Prediction of a 12-residue loop in bovine pancreatic trypsin inhibitor: effects of buried water.

The x-ray conformations of 5-, 7-, 9-, and 12-residue loops in bovine pancreatic trypsin inhibitor (BPTI) were predicted by the use of multiple independent Monte Carlo simulating annealing (MCSA) runs starting from random conformations. Four buried water molecules interacted with a 12-residue loop that started at residue 8 and ended at residue 19, and that included the binding region. The final conformation at the end of an MCSA run was characterized. Solvation free energy based on the solvent accessible surface area was included in the energy function at low simulated annealing temperatures. Conformational states were interactively separated by a recently developed algorithm. Computed loops were characterized in terms of total energy, and backbone and side chain root mean square deviations (RMSDs) between computed native loop conformations and the x-ray conformation. The 12-residue loop was computed with and without buried water [called WL12(8-19) and L12(8-19), respectively]. The backbone was reliably and reproducibly computed to within 1.1 A in L12(8-19) and 0.9 A in WL12(8-19). L12(8-19) required significantly more MCSA runs to achieve the same level of reproducibility as WL12(8-19). Based on the size of the cluster of low energy native loop conformations, and the computational effort, WL12(8-19) had greater entropy. In calculations of 7-, 9-, and 12-residue loops without buried water, the effects of buried water became obvious in the 12-residue loop calculation, which interacted with all four buried water molecules. Nearly all conformations of the native loop conformer had a hydrogen bond between the Lys 15 side chain and the backbone of Gly 12, Pro 13, and Cys 14, which may have implications in the rate of exchange of buried water with bulk solvent and in protein folding. The present version of MCSA program was more efficient than earlier versions.

Computational analysis of two similar neuropeptides yields distinct conformational ensembles.

Conformational states and thermodynamic properties for two similar neuropeptides, GDPFLRF-NH(2) and GYPFLRF-NH(2), have been computed by Monte Carlo simulated annealing (MCSA) conformational searches and Metropolis Monte Carlo (MMC) calculations. These peptides were recently shown to have dramatically different conformations in solution by NMR [Edison et al., J Neuroscience 1999;19:6318-6326]. Final conformations of multiple independent MCSA runs were the starting points for MMC calculations, and conformations saved at intervals during MMC runs were characterized in terms of total energy, configuration entropy, side-chain fraction population, and ensemble average inter-nuclear distances. Without the use of any NMR data-generated pseudo-potentials, the present calculations were in excellent qualitative agreement with all previous NMR experimental data and provided a foundation by which to more quantitatively interpret the experimental NMR results. Proteins 2000;40:367-377.

Conformational analysis of a farnesyltransferase peptide inhibitor, CVIM.

The conformational states of the peptide Cys-Val-Ile-Met (or CVIM) were computed and characterized. CVIM inhibits farnesylation of the Ras oncogene product, p21ras, at the cysteine residue of the C-terminal segment. CVIM is active in an extended conformation. A similar peptide (KTKCVFM) appears to bind the enzyme in the Type I bend conformation. In the present study, the conformations of CVIM were computed in an aqueous environment with the peptide in the zwitterionic state. Solvation free energy based on solvent accessible surface area and a distance dependent dielectric were used in the calculations. Final conformations of multiple independent Monte Carlo simulated annealing (MCSA) conformational searches were used as starting points for Metropolis Monte Carlo (MMC) runs. Conformations saved at intervals during MMC runs were analyzed. Conformers were separated by interactive clustering in dihedral angle coordinates. The four lowest energy conformers corresponding to a Type I bend, extended, AB-bend, and BA-bend were within 0.3 kcal/mol of each other, and dominant in terms of population. The Type I bend and extended conformers were supported by the binding studies. The extended conformer was the most populated. In the AB-bend conformer, 'A' indicates the alpha-helix conformation of Val, and 'B' indicates the beta-strand conformation of Ile. The AB- and BA-bend conformations differed from the extended conformation in the value of Val psi and Ile psi, respectively, and from the Type I bend conformation in the value of Ile psi and Val psi, respectively. The four lowest energy conformers were characterized in terms of energy, density of low energy conformations (or entropy), structure, side chain rotamer fraction population, and interatomic distances.

Conformational analysis of [Met5]-enkephalin: solvation and ionization considerations.

