Search

Recent improvements in the protein-structure prediction method developed in our laboratory, based on the thermodynamic hypothesis, are described. The conformational space is searched extensively at the united-residue level by using our physics-based UNRES energy function and the conformational space annealing method of global optimization. The lowest-energy coarse-grained structures are then converted to an all-atom representation and energy-minimized with the ECEPP/3 force field. The procedure was assessed in two recent blind tests of protein-structure prediction. During the first blind test, we predicted large fragments of alpha and alpha+beta proteins [60-70 residues with C(alpha) rms deviation (rmsd) <6 A]. However, for alpha+beta proteins, significant topological errors occurred despite low rmsd values. In the second exercise, we predicted whole structures of five proteins (two alpha and three alpha+beta, with sizes of 53-235 residues) with remarkably good accuracy. In particular, for the genomic target TM0487 (a 102-residue alpha+beta protein from Thermotoga maritima), we predicted the complete, topologically correct structure with 7.3-A C(alpha) rmsd. So far this protein is the largest alpha+beta protein predicted based solely on the amino acid sequence and a physics-based potential-energy function and search procedure. For target T0198, a phosphate transport system regulator PhoU from T. maritima (a 235-residue mainly alpha-helical protein), we predicted the topology of the whole six-helix bundle correctly within 8 A rmsd, except the 32 C-terminal residues, most of which form a beta-hairpin. These and other examples described in this work demonstrate significant progress in physics-based protein-structure prediction.

A new global optimization method, Conformation-family Monte Carlo, has been developed recently for searching the conformational space of macromolecules. In the present paper, we adapted this method for prediction of crystal structures of organic molecules without assuming any symmetry constraints except the number of molecules in the unit cell. This method maintains a database of low energy structures that are clustered into families. The structures in this database are improved iteratively by a Metropolis-type Monte Carlo procedure together with energy minimization, in which the search is biased toward the regions of the lowest energy families. The Conformation-family Monte Carlo method is applied to a set of nine rigid and flexible organic molecules by using two popular force fields, AMBER and W99. The method performed well for the rigid molecules and reasonably well for the molecules with torsional degrees of freedom.

Recent improvements of a hierarchical ab initio or de novo approach for predicting both alpha and beta structures of proteins are described. The united-residue energy function used in this procedure includes multibody interactions from a cumulant expansion of the free energy of polypeptide chains, with their relative weights determined by Z-score optimization. The critical initial stage of the hierarchical procedure involves a search of conformational space by the conformational space annealing (CSA) method, followed by optimization of an all-atom model. The procedure was assessed in a recent blind test of protein structure prediction (CASP4). The resulting lowest-energy structures of the target proteins (ranging in size from 70 to 244 residues) agreed with the experimental structures in many respects. The entire experimental structure of a cyclic alpha-helical protein of 70 residues was predicted to within 4.3 A alpha-carbon (C(alpha)) rms deviation (rmsd) whereas, for other alpha-helical proteins, fragments of roughly 60 residues were predicted to within 6.0 A C(alpha) rmsd. Whereas beta structures can now be predicted with the new procedure, the success rate for alpha/beta- and beta-proteins is lower than that for alpha-proteins at present. For the beta portions of alpha/beta structures, the C(alpha) rmsd's are less than 6.0 A for contiguous fragments of 30-40 residues; for one target, three fragments (of length 10, 23, and 28 residues, respectively) formed a compact part of the tertiary structure with a C(alpha) rmsd less than 6.0 A. Overall, these results constitute an important step toward the ab initio prediction of protein structure solely from the amino acid sequence.

The diffusion equation method of global minimization is applied to compute the crystal structure of S6, with no a priori knowledge about the system. The experimental lattice parameters and positions and orientations of the molecules in the unit cell are predicted correctly.

The low-energy conformations for a series of peptides based on the sequence of the ras P21 protein from position 55 to position 67 have been computed using conformational energy analysis. These sequences differed at position 61 and contained Gln, Pro, Leu, Lys, and Arg at this position. P21 proteins with Gln, Glu, or Pro at this position do not cause cell transformation at normal levels of expression; proteins with substitutions of at least 14 other amino acids at this position (Leu, Lys, and Arg having been found in tumors in place of the normally occurring Gln-61) do cause malignant transformation of cells in culture. We find that the segments of residues 55-67 from the nontransforming proteins (Gln- or Pro-61) adopt a structure that is energetically unfavorable for the same segment with Leu, Lys, or Arg at position 61. The critical feature of this structure is an alpha-helix from residues 62 to 68. Residue 61 (Gln or Pro) adopts an extended conformation. On the other hand, the segment from transforming proteins can adopt two structures, one all alpha-helical from residue 61 to residue 68 and the other a less-regular, higher-energy structure. The segments from the normal protein can adopt the all alpha-helical structure, a finding that can explain the fact that elevated intracellular levels of the normal protein also cause cell transformation. The results of the calculations suggest that specific changes in the structure of this region can account for the oncogenic effect of the proteins in which substitutions occur.

