Side-chain Packing Research Articles

Functional Coiled-Coil-like Assembly by Knob-into-Hole Packing of Single Heptad Repeat.

Coiled-coil peptides represent the principal building blocks for structure-based design of bionanomaterials. The sequence-structure relationship and precise nanoscale ordering of the coiled-coil helices originate from the knob-into-hole (KIH) packing of side chains. The helical interface stabilized by the KIH interaction is known to have chain lengths ranging from 30 to 1000 residues. Yet the shortest peptide required for oligomerization through KIH assembly is still unknown. Here, we report that through atomic resolution a minimal seven-residue amphipathic helix forms a different type of KIH motif, termed "supramolecular KIH packing", which confers an exceptional stability to the helical dimers. Significantly, at a low pH, the peptide self-assembles into nanofibers with coiled-coil architecture resembling the natural fibrous proteins. Furthermore, hierarchical ordering of the nanofibers affords lyotropic liquid crystals composed of a shortest natural helical sequence. Thus, this study expands the sequence space for a coiled-coil folding manifold and provides another paradigm for designer nanomaterials from minimal helical sequences.

The Effect of Ring Expansion in Thienobenzo[b]indacenodithiophene Polymers for Organic Field-Effect Transistors.

A fused donor, thienobenzo[b]indacenodithiophene (TBIDT), was designed and synthesized using a novel acid-promoted cascade ring closure strategy, and then copolymerized with a benzothiadiazole (BT) monomer. The backbone of TBIDT is an expansion of the well-known indacenodithiophene (IDT) unit and was expected to enhance the charge carrier mobility by improving backbone planarity and facilitating short contacts between polymer chains. However, the optimized field-effect transistors demonstrated an average saturation hole mobility of 0.9 cm2 V-1 s-1, lower than the performance of IDT-BT (∼1.5 cm2 V-1 s-1). Mobilities extracted from time-resolved microwave conductivity measurements were consistent with the trend in hole mobilities in organic field-effect transistor devices. Scanning tunneling microscopy measurements and computational modeling illustrated that TBIDT-BT exhibits a less ordered microstructure in comparison to IDT-BT. This reveals that a regular side-chain packing density, independent of conformational isomers, is critical to avoid local free volume due to irregular packing, which can host trapping impurities. DFT calculations indicated that TBIDT-BT, despite containing a larger, planar unit, showed less stabilization of planar backbone geometries in comparison to IDT-BT. This is due to the reduced electrostatic stabilizing interactions between the peripheral thiophene of the fused core and the BT unit, resulting in a reduction of the barrier to rotation around the single bond. These insights provide a greater understanding of the general structure-property relationships required for semiconducting polymer repeat units to ensure optimal backbone planarization, as illustrated with IDT-type units, guiding the design of novel semiconducting polymers with extended fused backbones for high-performance field-effect transistors.

Loosening of Side-Chain Packing Associated with Perturbations in Peripheral Dynamics Induced by the D76N Mutation of β2-Microglobulin Revealed by Pressure-NMR and Molecular Dynamic Simulations.

β2-Microglobulin (β2m) is the causative protein of dialysis-related amyloidosis, and its D76N variant is less stable and more prone to aggregation. Since their crystal structures are indistinguishable from each other, enhanced amyloidogenicity induced by the mutation may be attributed to changes in the structural dynamics of the molecule. We examined pressure and mutation effects on the β2m molecule by NMR and MD simulations, and found that the mutation induced the loosening of the inter-sheet packing of β2m, which is relevant to destabilization and subsequent amyloidogenicity. On the other hand, this loosening was coupled with perturbed dynamics at some peripheral regions. The key result for this conclusion was that both the mutation and pressure induced similar reductions in the mobility of these residues, suggesting that there is a common mechanism underlying the suppression of inherent fluctuations in the β2m molecule. Analyses of data obtained under high pressure conditions suggested that the network of dynamically correlated residues included not only the mutation site, but also distal residues, such as those of the C- and D-strands. Reductions in these local dynamics correlated with the loosening of inter-sheet packing.

