Importance of chirality and reduced flexibility of protein side chains: a study with square and tetrahedral lattice models

Side chains of amino acid residues are the determining factor that distinguishes proteins from other unstable chain polymers. In simple models they are often represented implicitly (e.g., by spin states) or simplified as one atom. Here we study side chain effects using two-dimensional square lattice and three-dimensional tetrahedral lattice models, with explicitly constructed side chains formed by two atoms of different chirality and flexibility. We distinguish effects due to chirality and effects due to side chain flexibilities, since residues in proteins are L residues, and their side chains adopt different rotameric states. For short chains, we enumerate exhaustively all possible conformations. For long chains, we sample effectively rare events such as compact conformations and obtain complete pictures of ensemble properties of conformations of these models at all compactness region. This is made possible by using sequential Monte Carlo techniques based on chain growth method. Our results show that both chirality and reduced side chain flexibility lower the folding entropy significantly for globally compact conformations, suggesting that they are important properties of residues to ensure fast folding and stable native structure. This corresponds well with our finding that natural amino acid residues have reduced effective flexibility, as evidenced by statistical analysis of rotamer libraries and side chain rotatable bonds. We further develop a method calculating the exact side chain entropy for a given backbone structure. We show that simple rotamer counting underestimates side chain entropy significantly for both extended and near maximally compact conformations. We find that side chain entropy does not always correlate well with main chain packing. With explicit side chains, extended backbones do not have the largest side chain entropy. Among compact backbones with maximum side chain entropy, helical structures emerge as the dominating configurations. Our results suggest that side chain entropy may be an important factor contributing to the formation of alpha helices for compact conformations.     

Simple physics-based analytical formulas for the potentials of mean force for the interaction of amino acid side chains in water. IV. Pairs of different hydrophobic side chains

The potentials of mean force of 21 heterodimers of the molecules modeling hydrophobic amino acid side chains: ethane (for alanine), propane (for proline), isobutane (for valine), isopentane (for leucine and isoleucine), ethylbenzene (for phenylalanine), methyl propyl sulfide (for methionine), and indole (for tryptophane) were determined by umbrella-sampling molecular dynamics simulations in explicit water as functions of distance and orientation. Analytical expressions consisting of the Gay-Berne term to represent effective van der Waals interactions and the cavity term proposed in our earlier work were fitted to the potentials of mean force. The positions and depths of the contact minima and the positions and heights of the desolvation maxima, including their dependence on the orientation of the molecules, are well represented by the analytical expressions for all systems; large deviations between the MD-determined PMF and the analytical approximations are observed for pairs involving the least spheroidal solutes: ethylbenzene, indole, and methyl propyl sulfide at short distances at which the PMF is high and, consequently, these regions are rarely visited. When data from the PMF within only 10 kcal/mol above the global minimum are considered, the standard deviation between the MD-determined and the fitted PMF is from 0.25 to 0.55 kcal/mol (the relative standard deviation being from 4% to 8%); it is larger for pairs involving nonspherical solute molecules. The free energies of contact computed from the PMF surfaces are well correlated with those determined from protein-crystal data with a slope close to that relating the free energies of transfer of amino acids (from water to n-octanol) to the average contact free energies determined from protein-crystal data. These observations justify future use of the determined potentials in coarse-grained protein-folding simulations.     

Importance of solvent accessibility and contact surfaces in modeling side-chain conformations in proteins

Contact surface area and chemical properties of atoms are used to concurrently predict conformations of multiple amino acid side chains on a fixed protein backbone. The combination of surface complementarity and solvent-accessible surface accounts for van der Waals forces and solvation free energy. The scoring function is particularly suitable for modeling partially buried side chains. Both iterative and stochastic searching approaches are used. Our programs (Sccomp-I and Sccomp-S), with relatively fast execution times, correctly predict chi1 angles for 92-93% of buried residues and 82-84% for all residues, with an RMSD of approximately 1.7 A for side chain heavy atoms. We find that the differential between the atomic solvation parameters and the contact surface parameters (including those between noncomplementary atoms) is positive; i.e., most protein atoms prefer surface contact with other protein atoms rather than with the solvent. This might correspond to the driving force for maximizing packing of the protein. The influence of the crystal packing, completeness of rotamer library and precise positioning of Cbeta atoms on the accuracy of side-chain prediction are examined. The Sccomp-S and Sccomp-I programs can be accessed through the Web (http://sgedg.weizmann.ac.il/sccomp.html) and are available for several platforms.     

