A new approach to the rapid determination of protein side chain conformations

Two efficient algorithms have been developed which allow amino acid side chain conformations to be optimized rapidly for a given peptide backbone conformation. Both these approaches are based on the assumption that each side chain can be represented by a small number of rotameric states. These states have been obtained by a dynamic cluster analysis of a large data base of known crystallographic structures. Successful applications of these algorithms to the prediction of known protein conformations are presented.     

RASP: rapid and robust backbone chemical shift assignments from protein structure

Chemical shift prediction has an unappreciated power to guide backbone resonance assignment in cases where protein structure is known. Here we describe Resonance Assignment by chemical Shift Prediction (RASP), a method that exploits this power to derive protein backbone resonance assignments from chemical shift predictions. Robust assignments can be obtained from a minimal set of only the most sensitive triple-resonance experiments, even for spectroscopically challenging proteins. Over a test set of 154 proteins RASP assigns 88 % of residues with an accuracy of 99.7 %, using only information available from HNCO and HNCA spectra. Applied to experimental data from a challenging 34 kDa protein, RASP assigns 90 % of manually assigned residues using only 40 % of the experimental data required for the manual assignment. RASP has the potential to significantly accelerate the backbone assignment process for a wide range of proteins for which structural information is available, including those for which conventional assignment strategies are not feasible.     

Antibody side chain conformations are position‐dependent

Side chain prediction is an integral component of computational antibody design and structure prediction. Current antibody modelling tools use backbone‐dependent rotamer libraries with conformations taken from general proteins. Here we present our antibody‐specific rotamer library, where rotamers are binned according to their immunogenetics (IMGT) position, rather than their local backbone geometry. We find that for some amino acid types at certain positions, only a restricted number of side chain conformations are ever observed. Using this information, we are able to reduce the breadth of the rotamer sampling space. Based on our rotamer library, we built a side chain predictor, position‐dependent antibody rotamer swapper (PEARS). On a blind test set of 95 antibody model structures, PEARS had the highest average χ₁ and accuracy (78.7% and 64.8%) compared to three leading backbone‐dependent side chain predictors. Our use of IMGT position, rather than backbone ϕ/ψ, meant that PEARS was more robust to errors in the backbone of the model structure. PEARS also achieved the lowest number of side chain–side chain clashes. PEARS is freely available as a web application at http://opig.stats.ox.ac.uk/webapps/pears .     

Preferred conformations and flexibility of aminoacyl side chain of penicillins

Possible conformations of penicillin G; d and l isomers of ampicillin; α-amino-α-methyl-benzyl penicillins and 3- pyridyl methyl penicillin have been studied by an energy minimization procedure using empirical potential functions. The preferred conformations of these antibiotics have been correlated with their biological activity. The conformational requirement of the antibiotic to be active against Gram-positive and Gram-negative (β-lactamase-negative) bacterial strains seems to be the same. The reduced activity of penicillin G against Gram-negative bacteria has been attributed to its lower ability to permeate the outer membrane. The flexibility of the sidechains of these antibiotics is also shown to be important for the desired biological activity.     

The backbone and side chain conformations of the cyclic tetrapeptide HC-toxin

A study of the conformational parameters of HC-toxin and its diacetyl derivative in chloroform solution has been carried out. Two-dimensional NMR spectroscopy and the nuclear Overhauser effect have been used in order to determine connectivities (assignments and sequence) and approximate torsion angles and interproton distances. The results are consistent with a bis-gamma-turn conformation previously reported for dihydrochlamydocin. Model building based upon NMR data supports a D configuration for Ala2 and Pro4 residues.     