Protein Docking Using a Single Representation for Protein Surface, Electrostatics, and Local Dynamics

Predicting the assembly of multiple proteins into specific complexes is critical to understanding their biological function in an organism and thus the design of drugs to address their malfunction. Proteins are flexible molecules, which inherently pose a problem to any protein docking computational method, where even a simple rearrangement of the side chain and backbone atoms at the interface of binding partners complicates the successful determination of the correct docked pose. Herein, we present a means of representing protein surface, electrostatics, and local dynamics within a single volumetric descriptor. We show that our representations can be physically related to the surface-accessible solvent area and mass of the protein. We then demonstrate that the application of this representation into a protein–protein docking scenario bypasses the need to compensate for, and predict, specific side chain packing at the interface of binding partners. This representation is leveraged in our de novo protein docking software, JabberDock, which can accurately and robustly predict difficult target complexes with an average success rate of >54%, which is comparable to or greater than the currently available methods.

Class I and II aminoacyl‐tRNA synthetase tRNA groove discrimination created the first synthetase–tRNA cognate pairs and was therefore essential to the origin of genetic coding

The genetic code likely arose when a bidirectional gene replicating as a quasi-species began to produce ancestral aminoacyl-tRNA synthetases (aaRS) capable of distinguishing between two distinct sets of amino acids. The synthetase class division therefore necessarily implies a mechanism by which the two ancestral synthetases could also discriminate between two different kinds of tRNA substrates. We used regression methods to uncover the possible patterns of base sequences capable of such discrimination and find that they appear to be related to thermodynamic differences in the relative stabilities of a hairpin necessary for recognition of tRNA substrates by Class I aaRS. The thermodynamic differences appear to be exploited by secondary structural differences between models for the ancestral aaRS called synthetase Urzymes and reinforced by packing of aromatic amino acid side chains against the nonpolar face of the ribose of A76 if and only if the tRNA CCA sequence forms a hairpin. The patterns of bases 1, 2, and 73 and stabilization of the hairpin by structural complementarity with Class I, but not Class II, aaRS Urzymes appear to be necessary and sufficient to have enabled the generation of the first two aaRS-tRNA cognate pairs, and the launch of a rudimentary binary genetic coding related recognizably to contemporary cognate pairs. As a consequence, it seems likely that nonrandom aminoacylation of tRNAs preceded the advent of the tRNA anticodon stem-loop. Consistent with this suggestion, coding rules in the acceptor-stem bases also reveal a palimpsest of the codon-anticodon interaction, as previously proposed. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1088-1098, 2019.

Packing of apolar side chains enables accurate design of highly stable membrane proteins.

The features that stabilize the structures of membrane proteins remain poorly understood. Polar interactions contribute modestly, and the hydrophobic effect contributes little to the energetics of apolar side-chain packing in membranes. Disruption of steric packing can destabilize the native folds of membrane proteins, but is packing alone sufficient to drive folding in lipids? If so, then membrane proteins stabilized by this feature should be readily designed and structurally characterized-yet this has not been achieved. Through simulation of the natural protein phospholamban and redesign of variants, we define a steric packing code underlying its assembly. Synthetic membrane proteins designed using this code and stabilized entirely by apolar side chains conform to the intended fold. Although highly stable, the steric complementarity required for their folding is surprisingly stringent. Structural informatics shows that the designed packing motif recurs across the proteome, emphasizing a prominent role for precise apolar packing in membrane protein folding, stabilization, and evolution.

Precise packing for membrane proteins

Protein Design Although nonpolar amino acid side chains pack efficiently in membrane proteins, it has been difficult to determine how much this contributes to membrane protein stability. Designed membrane proteins have largely relied on other stabilizing interactions such as metal-ligand interactions and hydrogen bonds. Mravic et al. uncovered a steric packing code underlying the folding of the natural protein phospholamban, which they used to design stable membrane proteins with nonpolar interfaces. They suggest that packing of nonpolar residues plays a role in the folding and stability of many membrane proteins. Science , this issue p. [1418][1] [1]: /lookup/doi/10.1126/science.aav7541

Analyses based on statistical thermodynamics for large difference between thermophilic rhodopsin and xanthorhodopsin in terms of thermostability.