An investigation of complexes of some model tripeptides containing phenylalanine and tyrosine residues with DNA by Fourier1H NMR spectroscopy

Complexes of native and denatured DNA with model tripeptides containing phenylalanine or tyrosine residues flanked by lysine or arginine residues, respectively have been investigated by pulsed Fourier¹H NMR spectroscopy. The existence of shifts into the strong-field region of the signals of aromatic protons of the model tripeptides in the complexes both with native and with denatured DNA has been shown. Results have been obtained that indicate the possibility of the intercalation of the side chains of aromatic amino acid residues into the DNA double helix.     

Molecular dynamics study of the solvation of an alpha-helical transmembrane peptide by DMSO

A 10-ns molecular dynamics study of the solvation of a hydrophobic transmembrane helical peptide in dimethyl sulfoxide (DMSO) is presented. The objective is to analyze how this aprotic polar solvent is able to solvate three groups of amino acid residues (i.e., polar, apolar, and charged) that are located in a stable helical region of a transmembrane peptide. The 25-residue peptide (sMTM7) used mimics the cytoplasmic proton hemichannel domain of the seventh transmembrane segment (TM7) from subunit a of H(+)-V-ATPase from Saccharomyces cerevisiae. The three-dimensional structure of peptide sMTM7 in DMSO has been previously solved by NMR spectroscopy. The radial and spatial distributions of the DMSO molecules surrounding the peptide as well as the number of hydrogen bonds between DMSO and the side chains of the amino acid residues involved are extracted from the molecular dynamics simulations. Analysis of the molecular dynamics trajectories shows that the amino acid side chains are fully embedded in DMSO. Polar and positively charged amino acid side chains have dipole-dipole interactions with the oxygen atom of DMSO and form hydrogen bonds. Apolar residues become solvated by DMSO through the formation of a hydrophobic pocket in which the methyl groups of DMSO are pointing toward the hydrophobic side chains of the residues involved. The dual solvation properties of DMSO cause it to be a good membrane-mimicking solvent for transmembrane peptides that do not unfold due to the presence of DMSO.     

Bifunctional reagents for the crosslinking of proteins

Proteins can be crosslinked by chemical compounds without any accompanying change in the complicated three-dimensional structure. Many bifunctional reagents that can react with functional groups in the side chains of the amino acids are now known. Not only can the distance between the linked amino acid residues be deduced from the known length of the bridge introduced, but information can also be obtained concerning changes in conformation during other reactions and concerning the arrangement of the components of a quaternary structure.     

Dual substrate recognition of aminotransferases

Pyridoxal 5'-phosphate-dependent aminotransferases reversibly catalyzes the transamination reaction in which the alpha-amino group of amino acid 1 is transferred to the 2-oxo acid of amino acid 2 (usually 2-oxoglutarate) to produce the 2-oxo acid of amino acid 1 and amino acid 2 (glutamate). An aminotransferase must thus be able to recognize and bind two kinds of amino acids (amino acids 1 and 2), the side chains of which are different in shape and properties, from among many other small molecules. The dual substrate recognition mechanism has been discovered based on three-dimensional structures of aromatic amino acids, histidinol phosphate, glutamine:phenylpyruvate, acetylornithine, and branched-chain amino acid aminotransferases. There are two representative strategies for dual substrate recognition. An aromatic amino acid aminotransferase prepares charged and neutral pockets for acidic and aromatic side chains, respectively, at the same place by a large-scale rearrangement of the hydrogen-bond network caused by the induced fit. In a branched-chain aminotransferase, the same hydrophobic cavity implanted with hydrophilic sites accommodates both hydrophobic and acidic side chains without side-chain rearrangements of the active-site residues, which is reminiscent of the lock and key mechanism. Dual substrate recognition in other aminotransferases is attained by combining the two representative methods.     

