β-sheet, the helical layer formed in the native structure by the two longest helices (α1 and α5) appears to consist of a native α1 interacting with the ends of an otherwise disordered α5. In this layer, α1 is packed onto the central β-sheet, forming native interactions with strands β1, β2 and β4 and also non-native interactions with β2. At the other side of the β-sheet, the additional helical layer formed by helices α2, α3 and α4 is also partially retained. The central helix of the layer, α3, is irregular and consists of one longer part, α3a, that appears to retain the native conformation including its N-capping interaction, and a shorter part, α3b, that is disordered. Helix α2 is quite short but its N-capping is also retained. Helix α4 is longer and seems to be natively formed towards the N-terminus. The packing of these helices among them and onto the central β-sheet seems rather loose. While α3a and α4 retain native packing contacts, the packing between α3a and α2 and that of any of these three helices onto the β-sheet appears to rely on non-native interactions.
The loops connecting secondary structure elements have also been analysed. In Hp apoFld the loops connecting C-terminals of β-strands with N-terminals of α-helices are typically longer than the others. Most of those longer loops (including the three loops that interact with the FMN cofactor in the holoprotein) have been probed and their native interactions appear generally lost or replaced by nonnative ones. Structurally, flavodoxins can be classified into long-chain and short-chain ones, the former ones carrying a 20-residue additional loop intercalated in strand β5. Hp apoFld is long-chained and its extra 20-residue loop has also been analysed. While it is mostly disordered, it nevertheless establishes both native and non-native contacts with α4.

Mutagenesis, Protein Expression and Purification. Site-directed mutagenesis was performed by
Mutagenex, Inc. into the pET28a expression plasmid, which contained the Hp flavodoxin gene. 6 All Hp Fld mutants were purified as described. 6 To obtain the corresponding apoflavodoxins, the FMN cofactor was removed from the holoproteins by treatment with trichloroacetic acid. 7 Apoflavodoxin concentration was determined using a molar extinction coefficient at 280 nm of 15.47 mM -1 ×cm -1 in MOPS 50 mM pH 7 8 for the wild-type protein and all other mutants, except for Y104F and Y121F, for which an extinction coefficient of 13.98 was used. 9 The far-UV CD spectra at pH 7 of all the mutants were similar to that of the wild-type protein, suggesting that all them could fold to the native structure.
Urea Denaturation. The conformational stability of wild-type and mutant apoflavodoxins was determined at pH 2 and at pH 7 using the linear extrapolation method (LEM) with urea as denaturant.
Samples of different urea concentrations were prepared by mixing 100-µL aliquots of buffered protein (20 µM apoflavodoxin in 500 mM MOPS pH 7, or in 230 mM sodium phosphate buffer pH 2) with 900-µL urea solutions ranging from 0 to 7.0 M (pH 7 experiments) or from 0 to 4.0 M (pH 2 experiments). The samples were allowed to reach thermodynamic equilibrium at 25.0 °C for at least 30 minutes before their fluorescence emission (excitation at 280 nm and emission at 320 nm) was recorded in a thermostated Cary Eclipse Fluorescence Spectrophotometer (Varian Medical Systems, Inc., USA). Data Fitting. Hp apoflavodoxin urea denaturation is two-state. 10 Urea unfolding curves were accordingly analysed using a two-state model, as previously explained in detail. 10,11 Briefly, ΔG is taken as a linear function of the denaturing concentration (Eq. 1): where R is the universal gas constant, T the absolute temperature and U1/2 the urea concentration of mid denaturation. Since the stability of Hp apoFld at pH 2 is low and the native baseline of the unfolding transition is not fully observed, the unfolding curves recorded at pH 2 were analysed constraining the value of mN to be zero. Curve fitting was done using Origin 8 software (Microcal LLC., Northampton, MA, USA). The U1/2 and m values reported are averages of two measurements ± SE.