Although the two membrane proteins, thermophilic rhodopsin (TR) and xanthorhodopsin (XR), share a high similarity in amino-acid sequence and an almost indistinguishable three-dimensional structure, TR is much more thermostable than XR. This is counterintuitive also because TR possesses only a smaller number of intramolecular hydrogen bonds (HBs) than XR. Here we investigate physical origins of the remarkable difference between XR and TR in the stability. Our free-energy function (FEF) is improved so that not only the portion within the transmembrane (TM) region but also the extracellular and intracellular portions within the water-immersed (WI) regions can be considered in assessing the stability. The assessment is performed on the basis of the FEF change upon protein folding, which is calculated for the crystal structure of XR or TR. Since the energetics within the TM region is substantially different from that within the WI regions, we determine the TM and WI portions of XR or TR by analyzing the distribution of water molecules using all-atom molecular dynamics simulations. The energetic component of the FEF change consists of a decrease in energy arising from the formation of intramolecular HBs and an increase in energy caused by the break of protein-water HBs referred to as "energetic dehydration penalty." The entropic component is a gain of the translational, configurational entropies of hydrocarbon groups within the lipid bilayer and of water molecules. The entropic component is calculated using the integral equation theory combined with our morphometric approach. The energetic one is estimated by a simple but physically reasonable method. We show that TR is much more stable than XR for the following reasons: The decrease in energy within the TM region is larger, and the energetic dehydration penalty within the WI regions is smaller, leading to higher energetic stabilization, and tighter packing of side chains accompanying the association of seven helices confers higher entropic stabilization on TR.

Effect of Alkyl Side Chain Length on Doping Kinetics, Thermopower, and Charge Transport Properties in Highly Oriented F4TCNQ-Doped PBTTT Films.

Doping of polymer semiconductors such as poly(2,5-bis(3-alkylthiophen-2-yl)thieno[3,2- b]thiophene) (PBTTT) with acceptor molecules such as 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane (F4TCNQ) is widely used to tune the charge transport and thermoelectric (TE) properties in thin films. However, the mechanism of dopant insertion in the polymer matrix, insertion kinetics, and the ultimate doping levels reached have been investigated only marginally. This contribution addresses the effect of alkyl side chain length on the doping mechanism of a series of PBTTTs with linear side chains ranging from n-octyl to n-octyldecyl. The study focuses on thin films oriented by high-temperature rubbing and sequentially doped in F4TCNQ solution. Structure-property correlations are established as a function of side chain length by a combination of transmission electron microscopy, polarized UV-vis-NIR spectroscopy, and charge transport/thermopower measurements. Intercalation of F4TCNQ into the layers of side chains results in the expansion of the lattice along the side chains and the contraction along the π-stacking direction for all polymers. The extent of lattice expansion decreases with the increasing side chain length. UV-vis-NIR spectroscopy demonstrates integer charge transfer for all investigated PBTTTs. The doping kinetics and the final doping level depend on both the side chain length and packing. Highly disordered n-octyl and crystalline n-octyldecyl side chain layers tend to hamper dopant diffusion in the side chain layers contrary to n-dodecyl side chains that can host the highest proportion of dopants. Consequently, the best TE properties are observed for C12-PBTTT films. Alignment of the polymers significantly enhances the TE performance by increasing the charge conductivity and the thermopower along the rubbing direction. Aligned films of C12-PBTTT show charge conductivities of 193 S cm-1 along the rubbing direction and power factors of approximately 100 μW m-1 K-2 versus a few μW m-1 K-2 for nonoriented films.