Minimal ensembles of side chain conformers for modeling protein–protein interactions

The goal of this article is to reduce the complexity of the side chain search within docking problems. We apply six methods of generating side chain conformers to unbound protein structures and determine their ability of obtaining the bound conformation in small ensembles of conformers. Methods are evaluated in terms of the positions of side chain end groups. Results for 68 protein complexes yield two important observations. First, the end‐group positions change less than 1 Å on association for over 60% of interface side chains. Thus, the unbound protein structure carries substantial information about the side chains in the bound state, and the inclusion of the unbound conformation into the ensemble of conformers is very beneficial. Second, considering each surface side chain separately in its protein environment, small ensembles of low‐energy states include the bound conformation for a large fraction of side chains. In particular, the ensemble consisting of the unbound conformation and the two highest probability predicted conformers includes the bound conformer with an accuracy of 1 Å for 78% of interface side chains. As more than 60% of the interface side chains have only one conformer and many others only a few, these ensembles of low‐energy states substantially reduce the complexity of side chain search in docking problems. This approach was already used for finding pockets in protein–protein interfaces that can bind small molecules to potentially disrupt protein–protein interactions. Side‐chain search with the reduced search space will also be incorporated into protein docking algorithms. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Improving ranking of models for protein complexes with side chain modeling and atomic potentials

An atomically detailed potential for docking pairs of proteins is derived using mathematical programming. A refinement algorithm that builds atomically detailed models of the complex and combines coarse grained and atomic scoring is introduced. The refinement step consists of remodeling the interface side chains of the top scoring decoys from rigid docking followed by a short energy minimization. The refined models are then re‐ranked using a combination of coarse grained and atomic potentials. The docking algorithm including the refinement and re‐ranking, is compared favorably to other leading docking packages like ZDOCK, Cluspro, and PATCHDOCK, on the ZLAB 3.0 Benchmark and a test set of 30 novel complexes. A detailed analysis shows that coarse grained potentials perform better than atomic potentials for realistic unbound docking (where the exact structures of the individual bound proteins are unknown), probably because atomic potentials are more sensitive to local errors. Nevertheless, the atomic potential captures a different signal from the residue potential and as a result a combination of the two scores provides a significantly better prediction than each of the approaches alone. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Standard conformations of a polypeptide chain in irregular protein regions

A detailed stereochemical analysis of known protein structures has been made which shows that: (1) irregular regions of proteins consist of a limited number of standard structures formed by three, four of more residues; (2) an amino acid residue of a protein can adopt one of the six sterically allowed conformations designated here as alpha, alpha L, beta, gamma, delta, and epsilon. It is shown that there are two allowed conformations of a polypeptide chain at the N-end of an alpha-helix, beta alpha n- and beta gamma alpha n-conformations, where n is a number of residues in the alpha-helix. At the C-end of the alpha-helix there are two conformations as well, alpha n gamma beta- and alpha n gamma alpha L beta-ones. Two beta-strands in a beta-hairpin can be joined, for example, by standard structures with beta beta alpha L beta-, beta alpha gamma alpha L beta-, beta alpha alpha gamma alpha L beta-conformations which are referred to as turns. In the regions where a polypeptide chain passes from one layer to another there are standard structures with beta gamma beta-, beta alpha beta beta-, beta alpha gamma beta-conformations etc., referred to as cross-overs. A structure of any protein irregular region can be represented as a combination of these and other standard turns and cross-overs considered in the paper. The major part of the turns and cross-overs has residues in alpha L- or epsilon-conformations which must be glycine or other residues with small or flexible side chains. Massive hydrophobic residues must not occupy the first beta-positions of the most standard structures. The results obtained can be successfully applied for prediction of the location of the turns and cross-overs in proteins from their amino acid sequences and for interpretation of electron density maps.     

Correlated alternative side chain conformations in the RNA-recognition motif of heterogeneous nuclear ribonucleoprotein A1

The RNA-recognition motif (RRM) is a common and evolutionarily conserved RNA-binding module. Crystallographic and solution structural studies have shown that RRMs adopt a compact α/β structure, in which four antiparallel β-strands form the major RNA-binding surface. Conserved aromatic residues in the RRM are located on the surface of the β-sheet and are important for RNA binding. To further our understanding of the structural basis of RRM-nucleic acid interaction, we carried out a high resolution analysis of UP1, the N-terminal, two-RRM domain of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), whose structure was previously solved at 1.75–1.9 Å resolution. The two RRMs of hnRNP A1 are closely related but have distinct functions in regulating alternative pre-mRNA splice site selection. Our present 1.1 Å resolution crystal structure reveals that two conserved solvent-exposed phenylalanines in the first RRM have alternative side chain conformations. These conformations are spatially correlated, as the individual amino acids cannot adopt each of the observed conformations independently. These phenylalanines are critical for nucleic acid binding and the observed alternative side chain conformations may serve as a mechanism for regulating nucleic acid binding by RRM-containing proteins.     