[Met5]-Enkephalin has the sequence Tyr-Gly-Gly-Phe-Met. Only the extended conformation of the peptide has been observed by X-ray crystallography. Nuclear magnetic resonance spectroscopy supports the presence of a turn at Gly 3 and Phe 4 in dimethyl sulfoxide. In this study, the peptide conformational states and thermodynamic properties are understood in terms of ionization state and solvent environment. In the calculation, final conformations obtained from multiple independent Monte Carlo simulated annealing conformational searches are starting points for molecular dynamics simulations. In an aqueous environment given by the use of solvation free energy and the zwitterionic state, dominant structural motifs computed are G-P Type II' bend, G-G Type II' bend, and G-G Type I' bend motifs, in order of increasing free energy. In the calculation of the peptide with neutral N- and C-termini and solvation free energy, the extended conformer dominates (by at least a factor of 2.5), and the conformation of another low free energy conformer superimposes well on the pharmacophoric groups of morphine. Neutralization of charge and solvation induce and stabilize the extended conformation, respectively. A mechanism of inter-conversion between the extended conformer and three bent conformers is supported by phi/psi-scatter plots, and by the conformer relative free energies. An estimate of the entropy change of receptor unbinding is 8.3 cal K-1 mol-1, which gives rise to a -2.5 kcal/mol entropy contribution to the free energy of unbinding at 25 degrees C. The conformational analysis methodology described here should be useful in studies on short peptides and flexible protein surface loops that have important biological implications.

The loop problem in proteins: a Monte Carlo simulated annealing approach.

A Monte Carlo simulated annealing (MCSA) algorithm was used to generate the conformations of local regions in bovine pancreatic trypsin inhibitor (BPTI) starting from random initial conformations. In the approach explored, only the conformation of the segment is computed; the rest of the protein is fixed in the known native conformation. Rather than follow a single simulation exhaustively, computer time is better used by performing multiple independent MCSA simulations in which different starting temperatures are employed and the number of conformations sampled is varied. The best computed conformation is chosen on the basis of lowest total energy and refined further. The total energy used in the annealing is the sum of the intrasegment energy, the interaction energy of the segment with the local surrounding region, and a distance constraint to generate a smooth connection of the initially randomized segment with the rest of the protein. The rms deviations between the main-chain conformations of the computed segments in BPTI and those of the native x-ray structure are 0.94 A for a 5-residue alpha-helical segment, 1.11 A for a 5-residue beta-strand segment, and 1.03, 1.61, and 1.87 A for 5-, 7-, and 9-residue loop segments. Side-chain deviations are comparable to the main-chain deviations for those side chains that interact strongly with the fixed part of the protein. A detailed view of the deviations at an atom-resolved level is obtained by comparing the predicted segments with their known conformations in the crystal structure of BPTI. These results emphasize the value of predetermined fixed structure against which the computed segment can nest.

An energy-based approach to packing the 7-helix bundle of bacteriorhodopsin.

Based on the heavy-atom coordinates determined by the electron microscopy for the seven main helical regions of bacteriorhodopsin with the all-trans retinal isomer, energy optimizations were carried out for helix bundles containing the all-trans retinal and 13-cis retinal chromophores, respectively. A combination of simulated annealing and energy minimization was utilized during the process of energy optimization. It was found that the 7-helix bundle containing the all-trans isomer is about 10 kcal/mol lower in conformational energy than that containing the 13-cis isomer. An energetic analysis indicates that such a difference in energy is consistent with the observation that absorption of a 570-nm proton is required for the conversion of a bacteriorhodopsin from its all-trans to 13-cis form. It was also found that the above conversion process is accompanied by a significant conformational perturbation around the chromophore, as reflected by the fact that the beta-ionone ring of retinal moves about 5.6 A along the direction perpendicular to the membrane plane. This is consistent with the observation by Fodor et al. (Fodor, S.P.A., Ames, J.B., Gebhard, R., van der Berg, E.M.M., Stoeckenius, W., Lugtenburg, J., & Mathies, R.A., 1988, Biochemistry 27, 7097-7101). Furthermore, it is interesting to observe that although the retinal chromophore undergoes a significant change in its spatial position, the orientation of its transition dipole changes only slightly, in accord with experimental observations. In other words, even though orientation of the retinal transition dipole is very restricted, there is sufficient room, and degrees of freedom, for the retinal chromophore to readjust its position considerably. This finding provides new insight into the subtle change of the retinal microenvironment, which may be important for revealing the proton-pumping mechanism of bacteriorhodopsin. The importance of electrostatic and nonbonded interactions in stabilizing the 7-helix bundle structure has also been analyzed. Electrostatic interactions favor an antiparallel arrangement among adjacent helices. Nonbonded interactions, however, drive most of the closely packed helices into an arrangement in which the packing angles lie around -160 degrees, a value very near the -154 degrees value computed earlier as the most favorable packing arrangement of two poly(Ala) alpha-helices (Chou, K.-C., Némethy, G., & Scheraga, H.A., 1983, J. Phys. Chem. 87, 2869-2881). The structural features of the 7-helix bundle and their relationship to those found in typical 4-helix bundle proteins are also discussed.(ABSTRACT TRUNCATED AT 400 WORDS)

Geometric and energy parameters in lysine-retinal chromophores.