Statistically based reduced representation of amino acid side chains

Preferred conformations of amino acid side chains have been well established through statistically obtained rotamer libraries. Typically, these provide bond torsion angles allowing a side chain to be traced atom by atom. In cases where it is desirable to reduce the complexity of a protein representation or prediction, fixing all side-chain atoms may prove unwieldy. Therefore, we introduce a general parametrization to allow positions of representative atoms (in the present study, these are terminal atoms) to be predicted directly given backbone atom coordinates. Using a large, culled data set of amino acid residues from high-resolution protein crystal structures, anywhere from 1 to 7 preferred conformations were observed for each terminal atom of the non-glycine residues. Side-chain length from the backbone C(alpha) is one of the parameters determined for each conformation, which should itself be useful. Prediction of terminal atoms was then carried out for a second, nonredundant set of protein structures to validate the data set. Using four simple probabilistic approaches, the Monte Carlo style prediction of terminal atom locations given only backbone coordinates produced an average root mean-square deviation (RMSD) of approximately 3 A from the experimentally determined terminal atom positions. With prediction using conditional probabilities based on the side-chain chi(1) rotamer, this average RMSD was improved to 1.74 A. The observed terminal atom conformations therefore provide reasonable and potentially highly accurate representations of side-chain conformation, offering a viable alternative to existing all-atom rotamers for any case where reduction in protein model complexity, or in the amount of data to be handled, is desired. One application of this representation with strong potential is the prediction of charge density in proteins. This would likely be especially valuable on protein surfaces, where side chains are much less likely to be fixed in single rotamers. Prediction of ensembles of structures provides a method to determine the probability density of charge and atom location; such a prediction is demonstrated graphically.     

Potentials of mean force between ionizable amino acid side chains in water

Potentials of mean force (PMF) between all possible ionizable amino acid side chain pairs in various protonation states were calculated using explicit solvent molecular dynamics simulations with umbrella sampling and the weighted histogram analysis method. The side chains were constrained in various orientations inside a spherical cluster of 200 water molecules. Beglov and Roux's Spherical Solvent Boundary Potential was used to account for the solvent outside this sphere. This approach was first validated by calculating PMFs between monatomic ions (K(+), Na(+), Cl(-)) and comparing them to results from the literature and results obtained using Ewald summation. The strongest interaction (-4.5 kcal/mol) was found for the coaxial Arg(+).Glu(-) pair. Many like-charged side chains display a remarkable lack of repulsion, and occasionally a weak attraction. The PMFs are compared to effective energy curves obtained with common implicit solvation models, namely Generalized Born (GB), EEF1, and uniform dielectric of 80. Overall, the EEF1 curves are too attractive, whereas the GB curves in most cases match the minima of the PMF curves quite well. The uniform dielectric model, despite some fortuitous successes, is grossly inadequate.     

Hydroxyl radical probe of the surface of lysozyme by synchrotron radiolysis and mass spectrometry.

A new approach is reported that combines synchrotron radiolysis and mass spectrometry to probe the surface of proteins. Hydroxyl radicals produced upon the radiolysis of protein solutions with synchrotron light for several milliseconds result in the reaction of amino acid side chains. This results in the formation of stable oxidation products where the level of oxidation at the reactive residues is influenced by the accessibility of their side chains to the bulk solvent. The aromatic and sulfur-containing residues have been found to react preferentially in accord with previous peptide studies. The sites of oxidation have been determined by tandem mass spectrometry. The rate of oxidation at these reactive markers has been measured for each of the proteolytic peptides as a function of exposure time based on the relative proportion of modified and unmodified peptide ions detected by mass spectrometry. Oxidation rates have been found to correlate closely with a theoretical measure of the accessibility of residue side chains to the bulk solvent in the native protein structure. The synchrotron-based approach is able to distinguish the relative accessibility of the tryptophan residue side chains of lysozyme at positions 62 and 123 from each other and all other tryptophan residues based on their rates of oxidation.     

