Aromatic side-chain interactions in proteins. I. Main structural features

In a data set of 593 nonhomologous proteins from the PDB, we have analyzed the pairing of phenylalanine, tyrosine, tryptophan, and histidine residues with their closest aromatic partner. The frequency distribution of the shortest interatomic distance of partners is bimodal with a sharp peak at approximately 3.8 A and a wider one at a longer distance. Only the 3.8 A peak corresponds to direct ring-ring interactions thus aromatic pairs. The aromatic pairs were separated into two classes, near-sequence pairs and far-sequence pairs. Near sequence pairs stabilize local structure, and far-sequence pairs stabilize tertiary structure. Far-sequence pairs (74% of all pairs) mainly bridge two beta-strands, followed by pairs that bridge a beta-strand and a helix, and pairs that bridge a beta-strand and a random coil structure. Pairs that bridge helices are rare. The secondary structure of the near-sequence pairs depends on the partner distance in the sequence. When the partners are 1, 3, or 4 residues apart in the sequence, pairs are mostly found in helical structures. When the partners are two apart, pairs are mostly found in the same beta-strand. Analysis of the frequency of near sequence pairs supports the hypothesis that aromatic pairing occurs after, rather than before, the formation of secondary structures.     

The G protein-coupled 5-HT1A receptor causes suppression of caspase-3 through MAPK and protein kinase Calpha

Several studies have analysed aromatic interactions, involving mostly phenylalanine, tyrosine and tryptophan. Only a few studies have considered histidine as an interacting aromatic residue. An extensive analysis of aromatic His-X interactions is performed here on a data set of 593 PDB structures: 68% of the histidine are involved in aromatic pairs and 1271 non-redundant His-X pairs were analysed. Thirty percent of these pairs involve an aromatic partner less than 6 apart in the sequence. These near-sequence pairs correspond to conformations which stabilise secondary structures, mainly alpha-helices when the residues are 4 apart and beta-strands when they are 2 apart in the sequence. The partners of the other His-X pairs (887, 70%) are more than 5 apart in the sequence. Of these far-sequence pairs, 35% bridge beta strands and only 9% helices. The near-sequence pairs are sterically constrained as supported by conformer distribution. The X partners of far-sequence His-X pairs are mainly "above" the histidine ring with tilted and normal rings, corresponding to a "T shape; face to edge" orientation. Phenylalanine, the only aromatic residue with no heteroatom, is a disfavoured partner, whereas histidine is the preferred one. Heteroatom-heteroatom interactions are favoured in near-sequence as well as in far-sequence His-His, His-Trp and His-Tyr pairs.     

Aromatic side-chain interactions in proteins. Near- and far-sequence His–X pairs

Several studies have analysed aromatic interactions, involving mostly phenylalanine, tyrosine and tryptophan. Only a few studies have considered histidine as an interacting aromatic residue. An extensive analysis of aromatic His–X interactions is performed here on a data set of 593 PDB structures: 68% of the histidine are involved in aromatic pairs and 1271 non-redundant His–X pairs were analysed. Thirty percent of these pairs involve an aromatic partner less than 6 apart in the sequence. These near-sequence pairs correspond to conformations which stabilise secondary structures, mainly α-helices when the residues are 4 apart and β-strands when they are 2 apart in the sequence. The partners of the other His–X pairs (887, 70%) are more than 5 apart in the sequence. Of these far-sequence pairs, 35% bridge beta strands and only 9% helices. The near-sequence pairs are sterically constrained as supported by conformer distribution. The X partners of far-sequence His–X pairs are mainly “above” the histidine ring with tilted and normal rings, corresponding to a “T shape; face to edge” orientation. Phenylalanine, the only aromatic residue with no heteroatom, is a disfavoured partner, whereas histidine is the preferred one. Heteroatom–heteroatom interactions are favoured in near-sequence as well as in far-sequence His–His, His–Trp and His–Tyr pairs.     