Slow Motion Protein Dance Visualized Using Red-Edge Excitation Shift of a Buried Fluorophore.

It has been extremely challenging to detect protein structures with a dynamic core, such as dry molten globules, that remain in equilibrium with the tightly packed native (N) state and that are important for a myriad of entropy-driven protein functions. Here, we detect the higher entropy conformations of a human serum protein, using red-edge excitation shift experiments. We covalently introduced a fluorophore inside the protein core and observed that in a subset of native population, the side chains of the polar and buried residues have different spatial arrangements than the mean population and that they solvate the fluorophore on a timescale much slower than the nanosecond timescale of fluorescence. Our results provide direct evidence for the dense fluidity of protein core and show that alternate side-chain packing arrangements exist in the core that might be important for multiple binding functions of this protein.

Accurate calculation of side chain packing and free energy with applications to protein molecular dynamics.

To address the large gap between time scales that can be easily reached by molecular simulations and those required to understand protein dynamics, we present a rapid self-consistent approximation of the side chain free energy at every integration step. In analogy with the adiabatic Born-Oppenheimer approximation for electronic structure, the protein backbone dynamics are simulated as preceding according to the dictates of the free energy of an instantaneously-equilibrated side chain potential. The side chain free energy is computed on the fly, allowing the protein backbone dynamics to traverse a greatly smoothed energetic landscape. This computation results in extremely rapid equilibration and sampling of the Boltzmann distribution. Our method, termed Upside, employs a reduced model involving the three backbone atoms, along with the carbonyl oxygen and amide proton, and a single (oriented) side chain bead having multiple locations reflecting the conformational diversity of the side chain’s rotameric states. We also introduce a novel, maximum-likelihood method to parameterize the side chain interactions using protein structures. We demonstrate state-of-the-art accuracy for predicting χ1 rotamer states while consuming only milliseconds of CPU time. Our method enables rapidly equilibrating coarse-grained simulations that can nonetheless contain significant molecular detail. We also show that the resulting free energies of the side chains are sufficiently accurate for de novo folding of some proteins.

Mispacking of the Phe87 Side Chain Reduces the Kinetic Stability of Human Transthyretin.

Aggregation of transthyretin (TTR) causes TTR amyloidoses. The native TTR tetramer (a dimer of dimers) is stabilized by packing of phenylalanine 87 (F87) into a hydrophobic cavity of a neighboring protomer across the strong dimer interface. X-ray structures at acidic pH show that the side chain of F87 can be displaced from its binding pocket, but the resultant solution conformations remain unknown. Here we used 19F nuclear magnetic resonance (NMR) and 19F-labeled C10S-S85C TTR to characterize two local conformations of the loop containing F87. At neutral pH, F87 packs correctly into the interprotomer cavity in the dominant conformational state (93% population, T) whereas the remaining minor population is a mispacked tetramer (T*). The population of T* can be enhanced in heterotetramers by mixing C10S-S85C TTR with increasing molar ratios of A120L TTR, where a bulky leucine residue is introduced to disfavor the T state by steric hindrance. Exchange between the T and T* states in the presence of A120L is mediated by subunit exchange from the C10S-S85C tetramer. Compared to the TTR tetramer in which the dimers are correctly packed, mispacking of one or both dimer pairs leads to an increase in the urea unfolding rate of 4-fold or at least 15-fold, respectively. Consistent acid-mediated tetramer dissociation was observed by 19F NMR aggregation assays. Our results highlight the important role of the interprotomer F87 side chain packing in determining the kinetic stability of the TTR tetramer; mispacking of F87 in the T* state predisposes it for rapid dissociation and entry into the aggregation pathway.