Contact pair dynamics during folding of two small proteins: chicken villin head piece and the Alzheimer protein beta-amyloid

The folding of an extended protein to its unique native state requires establishment of specific, predetermined, often distant, contacts between amino acid residue pairs. The dynamics of contact pair formation between various hydrophobic residues during folding of two different small proteins, the chicken villin head piece (HP-36) and the Alzheimer protein beta-amyloid (betaA-40), are investigated by Brownian dynamics (BD) simulations. These two proteins represent two very different classes-HP-36 being globular while betaA-40 is nonglobular, stringlike. Hydropathy scale and nonlocal helix propensity of amino acids are used to model the complex interaction potential among the various amino acid residues. The minimalistic model we use here employs a connected backbone chain of atoms of equal size while an amino acid is attached to each backbone atom as an additional atom of differing sizes and interaction parameters, determined by the characteristics of each amino acid. Even for such simple models, we find that the low-energy structures obtained by BD simulations of both the model proteins mimic the native state of the real protein rather well, with a best root-mean-square deviation of 4.5 A for HP-36. For betaA-40 (where a single well-defined structure is not available), the simulated structures resemble the reported ensemble rather well, with the well-known beta-bend correctly reproduced. We introduce and calculate a contact pair distance time correlation function, C(P) (ij)(t), to quantify the dynamical evolution of the pair contact formation between the amino acid residue pairs i and j. The contact pair time correlation function exhibits multistage dynamics, including a two stage fast collapse, followed by a slow (microsecond long) late stage dynamics for several specific pairs. The slow late stage dynamics is in accordance with the findings of Sali et al. Analysis of the individual trajectories shows that the slow decay is due to the attempt of the protein to form energetically more favorable pair contacts to replace the less favorable ones. This late stage contact formation is a highly cooperative process, involving participation of several pairs and thus entropically unfavorable and expected to face a large free energy barrier. This is because any new pair contact formation among hydrophobic pairs will require breaking of several contacts, before the favorable ones can be formed. This aspect of protein folding dynamics is similar to relaxation in glassy liquids, where also alpha relaxation requires highly cooperative process of hopping. The present analysis suggests that waiting time for the necessary pair contact formation may obey the Poissonian distribution. We also study the dynamics of Forster energy transfer during folding between two tagged amino acid pairs. This dynamics can be studied by fluorescence resonance energy transfer (FRET). It is found that suitably placed donor-acceptor pairs can capture the slow dynamics during folding. The dynamics probed by FRET is predicted to be nonexponential.     

An investigation of complexes of some model tripeptides containing phenylalanine and tyrosine residues with DNA by Fourier1H NMR spectroscopy

Complexes of native and denatured DNA with model tripeptides containing phenylalanine or tyrosine residues flanked by lysine or arginine residues, respectively have been investigated by pulsed Fourier¹H NMR spectroscopy. The existence of shifts into the strong-field region of the signals of aromatic protons of the model tripeptides in the complexes both with native and with denatured DNA has been shown. Results have been obtained that indicate the possibility of the intercalation of the side chains of aromatic amino acid residues into the DNA double helix.All-Union Scientific-Research Institute of the Genetics and Breeding of Industrial Microorganisms, Moscow. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 751–755, November–December, 1986.     

A novel synthesis of N-methyl asparagine,arginine, histidine,and tryptophan

[reaction: see text] N-Methyl amino acid residues in peptides modify several pharmacologically useful parameters, but synthesis of alkylated peptides is hampered by unavailability of N-methylated monomers. The syntheses of four N-methyl amino acids with basic side chains are presented. The side chains of these basic amino acids needed to be specially protected or constructed. This completes the set of 20 common L-amino acid N-methyl derivatives prepared via 5-oxazolidinone intermediates by our group.     

Energy barriers of proton transfer reactions between amino acid side chain analogs and water from ab initio calculations

Proton transfer reactions were studied in all titratable pairs of amino acid side chains where, under physiologically reasonable conditions, one amino acid may function as a donor and the other one as an acceptor. Energy barriers for shifting the proton from donor to acceptor atom were calculated by electronic structure methods at the MP2/6-31++G(d,p) level, and the well-known double-well potentials were characterized. The energy difference between both minima can be expressed by a parabola using as argument the donor-acceptor distance R(DA). In this work, the fit parameters of the quadratic expression are determined for each donor-acceptor pair. Moreover, it was found previously that the energy barriers of the reactions can be expressed by an analytical expression depending on the distance between donor and acceptor and the energy difference between donor and acceptor bound states. The validity of this approach is supported by the extensive new data set. This new parameterization of proton transfer barriers between titratable amino acid side chains allows us to very efficiently estimate proton transfer probabilities in molecular modelling studies or during classical molecular dynamics simulation of biomolecular systems.     