BMD Simulations. The HQBM module with the reaction coordinate RC3 12,13 (CHARMM package) 14,15 that has been used is designed to perform BMD simulations considering multiple restraints, such as experimental Φ values. In this case, 32 of the 33 reliable Φ values shown in Table 1 and Figure 1 of the main text were taken to set the restraints (out of the two Φ values corresponding to residue 148, that of T148A was used). Slightly negative Φ values (i.e. -0.11≤Φ≤0) were taken as zero (i.e. Φ = 0) to indicate full disruption of the probed interaction. HQBM introduces a small energy perturbation on the starting system (the Protein Data Bank 2BMV structure) 2 that forces it to decrease the average difference between experimental and calculated Φ values (see Figure S3a1-a3). The difference is the reaction coordinate (RC) of the simulations, and it is reduced along the time (t) by a cost function introduced in the force field as described in: 12 with ( ) = min 0≤ ≤ ( ), and: where I is the list of NΦ available experimental Φ values, Φi exp , and Φi calc is calculated at every time frame for residue i, as defined by Paci et al.: 12 Ni(t) being the number of native contacts made by residue i at time t, and Ni nat the number of native contacts made by the same residue in the native state reference structure. The number of native contacts made by residue i, is obtained by: with: where M is the number of selected atoms, e.g. C α atoms or the heavy atoms in sidechains, rij is the distance between the atoms i and j, and ∆ij(Q) is equal to 1 if atoms i and j are closer than the rc cutoff distance set to count the contacts in the reference structure and belong to residues at least Q residues apart in the sequence, and is equal to zero, otherwise. Such a theoretical Φ value definition has been shown to be a good approximation to the experimental Φ values in modelling both transition state 12 and intermediate ensembles. 16,17 BMD simulations were run using the modified version of the effective potential function EEF1 (including the extra parameter type CR) which combines the Charmm19 polar hydrogen energy function 18 with an excluded volume implicit solvation model. [19][20][21] The non-bonded setup parameters for EEF1 were CTONNB 7, CTOFNB 9, CUTNB 14, and GROUP RDIE. A preparation phase including a minimization step (Adopted Basis Newton-Raphson, 500 steps), 400ps-heating (using a ramp from 98 to 298 K with an increment of 5 K every 10 ps), and 6ns-equilibration step (2fs-timestep, Hoover thermostat) were performed before initiating the targeted 5ns-BMD productive simulations. The starting conformations for the 21 replicas run for each set of BMD simulations were frames evenly extracted (every 50 ps) from the last nanosecond of the 6ns-equilibration trajectory The productive phase was initiated by setting the coupling constant α (see Eq. 3) to 5000, a value that was quadruplicated after every ns. We set the parameter β (see Eq. 7) to 5 Å -1 , the cutoff distance for accounting native contacts to 6 Å, and the parameter Q (see Eq. 6) to 4. Three sets of independent simulations (each one encompassing 21 replicas) were run, which differed in the selection of atoms used to compute Φ values. The side chain heavy atom set considered only non-hydrogen side chain atoms, the whole side chain set considered all side chain atoms, while the all-atom set considered all residue atoms.
Unbiased MD Simulations. GROMACS package (v.2018) 22 was used for running and analyzing trajectories. Two different protonation states were simulated: pH 2 (with all ionizable residues in their protonated form) and pH 7 (with residues protonated as default in GROMACS), starting -as for the BMD simulations-from the native Hp apoFld conformation (PDB ID 2BMV). 2 The simulations were performed on periodic boundary conditions (PBC) of a truncated dodecahedral box in which the minimum distance between the protein and the box edge was 1 nm. This box was filled either with Tip3p 23 (for Charmm22/CMAP force field) or Tip4p-d-disp 24 (for Amber99SB-disp force field) water molecules. Sodium and chlorine ions were added to neutralize the systems, and at the same time to fit an ionic strength of 250 mM. A short minimization step of the solvated system was performed (20,000 steps of 1 fs with the steepest descent algorithm) 25 . Then, ten replicas for each forcefield/water-model setup, i.e. five for each protonation state here simulated (pH 2 and pH 7), were gradually heated through a heating ladder starting at 38 K that increases nine times the temperature by 40 K (every 50 ns, using a 2fs-timestep) until reaching 398 K. An NVT ensemble was set up through the V-rescale thermostat. 26 Then, two consecutive NPT equilibration steps were followed by setting in both the V-rescale thermostat 26 at 398 K, the Parrinello-Rahman barostat 27 at 1 atm pressure and a timestep of 2 fs. The first of these equilibration steps ran for 250 ps with restraints imposed on the heavy atoms, whereas the second one ran for 500 ps without any restraint. The Verlet cutoff-scheme algorithm 28,29 was used for van der Waals interactions and the PME method 30 for electrostatic interactions, both with a cutoff of 1 nm. The productive phase was collected under the same conditions and settings, and 3µs-or 5µs-long simulations were run for each replica with the Charmm22/CMAP + Tip3p setup, while with the Amber99SB-disp + Tip4p-d-disp setup 2µs-long simulations were also run per replica.