2,6-Bis(pyrazol-1-yl)pyridine-4-carboxylate Esters with Alkyl Chain Substituents and Their Iron(II) Complexes

Two series of 4-(alkoxyphenyl) 2,6-bis{pyrazol-1-yl}pyridine-4-carboxyate (L3R) or alkyl 2,6-bis{pyrazol-1-yl}pyridine-4-carboxyate (L4R) esters have been synthesized and complexed to iron(II), where R = C nH2 n+1 ( n = 6, 12, 14, 16, 18); two other derivatives related to L3R are also reported. While the solid [Fe(L4R)2][BF4]2 compounds are isostructural by powder diffraction and show similar spin state behaviors, the [Fe(L3R)2][BF4]2 series shows more varied structures and magnetic properties. This was confirmed by solvated crystal structures of [Fe(L3R)2][BF4]2 with n = 6, 14, 16, which all adopt the P1̅ space group but show significantly different side-chain conformations and/or crystal packing. The solid complexes are mostly low spin at room temperature, with many exhibiting the onset of thermal spin crossover (SCO) upon warming. Heating the complexes with n ≥ 14 significantly above their SCO temperature transforms them irreversibly into a predominantly high spin state, which is accompanied by structure changes and loss of crystallinity by powder diffraction. These transformations do not coincide with lattice solvent loss and may reflect melting and refreezing of their alkyl chain conformations during the thermal cycle. Four of the complexes exhibit SCO in CD3CN solution with T1/2 = 273-277 K, which is apparently unaffected by their alkyl chain substituents.

Maintaining and breaking symmetry in homomeric coiled-coil assemblies

In coiled-coil (CC) protein structures α-helices wrap around one another to form rope-like assemblies. Most natural and designed CCs have two–four helices and cyclic (Cn) or dihedral (Dn) symmetry. Increasingly, CCs with five or more helices are being reported. A subset of these higher-order CCs is of interest as they have accessible central channels that can be functionalised; they are α-helical barrels. These extended cavities are surprising given the drive to maximise buried hydrophobic surfaces during protein folding and assembly in water. Here, we show that α-helical barrels can be maintained by the strategic placement of β-branched aliphatic residues lining the lumen. Otherwise, the structures collapse or adjust to give more-complex multi-helix assemblies without Cn or Dn symmetry. Nonetheless, the structural hallmark of CCs—namely, knobs-into-holes packing of side chains between helices—is maintained leading to classes of CCs hitherto unobserved in nature or accessed by design.

A thing of beauty: Structure and function of insulin's "aromatic triplet".

The classical crystal structure of insulin was determined in 1969 by D.C. Hodgkin et al. following a 35-year program of research. This structure depicted a hexamer remarkable for its self-assembly as a zinc-coordinated trimer of dimer. Prominent at the dimer interface was an "aromatic triplet" of conserved residues at consecutive positions in the B chain: PheB24 , PheB25 and TyrB26 . The elegance of this interface inspired the Oxford team to poetry: "A thing of beauty is a joy forever" (John Keats as quoted by Blundell, T.L., et al. Advances in Protein Chemistry 26:279-286 [1972]). Here, we revisit this aromatic triplet in light of recent advances in the structural biology of insulin bound as a monomer to fragments of the insulin receptor. Such co-crystal structures have defined how these side chains pack at the primary hormone-binding surface of the receptor ectodomain. On receptor binding, the B-chain β-strand (residues B24-B28) containing the aromatic triplet detaches from the α-helical core of the hormone. Whereas TyrB26 lies at the periphery of the receptor interface and may functionally be replaced by a diverse set of substitutions, PheB24 and PheB25 engage invariant elements of receptor domains L1 and αCT. These critical contacts were anticipated by the discovery of diabetes-associated mutations at these positions by Donald Steiner et al. at the University of Chicago. Conservation of PheB24 , PheB25 and TyrB26 among vertebrate insulins reflects the striking confluence of structure-based evolutionary constraints: foldability, protective self-assembly and hormonal activity.

Universal effects of solvent species on the stabilized structure of a protein.