Aromatic side-chain interactions in proteins: near- and far-sequence Tyr-X pairs

In the present study, an extensive analysis of the aromatic Tyr-X interactions is performed on a data set of 593 PDB structures, X being Phe, His, Tyr, and Trp. The nonredundant Tyr-X pairs (2645) were retained and separated by both the residue distance in the sequence and the secondary structures they bridge. Similar to the Phe-X and His-X pairs, the far-sequence Tyr-X pairs (X partner > five apart in the sequence: 74%) show comparable secondary structures and conformers for either type of X partner, in contrast with the near-sequence Tyr-X pairs (26%). As the Phe-X pairs, the near-sequence Tyr-X pairs stabilize secondary structures, mainly the alpha- helices (positions 1, 3, and 4) and the beta-strands (position 2). Like the Phe-X and His-X pairs, most far-sequence Tyr-X pairs (34%) bridge beta-strands and only 11% bridge helices. As for the Phe-X and the His-X pairs, the X partners of the far-sequence Tyr-X pairs are frequently "above" the tyrosine ring with tilted and normal rings, whereas the X partner of the near-sequence Tyr-X pairs gradually moves from the "aside" to the "above" location, together with a progressive decrease of normal and increase of parallel rings, respectively. Unlike the His-X pairs, the interactions of the hetroatom in Tyr-X pairs are only favored with a sequence position +4 and over, owing to the spatial accessibility of the heteroatom.     

Calculation of conformational ensembles from potentials of mean force. An approach to the knowledge-based prediction of local structures in globular proteins

We present a prototype of a new approach to the folding problem of polypeptide chains. This approach is based on the analysis of known protein structures. It derives the energy potentials for the atomic interactions of all amino acid residue pairs as a function of the distance between the involved atoms. These potentials are then used to calculate the energies of all conformations that exist in the data base with respect to a given sequence. Then, by using only the most stable conformations, clusters of the most probable conformations for the given sequence are obtained. To discuss the results properly we introduce a new classification of segments based on their conformational stability. Special care is taken to allow for sparse data sets. The use of the method is demonstrated in the discussion of the identical oligopeptide sequences found in different conformations in unrelated proteins. VNTFV, for example, adopts a beta-strand in ribonuclease but it is found in an alpha-helical conformation in erythrocruorin. In the case of VNTFV the ensemble obtained consists of a single cluster of beta-strand conformations, indicating that this may be the preferred conformation for the pentapeptide. When the flanking residues are included in the calculation the hepapeptide P-VNTFV-H (ribonuclease) again yields an ensemble of beta-strands. However, in the ensemble of D-VNTFV-A (erythrocruorin) the major cluster is of alpha-helical type. In the present study we concentrate on the local aspects of protein conformations. However, the theory presented is quite general and not restricted to oligopeptides. We indicate extensions of the approach to the calculation of global conformations of proteins as well as conceivable applications to a number of molecular systems.     

Salt bridges do not stabilize polyproline II helices

Interactions between side chains, and in particular salt bridges, have been shown to be important in the stabilization of secondary structure. Here we investigate the contribution of a salt bridge formed between a lysine and a glutamate to the polyproline II (P(II)) helical content of proline-rich peptides. Since this structure has precisely three residues per turn, charged residues spaced three residues apart are on the same side of the helix and are best situated to interact. By contrast, computer simulations show that charged residues spaced four residues apart are both too far apart to interact strongly and are oriented such that interactions are unlikely. We have measured the P(II) content of peptides containing a lysine and glutamate pair spaced three or four residues apart using circular dichroism spectroscopy. Somewhat surprisingly we find that the P(II) content is insensitive to both the spacing and the pH. These findings indicate that i --> i + 3 salt bridges do not stabilize the P(II) helical conformation. The implications of these observations for both P(II) helix formation and denatured protein conformations are discussed.     