Electron detachment dissociation for top-down mass spectrometry of acidic proteins

Electron detachment dissociation (EDD) is an emerging mass spectrometry (MS) technique for the primary structure analysis of peptides, carbohydrates, and oligonucleotides. Herein, we explore the potential of EDD for sequencing of proteins of up to 147 amino acid residues by using top-down MS. Sequence coverage ranged from 72% for Melittin, which lacks carboxylic acid functionalities, to 19% for an acidic 147-residue protein, to 12% for Ferredoxin, which showed unusual backbone fragmentation next to cysteine residues. A limiting factor for protein sequencing by EDD is the facile loss of small molecules from amino acid side chains, in particular CO(2). Based on the types of fragments observed and fragmentation patterns found, we propose detailed mechanisms for protein backbone cleavage and side chain dissociation in EDD. The insights from this study should further the development of EDD for top-down MS of acidic proteins.     

Metal ligand aromatic cation-pi interactions in metalloproteins: ligands coordinated to metal interact with aromatic residues

Cation-pi interactions between aromatic residues and cationic amino groups in side chains and have been recognized as noncovalent bonding interactions relevant for molecular recognition and for stabilization and definition of the native structure of proteins. We propose a novel type of cation-pi interaction in metalloproteins; namely interaction between ligands coordinated to a metal cation--which gain positive charge from the metal--and aromatic groups in amino acid side chains. Investigation of crystal structures of metalloproteins in the Protein Data Bank (PDB) has revealed that there exist quite a number of metalloproteins in which aromatic rings of phenylalanine, tyrosine, and tryptophan are situated close to a metal center interacting with coordinated ligands. Among these ligands are amino acids such as asparagine, aspartate, glutamate, histidine, and threonine, but also water and substrates like ethanol. These interactions play a role in the stability and conformation of metalloproteins, and in some cases may also be directly involved in the mechanism of enzymatic reactions, which occur at the metal center. For the enzyme superoxide dismutase, we used quantum chemical computation to calculate that Trp163 has an interaction energy of 10.09 kcal mol(-1) with the ligands coordinated to iron.     

The effect of histidine oxidation on the dissociation patterns of peptide ions

Oxidative modifications to amino acid side chains can change the dissociation pathways of peptide ions, although these variations are most commonly observed when cysteine and methionine residues are oxidized. In this work we describe the very noticeable effect that oxidation of histidine residues can have on the dissociation patterns of peptide ions containing this residue. A common product ion spectral feature of doubly charged tryptic peptides is enhanced cleavage at the C-terminal side of histidine residues. This preferential cleavage arises as a result of the unique acid/base character of the imidazole side chain that initiates cleavage of a proximal peptide bond for ions in which the number of protons does not exceed the number of basic residues. We demonstrate here that this enhanced cleavage is eliminated when histidine is oxidized to 2-oxo-histidine because the proton affinity and nucleophilicity of the imidazole side chain are lowered. Furthermore, we find that oxidation of histidine to 2-oxo-histidine can cause the misassignment of oxidized residues when more than one oxidized isomer is simultaneously subjected to tandem mass spectrometry (MS/MS). These spectral misinterpretations can usually be avoided by using multiple stages of MS/MS (MS(n)) or by specially optimized liquid chromatographic separation conditions. When these approaches are not accessible or do not work, N-terminal derivatization with sulfobenzoic acid avoids the problem of mistakenly assigning oxidized residues.     

Versatile synthesis of fluorescent, cross-linked peptides as biological probes with the advantage of high helix content

A variety of fluorophores were introduced at the N-termini of short peptides for use as biological-probes. The fluorescent peptides were cross-linked with a diacetylenic cross-linking agent between the amino acid side chains of ornithine (Orn) residues to produce peptides with high helix content.