We investigate the effects of solvent specificities on the stability of the native structure (NS) of a protein on the basis of our free-energy function (FEF). We use CPB-bromodomain (CBP-BD) and apoplastocyanin (apoPC) as representatives of the protein universe and water, methanol, ethanol, and cyclohexane as solvents. The NSs of CBP-BD and apoPC consist of 66% α-helices and of 35% β-sheets and 4% α-helices, respectively. In order to assess the structural stability of a given protein immersed in each solvent, we contrast the FEF of its NS against that of a number of artificially created, misfolded decoys possessing the same amino-acid sequence but significantly different topology and α-helix and β-sheet contents. In the FEF, we compute the solvation entropy using the morphometric approach combined with the integral equation theories, and the change in electrostatic (ES) energy upon the folding is obtained by an explicit atomistic but simplified calculation. The ES energy change is represented by the break of protein-solvent hydrogen bonds (HBs), formation of protein intramolecular HBs, and recovery of solvent-solvent HBs. Protein-solvent and solvent-solvent HBs are absent in cyclohexane. We are thus able to separately evaluate the contributions to the structural stability from the entropic and energetic components. We find that for both CBP-BD and apoPC, the energetic component dominates in methanol, ethanol, and cyclohexane, with the most stable structures in these solvents sharing the same characteristics described as an association of α-helices. In particular, those in the two alcohols are identical. In water, the entropic component is as strong as or even stronger than the energetic one, with a large gain of translational, configurational entropy of water becoming crucially important so that the relative contents of α-helix and β-sheet and the content of total secondary structures are carefully selected to achieve sufficiently close packing of side chains. If the energetic component is excluded for a protein in water, the priority is given to closest side-chain packing, giving rise to the formation of a structure with very low α-helix and β-sheet contents. Our analysis, which requires minimal computational effort, can be applied to any protein immersed in any solvent and provides robust predictions that are quite consistent with the experimental observations for proteins in different solvent environments, thus paving the way toward a more detailed understanding of the folding process.

Applying graph theory to protein structures: an Atlas of coiled coils

MotivationTo understand protein structure, folding and function fully and to design proteins de novo reliably, we must learn from natural protein structures that have been characterized experimentally. The number of protein structures available is large and growing exponentially, which makes this task challenging. Indeed, computational resources are becoming increasingly important for classifying and analyzing this resource. Here, we use tools from graph theory to define an Atlas classification scheme for automatically categorizing certain protein substructures.ResultsFocusing on the α-helical coiled coils, which are ubiquitous protein-structure and protein–protein interaction motifs, we present a suite of computational resources designed for analyzing these assemblies. iSOCKET enables interactive analysis of side-chain packing within proteins to identify coiled coils automatically and with considerable user control. Applying a graph theory-based Atlas classification scheme to structures identified by iSOCKET gives the Atlas of Coiled Coils, a fully automated, updated overview of extant coiled coils. The utility of this approach is illustrated with the first formal classification of an emerging subclass of coiled coils called α-helical barrels. Furthermore, in the Atlas, the known coiled-coil universe is presented alongside a partial enumeration of the ‘dark matter’ of coiled-coil structures; i.e. those coiled-coil architectures that are theoretically possible but have not been observed to date, and thus present defined targets for protein design.Availability and implementationiSOCKET is available as part of the open-source GitHub repository associated with this work (https://github.com/woolfson-group/isocket). This repository also contains all the data generated when classifying the protein graphs. The Atlas of Coiled Coils is available at: http://coiledcoils.chm.bris.ac.uk/atlas/app.

Loss of function mutations in VARS encoding cytoplasmic valyl-tRNA synthetase cause microcephaly, seizures, and progressive cerebral atrophy.