Electronic and vibrational circular dichroism of aromatic amino acids by density functional theory

Structures of model compounds mimicking aromatic amino acid residues in proteins are optimized by density functional theory (DFT), assuming that the main-chain conformation was a random coil. Excitation energies and dipole and rotational strengths for the optimized structures were calculated based on time-dependent DFT (TD-DFT). The electronic circular dichroism (ECD) bands of the models were significantly affected by side-chain conformations. Hydration models of the aromatic residues were also subjected to TD-DFT calculations, and the ECD bands of these models were found to be highly perturbed by the hydration of the main-chain amide groups. In addition to calculating the random-coil conformation, we also performed TD-DFT calculations of the aromatic residue models, assuming that the main-chain conformation was an alpha-helix or beta-strand. As expected, the overall feature of the ECD bands was also perturbed by the main-chain conformations. Moreover, vibrational circular dichroism (VCD) spectra of the hydration models in a random-coil structure were simulated by DFT, which showed that the VCD spectra are more sensitive to the side-chain conformations than the ECD spectra. The present results show that analyses combining ECD and VCD spectroscopy and using DFT calculations can elucidate the main- and side-chain conformations of aromatic residues in proteins.     

Free energy of imperfect nucleic acid helices. 3. Small internal loops resulting from mismatches

Physical studies of enzymioally synthesized oligoribonucleotides of defined sequence are used to evaluate quantitatively the destabilizing influence of mismatched bases in a double helix. The series (A-)₄G(-C)_n(-U)₄, N = 1 to 6, exist as imperfect dimer helices when N is equal to or less than 4, and as monomolecular hairpin helices when N is 5 and 6. Internal loops become progressively more destabilizing as their size increases from 2 to 4 to 6 nucleotides resulting from 1, 2 and 3 consecutive mismatched base pairs. However, the stability of a helix will generally be greater if a given number of mismatched pairs occur consecutively rather than in isolation from one another.These data may be used for improved calculations of stability of RNA secondary structure, to estimate the frequency of structural fluctuations in a double helix and to assess the stability of modified polynucleotide helices. An unmodified double helix of one million randomly arranged base pairs should contain on the time average approximately 10 G.C and 500 A.U pairs in non-hydrogen bonded, unstacked conformations at 25 °C. Our estimate of the effect of mismatching on T_m values of high polymers is less precise because of the long temperature extrapolation required. However, we estimate that DNA or RNA treated with mutagens which interrupt up to 20% of the nucleotide pairs will show a drop of about 1.2 deg. C in melting temperature with each unit per cent of modification.     

A pH-dependent variation in alpha-helix structure of the S-peptide of ribonuclease A studied by Monte Carlo simulated annealing.

Low-energy conformations of the S-peptide fragment (20 amino acid residues long) of ribonuclease A were studied by Monte Carlo simulated annealing. The obtained lowest-energy structures have alpha-helices with different size and location, depending distinctively on the ionizing states of acidic amino acid residues. The simulation started from completely random initial conformation and was performed without any bias toward a particular structure. The most conspicuous alpha-helices arose from the simulation when both Glu 9 and Asp 14 were assumed to be electrically neutral, whereas the resulting conformations became much less helical when Asp 14 rather than Glu 9 was allowed to have a negative charge. Together with experimental evidence that the alpha-helix in the S-peptide is most stable at pH 3.8, we consider the helix formation need the carboxyl group of Asp 14 to be electrically neutral in this weakly acidic condition. In contrast, a negative charge at Asp 14 appears to function in support of a view that this residue is crucial to helix termination owing to its possibility to form a salt bridge with His 12. These results indicate that the conformation of the S-peptide depends considerably on the ionizing state of Asp 14.     

Synthesis and study of peptides with semirigid i and i + 7 side-chain bridges designed for alpha-helix stabilization