Analyses Implemented for the Simulated Trajectories and the Selected MG Ensembles. The last nanosecond of the 5ns-productive BMD trajectories (Figure S3a1-a3) was taken as the sampling space to extract representative Hp apoFld MG ensembles for each BMD simulations set. A variety of analyses was implemented on these 1ns-BMD trajectories, as well as on the metatrajectories and selected ensembles extracted from the unbiased MD simulations. Details on the 2D-RMSD-based clustering and the subsequent conformations filtering applied on these simulations are given in the next subsection and in Figure 4 (of the main text), Figure S7 and Figure S9. Plots of RC evolution ( Figure S3a1-a3), Rgyr (Figure S3b1-b3) and RMSD (Figure S3c1-c3), as well as interaction distances (for methyl/aromatic interactions, Tables S7, S8 and S17) were obtained over the trajectories or ensembles using either CHARMM scripts (in BMD) or analysis tools from the GROMACS package (in unbiased MD). In the BMD last-ns trajectories, after calculating the average RMSD values of all frames against all (2D-RMSD, not shown), the centroid structure was identified in each of the 21 replicas as that with the lowest 2D-RMSD average (see in Figure S3b1-b3). Such centroids conformed the 21-structure MG ensembles here analysed for each BMD simulation set.
Secondary structure was assigned and calculated using DSSP (63) (Tables S3 and S11). The PDBMD2CD server 31 was used to predict circular dichroism (CD) spectra in order to compare them with corresponding spectra determined experimentally (Figures 3b,c, S5, S8 and S10a). Solventaccessible surface area (SASA) analyses were performed using the ProtSA server. 32,33 A probe radius of 1.4 Å and 2,000 unfolded conformations were used to calculate SASAs in the unfolded state (Table S5). Theoretical hydrodynamics radii, Rh, of the ensembles were calculated as described 10 from the translational self-diffusion coefficients, D, calculated by the program HYDROPRO v.10. 34,35 Input parameters for this program are summarized in a footnote of Tables S4, S12 and S13.
2D-RMSD Clustering Analysis on Unbiased MD Trajectories. For unbiased MD simulations, a 2D-RMSD-based clustering analysis was performed to compare the behavior observed for Hp apoFld at pH 2 with the basal stability shown by the protein at pH 7. For this purpose, a metatrajectory joining all the replicas for each simulated condition was built by taking 1/50 frames of the original trajectories (one frame every 5 ns). A 2D-RMSD matrix for each of these metatrajectories was obtained using the gmx rms tool from GROMACS package, 22 and both were combined into one distance matrix that was then used to perform the comparative structural clustering. In the analysis, the Agglomerative Clustering algorithm 36 with a linkage distance threshold of 0.6 nm (6 Å) was used as implemented in the scikit-learn Python package. 37 The results of the clustering analysis are shown as individual conformations (frames) belonging to clusters observed over the simulation time for each metatrajectory (pH 2 and pH 7, see Figure S7).    Fractional presence of residues from the native minimized initial structure (PDB ID 2BMV) 2 in same secondary structure elements of the 21 ensemble structures is shown, correlatively, as thin bars (pale blue for α-helices; pale green forβ-strands) over the specified secondary structure element. The average fraction in the ensemble (±SE) for the secondary structure element is given on top of it. Overall α-helix and β-strand average fractions are highlighted with a gray box background. Secondary structure assignments were made by the DSSP program. 38 Residues assigned as H and E are counted as α-helices and β-strands, respectively.

Figure S5. Predicted vs. experimental far-UV CD spectra. (a)
Far-UV CD profiles predicted by the PDBMD2CD server 31 for the BMD whole side chain ensemble (thin light blue lines) compared to the experimental one of Hp apoFld MG 39 (pH 2, thick blue line). The predicted far-UV CD profile of the minimized Hp apoFld structure (native, thin red line) and the experimental one recorded at pH 7 39 (thick red line) are also depicted for comparison. As the experimental spectrum of the native conformation (pH 7) may contain contributions from aromatic residues that alter the shape of the spectrum, the spectrum calculated for the minimized native structure, rather than the experimental native spectrum, should be taken as the reference to compare with the MG data (either experimental or not). RMSDs (in ∆ε units) for the 10 best-matching predicted CD profiles (i.e. those with the lowest RMSD) vs. the experimental one of the Hp apoFld MG range from 0.51 to 0.74.   