Backbone conformations and side chain flexibility of two somatostatin mimics investigated by molecular dynamics simulations

Molecular dynamics simulations with two designed somatostatin mimics, SOM230 and SMS 201‐995, were performed in explicit water for a total aggregated time of 208 ns. Analysis of the runs with SOM230 revealed the presence of two clusters of conformations. Strikingly, the two sampled conformers correspond to the two main X‐ray structures in the asymmetric unit of SMS 201‐995. Structural comparison between the residues of SOM230 and SMS 201‐995 provides an explanation for the high binding affinity of SOM230 to four of five somatostatin receptors. Similarly, cluster analysis of the simulations with SMS 201‐995 shows that the backbone of the peptide interconverts between its two main crystallographic conformers. The conformations of SMS 201‐995 sampled in the two clusters violated two different sets of NOE distance constraints in agreement with a previous NMR study. Differences in side chain fluctuations between SOM230 and SMS 201‐995 observed in the simulations may contribute to the relatively higher binding affinity of SOM230 to most somatostatin receptors. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Phenylalanine side chain behavior of the intestinal fatty acid-binding protein: the effect of urea on backbone and side chain stability

The equilibrium unfolding behavior of the intestinal fatty acid-binding protein has been investigated by (19)F-NMR after incorporation of 4-fluorophenylalanine and by pulsed field gradient diffusion (1)H-NMR. At low urea concentrations (0-3 m) but prior to the global unfolding that begins at 4 m urea, the protein exhibits dynamic motion in the backbone and an expanded hydrodynamic radius with no major change in the side chain orientation. As monitored by two-dimensional (19)F-(19)F nuclear Overhauser effect, the distance between two phenylalanine residues (Phe(68) and Phe(93)) located in the two different beta-sheets that enclose the internal cavity did not change up to 4 m urea. Additionally, the chemical shifts of these two residues changed almost identically as a function of denaturant. At all urea concentrations, as well as in the native protein, multiple conformations exist. These conformers interconvert at different rates under different conditions, ranging from slow exchange by showing separate peaks in the native state to intermediate exchange at intermediate urea concentrations. Residual structure persisted around Phe(62) even at very high concentrations of denaturant, suggesting that region as a nucleation site during folding. The results were compared with previous studies examining the backbone behavior (Hodsdon, M. E., and Frieden, C. (2001) Biochemistry 40, 732-742) and suggest that the side chains show more stability than the backbone prior to global unfolding of the protein.     

The kinetics of side chain stabilization during protein folding

The rate of stabilization of side chains during protein folding has never been carefully studied. Recent developments in labeling proteins with (19)F-labeled amino acids coupled with real-time NMR measurements have allowed such measurements to be made. This paper describes the application of this method to the study of several proteins using 6-(19)F-tryptophan as the reporting group. It is found that these side chains adopt their final stable state at the last stages of the folding process and that the stabilization of side chains into their final conformation is a highly cooperative process. It is also possible to show the presence of intermediates in which the side chains are not correctly packed. The technique should be applicable to many systems.     

Structural basis of hierarchical multiple substates of a protein. III: Side chain and main chain local conformations

An analysis is carried out of differences in the minimum energy conformations obtained in the previous paper by energy minimization starting from conformations sampled by a Monte Carlo simulation of conformational fluctuations in the native state of a globular protein, bovine pancreatic trypsin inhibitor. Main conformational differences in each pair of energy minima are found usually localized in several side chains and in a few local main chain segments. Such side chains and local main chain segments are found to take a few distinct local conformations in the minimum energy conformations. Energy minimum conformations can thus be described in terms of combinations of these multiple local conformations.     