A search for conformational constraints on the peptide alpha-helical conformation indicated that para-substituted amino acid derivatives of a benzene ring might be suitable for linking pairs of side chains that are separated by two turns of the helix. A 14-residue synthetic, amphiphilic alpha-helical peptide model system has been used to study the helix stabilizing effects of a series of four such bridges having constitutionally isomeric structures. These bridges were used to link positions 3 and 10 of the model peptides. The peptides were synthesized in good yield by standard solid-phase methods, including cyclization on the solid support. They were then studied for their solution conformations and melting behavior by circular dichroism (CD) spectropolarimetry, and for their elution behavior on reversed-phase HPLC columns. In aqueous solution and in 50% (v/v) trifluoroethanol, the most effective bridge for helix stabilization consisted of a 4-(aminomethyl)phenylacetic acid residue (AMPA) linked by amide bonds to the side chain functional groups of a (S)-2,3-diaminopropionic acid residue (Dap) in position 3 of the model peptide and an aspartic acid residue in position 10. This Dap3(AMPA), Asp10 bridge was about as effective as two Lys(i), Asp(i+4) lactam bridges incorporated linking residues 3 and 7, and 10 and 14, in the same model peptide sequence. This suggests that it is worth about 1 kcal/mol of helix stabilization energy.     

Prediction of strand pairing in antiparallel and parallel beta-sheets using information theory

An information theory approach was developed to predict the alignment of interacting antiparallel and parallel beta-strands. Information scores were derived for the preference of a residue on a beta-strand to be opposite a sequence of residues on an adjacent beta-strand. These scores were used to predict the interstrand register of interacting beta-strands from 10 alternative offset positions either side of the experimentally observed beta-sheet register. The amino acid sequence of an internal beta-strand can be correctly aligned with two beta-strands in a fixed position either side of the strand in 45% of antiparallel and 48% of parallel arrangements. For comparison, when another beta-strand from a nonhomologous protein substitutes the internal beta-strand, the same register is predicted for only 24 and 36% of antiparallel and parallel arrangements. As expected, alignment of a single fixed strand with just a second beta-strand sequence was more difficult, and gave a correct register in 31 and 37% of antiparallel and parallel beta-pairs, respectively. These scores are 10% higher than for two randomly selected beta-strand sequences. In general, prediction accuracy was not improved by information tables that distinguished hydrogen-bonding patterns or beta-strand order. These results will contribute to predicting the arrangement of beta-strands in beta-pleated sheets and protein topology.     

Active TEM-1 beta-lactamase mutants with random peptides inserted in three contiguous surface loops

Engineering of alternative binding sites on the surface of an enzyme while preserving the enzymatic activity would offer new opportunities for controlling the activity by binding of non-natural ligands. Loops and turns are the natural substructures in which binding sites might be engineered with this purpose. We have genetically inserted random peptide sequences into three relatively rigid and contiguous loops of the TEM-1 beta-lactamase and assessed the tolerance to insertion by the percentage of active mutants. Our results indicate that tolerance to insertion could not be correlated to tolerance to mutagenesis. A turn between two beta-strands bordering the active site was observed to be tolerant to random mutagenesis but not to insertions. Two rigid loops comprising rather well-conserved amino acid residues tolerated insertions, although with some constraints. Insertions between the N-terminal helix and the first beta-strand generated active libraries if cysteine residues were included at both ends of the insert, suggesting the requirement for a stabilizing disulfide bridge. Random sequences were relatively well accommodated within the loop connecting the final beta-strand to the C-terminal helix, particularly if the wild-type residue was retained at one of the loops' end. This suggests two strategies for increasing the percentage of active mutants in insertion libraries. The amino acid distribution in the engineered loops was analyzed and found to be less biased against hydrophobic residues than in natural medium-sized loops. The combination of these activity-selected libraries generated a huge library containing active hybrid enzymes with all three loops modified.     

Determinants of strand register in antiparallel beta-sheets of proteins.