For each force field/water model setup a metatrajectory built from all the simulation replicas run for the two protonation states (pH 2 and pH 7) is clustered using a 0.6nm-distance cutoff. (a) Clusters plot of the metatrajectory obtained from Charmm22/CMAP simulations (includes 5 replicas of 3 µs at pH 7 (native, blue line) and 3 replicas of 3 µs (0 to 3, 3 to 6 and 16 to 19 µs) and 2 of 5 µs (6 to 11 and 11 to 16 µs) at pH 2 (MG, orange line)). The indicated clusters (C4, C5 and C58) are exclusive (only populated in pH 2 simulations) and persistent ones selected to accomplish next filtering steps of MG ensembles based on experimental-vs-calculated Φ value correlations and far-UV CD spectra comparisons (see also the scheme in Figure 4a of the main text). (b) Clusters plot of the metatrajectory obtained from Amber99SB-disp simulations (includes 5 replicas of 2 µs at pH 7 (native, blue line) and 5 of 2 µs at pH 2 (MG, orange line)). No persistent and exclusive cluster is populated in the pH 2 metatrajectory. Therefore, no filtering based on that clustering is performed for these force field simulations (see also the scheme in Figure 4b of the main text).   the Amber-10 ensemble depicted as fractions of residues in secondary structure elements of the native minimized initial structure (PDB ID 2BMV) 2 that remain in the same elements of the ensembles. Fractions are shown correlatively (for each of the 10 conformations in the ensembles), as thin bars (pale blue for α-helices; pale green for β-strands) over the specified secondary structure element. The average fraction in the ensemble (±SE) for the secondary structure element is given on top of it. Overall α-helix and β-strand average fractions are highlighted with a gray box background. Secondary structure assignments were made by the DSSP program. 38 Residues assigned as H and E are counted as αhelices and β-strands, respectively.         Figure 3b and 3c of the main text). j Structures clusters selected from the 2D-RMSD-based clustering on metatrajectories built from classical MD simulations (at pH 2)

Supplementary Tables
with Charmm22/CMAP force field and Tip3p water model (see Figure 4a of the main text and Figure S7a). k Charmm-10 refers to the 10 best conformations (with Φ-correlation ≥ 0.6 and the lowest RMSD of experimental vs. calculated far-UV CD spectra) filtered from pH 2 Charmm22/CMAP metatrajectory (see Figure 4a of the main text and Figure S8d). l Amber-69 refers to the 69 conformations with Φ-correlation ≥ 0.6 in pH 2 Amber99SB-disp metatrajectory (see Figure 4b of the main text and Figure S9b). m Amber-10 refers to the best 10 conformations selected from ensemble Amber-69 with the lowest RMSD vs. the experimental far-UV CD spectrum of MG (see Figure S11a).   (11), Leu (8), Thr (11) and Val (13) and the heavy atoms in the aromatic rings of Phe (11), Trp (2) and Tyr (3) residues recovered by a CHARMM script. Interactions counted after using a distance cutoff of 4.5 Å. b Between parentheses, total residues pairs whose distances are accounted for in the interaction statistics shown in this table. c The second half of the 6ns-trajectory of the equilibration step performed before BMD simulation was used for the analysis of native Hp apoFld.     a Interacting residue pairs obtained as described in footnote a of Table S7. b As done for the analysis shown in Table S7, the second half of the 6ns-trajectory of the equilibration step before BMD simulation was used for the analysis of the native Hp apoFld. c Only residue pairs with an interacting time prevalence (% t) higher than 50 % (i.e. > 50 % of trajectory frames being registered) are shown. Numbers between parentheses indicate %t, which is obtained as described in footnote f of Table S7. d 2,121 frames of a metatrajectory built from the 21 last-ns trajectories were analyzed for each BMD simulations set. 1,500 frames in the case of the native Hp apoFld (see footnote e in Table S7). Percentages of time prevalence are also shown between parentheses, colored in red when the value is significantly lower than that for the same pair in the native state (partial or complete loss of interaction). Underlined appear those residue pairs interacting de novo in the MG (non-native interactions) whose %t is higher than 25 and the change in %t (native to molten globule) is at least 15 (the %t change appears indicated between round brackets in green). e Interacting methyl-carrying residue/aromatic residue pairs including one out of the thirteen residues with non-native experimental Φ values (see Table 1 of the main text).       vs.   Table S2. Experimental Φ values were used as given in Table S2 (i.e. assuming that slightly negative Φ values are zero). f RMSD (∆ε units) given by PDBMD2CD server 31 from comparison of a predicted and the experimental spectrum of Hp apoFld MG (pH 2) (see Figures S8 and S10a).         Amber-69 and Amber-10, respectively; details in footnote e of Table S10). Thr (11) and Val (13) and the heavy atoms in the aromatic rings of Phe (11), Trp (2) and Tyr (3) residues recovered by a CHARMM script. Interactions counted after using a distance cutoff of 0.45 nm (4.5 Å). b Between parentheses, the total number or residue pairs of a type whose distances are accounted for in the interaction statistics shown in this table. c Ensembles collected as described in footnote e of Table S10.