Progressive microcephaly and neurodegeneration are genetically heterogenous conditions, largely associated with genes that are essential for the survival of neurons. In this study, we interrogate the genetic etiology of two siblings from a non-consanguineous family with severe early onset of neurological manifestations. Whole exome sequencing identified novel compound heterozygous mutations in VARS that segregated with the proband: a missense (c.3192G>A; p.Met1064Ile) and a splice site mutation (c.1577-2A>G).The VARS gene encodes cytoplasmic valyl-tRNA synthetase (ValRS), an enzyme that is essential during eukaryotic translation. cDNA analysis on patient derived fibroblasts revealed that the splice site acceptor variant allele led to nonsense mediated decay, thus resulting in anull allele. Three-dimensional modeling of ValRS predicts that the missense mutation lies in a highly conserved region and could alter side chain packing, thus affecting tRNA binding or destabilizing the interface between the catalytic and tRNA binding domains. Further quantitation of the expression of VARS showed remarkably reduced levels of mRNA and protein in skin derived fibroblasts. Aminoacylation experiments on patient derived cells showed markedly reduced enzyme activity of ValRS suggesting the mutations to be loss of function. Bi-allelic mutations in cytoplasmic amino acyl tRNA synthetases are well-known for their role in neurodegenerative disorders, yet human disorders associated with VARS mutations have not yet been clinically well characterized. Our study describes the phenotype associated with recessive VARS mutations and further functional delineation of the pathogenicity of novel variants identified, which widens the clinical and genetic spectrum of patients with progressive microcephaly.

Comparing side chain packing in soluble proteins, protein‐protein interfaces, and transmembrane proteins

We compare side chain prediction and packing of core and non-core regions of soluble proteins, protein-protein interfaces, and transmembrane proteins. We first identified or created comparable databases of high-resolution crystal structures of these 3 protein classes. We show that the solvent-inaccessible cores of the 3 classes of proteins are equally densely packed. As a result, the side chains of core residues at protein-protein interfaces and in the membrane-exposed regions of transmembrane proteins can be predicted by the hard-sphere plus stereochemical constraint model with the same high prediction accuracies (>90%) as core residues in soluble proteins. We also find that for all 3 classes of proteins, as one moves away from the solvent-inaccessible core, the packing fraction decreases as the solvent accessibility increases. However, the side chain predictability remains high (80% within 30°) up to a relative solvent accessibility, rSASA≲0.3, for all 3 protein classes. Our results show that ≈40% of the interface regions in protein complexes are "core", that is, densely packed with side chain conformations that can be accurately predicted using the hard-sphere model. We propose packing fraction as a metric that can be used to distinguish real protein-protein interactions from designed, non-binding, decoys. Our results also show that cores of membrane proteins are the same as cores of soluble proteins. Thus, the computational methods we are developing for the analysis of the effect of hydrophobic core mutations in soluble proteins will be equally applicable to analyses of mutations in membrane proteins.

Scoring of Side-Chain Packings: An Analysis of Weight Factors and Molecular Dynamics Structures.

The protein side-chain packing problem (PSCPP) is a central task in computational protein design. The problem is usually modeled as a combinatorial optimization problem, which consists of searching for a set of rotamers, from a given rotamer library, that minimizes a scoring function (SF). The SF is a weighted sum of terms, that can be decomposed in physics-based and knowledge-based terms. Although there are many methods to obtain approximate solutions for this problem, all of them have similar performances and there has not been a significant improvement in recent years. Studies on protein structure prediction and protein design revealed the limitations of current SFs to achieve further improvements for these two problems. In the same line, a recent work reported a similar result for the PSCPP. In this work, we ask whether or not this negative result regarding further improvements in performance is due to (i) an incorrect weighting of the SFs terms or (ii) the constrained conformation resulting from the protein crystallization process. To analyze these questions, we (i) model the PSCPP as a bi-objective combinatorial optimization problem, optimizing, at the same time, the two most important terms of two SFs of state-of-the-art algorithms and (ii) performed a preprocessing relaxation of the crystal structure through molecular dynamics to simulate the protein in the solvent and evaluated the performance of these two state-of-the-art SFs under these conditions. Our results indicate that (i) no matter what combination of weight factors we use the current SFs will not lead to better performances and (ii) the evaluated SFs will not be able to improve performance on relaxed structures. Furthermore, the experiments revealed that the SFs and the methods are biased toward crystallized structures.