Antiparallel beta-sheets present two distinct environments to inter-strand residue pairs: beta(A,HB) sites have two backbone hydrogen bonds; whereas at beta(A,NHB) positions backbone hydrogen bonding is precluded. We used statistical methods to compare the frequencies of amino acid pairs at each site. Only approximately 10% of the 210 possible pairs showed occupancies that differed significantly between the two sites. Trends were clear in the preferred pairs, and these could be explained using stereochemical arguments. Cys-Cys, Aromatic-Pro, Thr-Thr, and Val-Val pairs all preferred the beta(A,NHB) site. In each case, the residues usually adopted sterically favored chi1 conformations, which facilitated intra-pair interactions: Cys-Cys pairs formed disulfide bonds; Thr-Thr pairs made hydrogen bonds; Aromatic-Pro and Val-Val pairs formed close van der Waals contacts. In contrast, to make intimate interactions at a beta(A,HB) site, one or both residues had to adopt less favored chi1 geometries. Nonetheless, pairs containing glycine and/or aromatic residues were favored at this site. Where glycine and aromatic side chains combined, the aromatic residue usually adopted the gauche conformation, which promoted novel aromatic ring-peptide interactions. This work provides rules that link protein sequence and tertiary structure, which will be useful in protein modeling, redesign, and de novo design. Our findings are discussed in light of previous analyses and experimental studies.     

Side-chain entropy effects on protein secondary structure formation

Loss of conformational entropy is one of the primary factors opposing protein folding. Both the backbone and side-chain of each residue in a protein will have their freedom of motion restricted in the final folded structure. The type of secondary structure of which a residue is part will have a significant impact on how much side-chain entropy is lost. Side-chain conformational entropies have previously been determined for folded proteins, simple models of unfolded proteins, alpha-helices, and a dipeptide model for beta-strands, but not for polyproline II (PII) helices. In this work, we present side-chain conformational estimates for the three regular secondary structure types: alpha-helices, beta-strands, and PII helices. Entropies are estimated from Monte Carlo computer simulations. Beta-strands are modeled as two structures, parallel and antiparallel beta-strands. Our data indicate that restraining a residue to the PII helix or antiparallel beta-strand conformations results in side-chain entropies equal to or higher than those obtained by restraining residues to the parallel beta-strand conformation. Side-chains in the alpha-helix conformation have the lowest side-chain entropies. The observation that extended structures retain the most side-chain entropy suggests that such structures would be entropically favored in unfolded proteins under folding conditions. Our data indicate that the PII helix conformation would be somewhat favored over beta-strand conformations, with antiparallel beta-strand favored over parallel. Notably, our data imply that, under some circumstances, residues may gain side-chain entropy upon folding. Implications of our findings for protein folding and unfolded states are discussed.     

The effect of intrinsic curvature on conformational properties of circular DNA.

Both thermal fluctuations and the intrinsic curvature of DNA contribute to conformations of the DNA axis. We looked for a way to estimate the relative contributions of these two components of the double-helix curvature for DNA with a typical sequence. We developed a model and Monte Carlo procedure to simulate the Boltzmann distribution of DNA conformations with a specific intrinsic curvature. Two steps were used to construct the equilibrium conformation of the model chain. We first specified the equilibrium DNA conformation at the base pair level of resolution, using a set of the equilibrium dinucleotide angles and DNA sequence. This conformation was then approximated by the conformation of the model chain consisting of a reduced number of longer, straight cylindrical segments. Each segment of the chain corresponded to a certain number of DNA base pairs. We simulated conformational properties of nicked circular DNA for different sets of equilibrium dinucleotide angles, different random DNA sequences, and lengths. Only random sequences of DNA generated with equal probability of appearance for all types of bases at any site of the sequence were used. The results showed that for a broad range of intrinsic curvature parameters, the radius of gyration of DNA circles should be nearly independent of DNA sequence for all DNA lengths studied. We found, however, a DNA properly that should strongly depend on DNA sequence if the double helix has essential intrinsic curvature. This property is the equilibrium distribution of the linking number for DNA circles that are 300-1000 bp in length. We found that a large fraction of the distributions corresponding to random DNA sequences should have two separate maxima. The physical nature of this unexpected effect is discussed. This finding opens new opportunities for joined experimental and theoretical studies of DNA intrinsic curvature.     

Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. II. Plastocyanin.