Serum from hypothyroid hypophysectomized rats did not stimulate sulfation or incorporation of amino acids into chick embryo sterna. When such rats were treated for a short time with growth hormone (somatotropin), their serum stimulated incorporation both of sulfate and of amino acids. The different actions of the two types of sera were not due to changes in thyroid state. The results support the existence in serum of a sulfation factor for chick embryo cartilage that is dependent upon growth hormone. Highly purified preparations of nonsuppressible insulin-like activity from human serum stimulated incorporation of amino acids, and of uridine into RNA, in chick embryo sterna in vitro; chondrocytes prepared from this tissue had specific high-affinity binding sites for this insulin-like activity. However, sulfate incorporation was stimulated very little, unless serum from hypothyroid hypophysectomized rats was also present. When L-3,5,3'-triiodothyronine was added as well, the stimulation was enhanced further. From these and other experiments, we conclude that (i) nonsuppressible insulin-like activity or a closely related peptide is the growth-hormone-dependent growth and sulfation factor for chick embryo cartilage: (ii) a second, unidentified factor must be present for the insulin-like activity to stimulate sulfation; and (iii) stimulation of sulfation by thyroid hormones in vitro is additive to that of nonsuppressible insulin-like activity.

Somatomedin potencies of sera were assayed by following sulfation of mucopolysaccharides in chick embryo sterna in vitro. Apparent potencies of sera from hypophysectomized rats, maintained on a low-iodine diet, were restored to levels above normal by addition to the incubation medium of L-triiodothyronine at 10-7 mol/liter of serum. The potencies of normal rat, human, and fetal calf sera were raised 1.3- to 3-fold by addition of triiodothyronine at 10-9-10-7 mol/liter of serum. L-Thyroxine was about 10 times less effective than triiodothyronine. Triiodothyronine alone did not stimulate sulfation to nearly the same extent as triiodothyronine plus serum, even at higher concentrations. Serum could not be substituted in this system by any of six purified hormones, nor by trace metals or amino acids not included in the incubation mixture. It is concluded that triiodothyronine, in combination with a factor in serum, causes a rapid stimulation of sulfation in chick embryo cartilage, and that thyroid hormones may be involved in the action of normal serum on this tissue.

The circumsporozoite protein of the human malaria parasite Plasmodium falciparum contains multiple tandem repeats of the amino acid sequence Asn-Ala-Asn-Pro. The repeated sequence encompasses the immunodominant region of the protein, and antibodies raised against it are potent inhibitors of invasion and development of sporozoites in cultured hepatocytes. Using a modified build-up procedure, we have explored a large number of possible helical and near-helical conformations of a terminally blocked tetraicosapeptide, consisting of six repeats of the sequence Asn-Ala-Asn-Pro, and conclude that two helical conformations are energetically favored to the exclusion of all others. One of these conformations is longer, thinner, and left-handed and is likely to be adopted in nonpolar environments, while the other is shorter, broader, and right-handed and should be favored in aqueous solutions. We propose that the immunodominant region of the circumsporozoite protein of P. falciparum adopts one of these conformations in vivo.

A succession of apparent local energy minima, in which the energies decrease successively, have been located for deca-L-alanine and the octapeptide loop of ribonuclease. The lowest energy minima were approximately 40 kcal/mole lower than the highest ones. The results suggest that the computational methods used here might be of considerable use in searching for the global energy minimum of larger polypeptides.

The matrix of second derivatives of 21 apparent local energy minima of deca-L-alanine has been computed. The results show that all these conformations are true local energy minima and, therefore, potentially stable conformations for this peptide. Calculations of the relative statistical weights of these conformations confirm earlier theoretical considerations on the importance of the librational free energy of stable conformations of peptides. As predicted earlier, the relative statistical weights of the local energy minima do not always fall in the same order as the relative energies.