The parameters used in the computer program ECEPP (Empirical Conformational Energy Program for Peptides) have been expanded to cover some key elements in retinal-containing proteins. These elements are 'all-trans retinal lysine with unprotonated imine', 'all-trans retinal lysine with protonated imine', '13-cis retinal lysine with unprotonated imine' and '13-cis retinal lysine with protonated imine' respectively. The geometric parameters of these four new 'amino acid residues' were derived by optimizing their molecular structures with the AM1 Hamiltonian included in MOPAC (Molecular Orbital PACkage), and their partial atomic charges were determined with a CNDO/2 (Complete Neglect of Differential Overlap) calculation. The parameters for nonbonded interactions and torsional potentials were obtained from the existing ECEPP parameters through a logical extension. The augmented ECEPP system thus obtained can be employed to investigate the conformation of bacteriorhodopsin and its proton-pumping mechanism from an energetic point of view. The computer modeling study on bacteriorhodopsin and other seven-helix membrane proteins, e.g. serotonin receptor and dopamine receptor, is under way in the Upjohn Laboratories.

Simulated annealing approach to the study of protein structures.

One of the most difficult problems in predicting the three dimensional structure of proteins is how to deal with the local minimum problem. In many cases of practical interest this problem has been reduced to how to select an appropriate set of starting conformations for carrying out energy minimizations. How these starting conformations are selected, however, is often based on the physical intuition of the person doing the calculations, and hence it is hard to avoid bearing some sort of arbitrariness. To improve such a situation, we introduced the simulated annealing Monte Carlo algorithm to locate the optimal starting conformations for energy minimizations. The method developed here is valid for both single and multiple polypeptide chain systems. The annealing process can be conducted with respect to either the internal dihedral angles of a polypeptide chain or the external rotations and translations of various constituent polypeptide chains, and hence is particularly useful for studying the packing arrangements of secondary structures in proteins, such as helix/helix packing, helix/sheet packing and sheet/sheet packing. It was shown via a number of comparative calculations that the final structures obtained through the annealing process not only had lower energies than the corresponding energy-minimized structures reported previously, but also assumed the forms closer to the observations in proteins. All these results indicate that a better result can be obtained in search of low-energy structures of proteins by incorporating the simulated annealing approach.(ABSTRACT TRUNCATED AT 250 WORDS)

A heuristic approach to predicting the tertiary structure of bovine somatotropin.

A combination of a heuristic approach and energy minimization was used to predict the three-dimensional structure of bovine somatotropin (bSt), also known as bovine growth hormone, a protein of 191 amino acids. The starting points for energy minimizations were generated from the following two types of inputs: (a) the amino acid sequence and (b) the heuristic inputs, which were derived according to physical, chemical, and biological principles by piecing together all useful information available. The predicted 3-D structure of the bSt molecule has all the features observed in four-helix bundle proteins. The four alpha-helices in bSt are intimately packed to form an assembly with an approximately square cross section. All the adjacent alpha-helices are antiparallel, with a somewhat tilted angle between each of the adjacent pairs so that the assembly of the four helices looks like a left-handed twisted bundle. There are two disulfide bonds in the bSt structure: one "hooking" the middle of a long loop with helix 4 so as to pull the long loop onto the surface of the helix bundle and the other "hooking" the C-terminal segment with the same helix so as to force the C-terminal segment to bend toward the helix bundle. As a consequence, a considerable part of the surface of the four-helix bundle is closely packed or intimately embraced by the loop segments. The predicted bSt structure has a hydrophobic core and a hydrophilic exterior surface. The energetic analysis of the predicted bSt structure indicates that the interaction between helices and loops plays a dominant role in stabilizing the four-helix bundle structure from the viewpoint of both electrostatic and nonbonded interactions. A technique called FOLD was meanwhile developed, by which one can fold a polypeptide chain into any shape as desired. This tool proved to be very useful during the heuristic model-building process.

Energetic approach to the folding of alpha/beta barrels.

The folding of a polypeptide into a parallel (alpha/beta)8 barrel (which is also called a circularly permuted beta 8 alpha 8 barrel) has been investigated in terms of energy minimization. According to the arrangement of hydrogen bonds between two neighboring beta-strands of the central barrel therein, such an alpha/beta barrel structure can be folded into six different types: (1) left-tilted, left-handed crossover; (2) left-tilted, right-handed crossover; (3) nontilted, left-handed crossover; (4) nontilted, right-handed crossover; (5) right-tilted, left-handed crossover; and (6) right-tilted, right-handed crossover. Here "tilt" refers to the orientational relation of the beta-strands to the axis of the central beta-barrel, and "crossover" to the beta alpha beta folding connection feature of the parallel beta-barrel. It has been found that the right-tilted, right-handed crossover alpha/beta barrel possesses much lower energy than the other five types of alpha/beta barrels, elucidating why the observed alpha/beta barrels in proteins always assume the form of right tilt and right-handed crossover connection. As observed, the beta-strands in the energy-minimized right-tilted, right-handed crossover (alpha/beta)8-barrel are of strong right-handed twist. The value of root-mean-square fits also indicates that the central barrel contained in the lowest energy (alpha/beta)8 structure thus found coincides very well with the observed 8-stranded parallel beta-barrel in triose phosphate isomerase (TIM). Furthermore, an energetic analysis has been made demonstrating why the right-tilt, right-handed crossover barrel is the most stable structure. Our calculations and analysis support the principle that it is possible to account for the main features of frequently occurring folding patterns in proteins by means of conformational energy calculations even for very complicated structures such as (alpha/beta)8 barrels.