In an attempt to understand the earliest events in the protein folding pathway, the complete sequence of French bean plastocyanin has been synthesized as a series of short peptide fragments, and the conformational preferences of each peptide examined in aqueous solution using proton n.m.r. methods. Plastocyanin consists largely of beta-sheet, with reverse turns and loops between the strands of the sheet, and one short helix. The n.m.r. experiments indicate that most of the peptides derived from the plastocyanin sequence have remarkably little propensity to adopt folded conformations in aqueous solution, in marked contrast to the peptides derived from the helical protein, myohemerythrin (accompanying paper). For most plastocyanin peptides, the backbone dihedral angles are predominantly in the beta-region of conformational space. Some of the peptides show weak NOE connectivities between adjacent amide protons, indicative of small local populations of backbone conformations in the a region of (phi,psi) space. A conformational preference for a reverse turn is seen in the sequence Ala65-Pro-Gly-Glu68, where a turn structure is found in the folded protein. Significantly, the peptide sequences that populate the alpha-region of (phi,psi) space are mostly derived from turn and loop regions in the protein. The addition of trifluoroethanol does not drive the peptides into helical conformations. In one region of the sequence, the n.m.r. spectra provide evidence of the formation of a hydrophobic cluster involving aromatic and aliphatic side-chains. These results have significance for understanding the initiation of protein folding. From these studies of the fragments of plastocyanin (this paper) and myohemerythrin (accompanying paper), it appears that there is a pre-partitioning of the conformational space sampled by the polypeptide backbone that is related to the secondary structure in the final folded state.     

On the use of phasing experiments to measure helical repeat and bulge loop-associated twist in RNA.

In a phasing experiment, two bends are introduced into a long duplex RNA or DNA and the number of base pairs between them varied. When electrophoresed in a gel, the set of molecules may show a periodic variation in mobility that contains information about the twist associated with the bends and the intervening helix. We show how a set of three phasing experiments can be used to extract this information, and apply it to an RNA helix bend at the bulge sequence A2. The bulge introduces a negative (left-handed) twist of approximately 30 degrees; at low temperatures, it is mostly confined to the 5' side of the bulge. The apparent helical repeat of random sequence RNA measured in these experiments was 10.2 +/- 0.1 base pairs, an unexpectedly low value. It is likely that moderate curvative of the RNA helix axis (30-40 degrees over 80 bp) has affected the measurement.     

100ps molecular dynamic simulation of d(TATCACC)2.

A heptanucleotide sequence d(TATCACC)2 from OR3 region of bacteriophage lambda is considered sufficient for the recognition of Cro protein. We present here results on molecular dynamic simulations on this sequence for 100 ps in 0.02 ps interval. The simulations are done using computer program GROMOS. The conformational results are averaged over each ps. The IUPAC torsional parameters for 100 conformations are illustrated using a wheal and a dial systems. Several other stereochemical parameters such as H-bonding lengths and angles, sugar puckers, helix twist and roll angles as also distances between opposite strand phosphorus are depicted graphically. We find that there is rupture of terminal H-bonds. The bases are tilted and shifted away from the helix axis giving rise to bifurcated H-bonds. H-bonds are seen even in between different base pairs. The role of these dynamic structural changes in the recognition of OR3 operator by Cro protein is discussed in the paper.     

Molecular dynamics simulations of polypeptide conformations in water: A comparison of alpha, beta, and poly(pro)II conformations.

A significant fraction of the so-called "random coil" residues in globular proteins exists in the left-handed poly(Pro)II conformation. In order to compare the behavior of this secondary structure with that of the other regular secondary structures, molecular dynamics simulations, with the GROMOS suite of programs, of an alanine octapeptide in water, in alpha-helix, beta-strand, and left-handed poly(Pro)II conformations, have been performed. Our results indicate a limited flexibility for the alpha-helix conformation and a relatively larger flexibility for the beta-strand and poly(Pro)II conformations. The behavior of oligopeptides with a starting configuration of beta-strand and poly(Pro)II conformations, both lacking interchain hydrogen bonds, were similar. The (phi, psi) angles reflect a continuum of structures including both beta and P(II) conformations, but with a preference for local P(II) regions. Differences in the network of water molecules involved in hydrogen bonding with the backbone of the polypeptide were observed in local regions of beta and P(II) conformations. Such water bridges help stabilize the P(II) conformation relative to the beta conformation. Proteins 1999;36:400-406.