Patterns in ionizable side chain interactions in protein structures

In a selected set of 44 high-resolution, non-homologous protein structures, the intramolecular hydrogen bonds or salt bridges formed by ionizable amino acid side chains were identified and analyzed. The analysis was based on the investigation of several properties of the involved residues such as their solvent exposure, their belonging to a certain secondary structural element, and their position relative to the N- and C-termini of their respective structural element. It was observed that two-thirds of the interactions made by basic or acidic side chains are hydrogen bonds to polar uncharged groups. In particular, the majority (78%) of the hydrogen bonds between ionizable side chains and main chain polar groups (sch:mch bonds) involved at least one buried atom, and in 42% of the cases both interacting atoms were buried. In α-helices, the sch:mch bonds observed in the proximity of the C- and N-termini show a clear preference for acidic and basic side chains, respectively. This appears to be due to the partial charges of peptide group atoms at the termini of α-helices, which establish energetically favorable electrostatic interactions with side chain carrying opposite charge, at distances even greater than 4.5 Å. The sch:mch interactions involving ionizable side chains that belong either to β-strands or to the central part of α-helices are based almost exclusively on basic residues. This results from the presence of main chain carbonyl oxygen atoms in the protein core which have unsatisfied hydrogen bonding capabilities.     

The Dynameomics rotamer library: amino acid side chain conformations and dynamics from comprehensive molecular dynamics simulations in water

We have recently completed systematic molecular dynamics simulations of 807 different proteins representing 95% of the known autonomous protein folds in an effort we refer to as Dynameomics. Here we focus on the analysis of side chain conformations and dynamics to create a dynamic rotamer library. Overall this library is derived from 31,000 occurrences of each of 86,217 different residues, or 2.7 × 10(9) rotamers. This dynamic library has 74% overlap of rotamer distributions with rotamer libraries derived from static high-resolution crystal structures. Seventy-five percent of the residues had an assignable primary conformation, and 68% of the residues had at least one significant alternate conformation. The average correlation time for switching between rotamers ranged from 22 ps for Met to over 8 ns for Cys; this time decreased 20-fold on the surface of the protein and modestly for dihedral angles further from the main chain. Side chain S(2) axis order parameters were calculated and they correlated well with those derived from NMR relaxation experiments (R = 0.9). Relationships relating the S(2) axis order parameters to rotamer occupancy were derived. Overall the Dynameomics rotamer library offers a comprehensive depiction of side chain rotamer preferences and dynamics in solution, and more realistic distributions for dynamic proteins in solution at ambient temperature than libraries derived from crystal structures, in particular charged surface residues are better represented. Details of the rotamer library are presented here and the library itself can be downloaded at http://www.dynameomics.org.     

Probing protein side chain dynamics via 13C NMR relaxation

Protein side chain dynamics is associated with protein stability, folding, and intermolecular interactions. Detailed dynamics information is crucial for the understanding of protein function and biochemical and biophysical properties, which can be obtained using NMR relaxation techniques. In this review, (13)C relaxation of methine, methylene and methyl groups with and without (1)H decoupling are described briefly for a better understanding of how spin relaxation is associated with motional (dynamics) parameters. Developments in the measurement and interpretation of (13)C auto-relaxation and cross-correlated relaxation data are presented too. Finally, recent progress in the use of (13)C relaxation to probe the dynamics of protein side chains is detailed mainly for the dynamics of non-deuterated proteins on picoseconds-nanosecond timescales.     

Incorporation of side chain flexibility into protein binding pockets using MTflex

The plasticity of active sites plays a significant role in drug recognition and binding, but the accurate incorporation of ‘receptor flexibility’ remains a significant computational challenge. Many approaches have been put forward to address receptor flexibility in docking studies by generating relevant ensembles on the energy surface. Herein, we describe the Movable Type with flexibility (MT_flex) method that generates ensembles on the more relevant free energy surface in a computationally tractable manner. This novel approach enumerates conformational states based on side chain flexibility and then estimates their relative free energies using the MT methodology. The resultant conformational states can then be used in subsequent docking and scoring exercises. In particular, we demonstrate that using the MT_flex ensembles improves MT’s ability to predict binding free energies over docking only to the crystal structure.