Energetic approach to the folding of four alpha-helices connected sequentially.

The packing of four alpha-helices, which each consist of 12 Ala residues and are sequentially connected to each other by a segment of 10 Ala residues, has been investigated by means of energy minimizations. For the lowest energy structure thus obtained, the following features have been found: (i) the four alpha-helices are intimately packed to form an assembly with an approximately square section; (ii) the distances of closest approach between two adjacent interhelix axes are 7.7 +/- 0.2 A and those between two diagonal interhelix axes are 11.2 +/- 0.2 A; (iii) the adjacent interhelix angles are -163 +/- 2 degrees; and (iv) the diagonal interhelix angles are 24 +/- 4 degrees. These results indicate that the polypeptide chain, driven by energetics (nonbonded and electrostatic interactions), is folded into a typical left-handed twisted four-helix bundle with an approximately 4-fold symmetric array, as observed in most four alpha-helix proteins. Furthermore, it has been found that the interaction between the loops formed by the connecting segments and the other part of molecule plays a significant role in stabilizing such a bundle structure. The technology developed here and the relevant knowledge obtained through this study are very useful for the study of modeling four-helix bundle proteins.

Conformational and geometrical properties of idealized beta-barrels in proteins.

An equation for calculating the distances between the atoms involved in forming an idealized hydrogen bond in a parallel or antiparallel beta-barrel has been derived by adjusting the corresponding data given by Pauling and Corey for a beta-sheet. Based on these distances, a geometrical optimization method was developed, by which one can generate various idealized beta-barrels: parallel or antiparallel, tilted or non-tilted, right-tilted or left-tilted. For each type of idealized beta-barrel thus obtained, the corresponding conformation and characteristic geometric parameters as well as their relationship are analyzed and discussed. Since the strand in a tilted beta-barrel traces a curve rather than a straight line on a cylinder-like surface, a regular chain in which the dihedral angles of each residue are the same cannot form a tilted beta-barrel but only a non-tilted beta-barrel. As observed, the strands of a right-tilted beta-barrel possess a very strong right-handed twist. The radii of the idealized tilted parallel and antiparallel beta-barrels are greater than those of the corresponding non-tilted ones by approximately 1 A and approximately 1.5 A, respectively. Consequently, there is relatively more room for a tilted beta-barrel to accommodate the internal side-chains, suggesting that a conformational change from a non-tilted beta-barrel to a tilted one would ease the repulsion among the crowded internal side-chains so as to make the structure more stable. The values of root-mean-square fits indicate that the idealized right-tilted beta-barrels coincide quite well with the observed beta-barrels in both parallel and antiparallel cases.

Energetics of the structure and chain tilting of antiparallel beta-barrels in proteins.

The preferred structural pattern of antiparallel beta-barrels in proteins, described as the right-handed tilting of the peptide strands with respect to the axis of the barrel, is accounted for in terms of intra- and interchain interaction energies. It is related to the preference of beta-sheets for right-handed twisting. Conformational energy computations have been carried out on three eight-stranded antiparallel beta-barrels composed of six-residue strands, in which L-Val and Gly alternate, and having a right-handed, a left-handed, or no tilt. After energy minimization, the relative energies of these structures were 0.0, 8.6, and 46.1 kcal/mol, respectively; i.e., the right-tilted beta-barrel is favored energetically, in agreement with anti-parallel beta-barrels observed in proteins. Tilting of the barrel is favored, relative to the nontilted structure, by both intra- and interstrand interactions, because tilting allows better packing of the bulky side chains. On the other hand, the energy difference between the left- and right-tilted barrels arises essentially from intrachain interactions. This is a consequence of the preference of beta-sheets for a right-handed twist. Space limitations inside the barrel are satisfied if there is an alternation of bulky residues and residues with small or no side chain (preferably Gly) in neighboring positions on adjacent strands. Such a pattern is seen frequently in antiparallel beta-barrels of globular proteins. The computations indicate that a structure with Val...Gly pairs can be accommodated in a beta-barrel with no distortion.