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The dynamics of the GroEL-GroES complex is investigated with a coarse-grained model. This model is one in which single-residue points are connected to other such points, which are nearby, by identical springs, forming a network of interactions. The nature of the most important (slowest) normal modes reveals a wide variety of motions uniquely dependent upon the central cavity of the structure, including opposed torsional rotation of the two GroEL rings accompanied by the alternating compression and expansion of the GroES cap binding region, bending, shear, opposed radial breathing of the cis and trans rings, and stretching and contraction along the protein assembly's long axis. The intermediate domains of the subunits are bifunctional due to the presence of two hinges, which are alternatively activated or frozen by an ATP-dependent mechanism. ATP binding stabilizes a relatively open conformation (with respect to the central cavity) and hinders the motion of the hinge site connecting the intermediate and equatorial domains, while enhancing the flexibility of the second hinge that sets in motion the apical domains. The relative flexibilities of the hinges are reversed in the nucleotide-free form. Cooperative cross-correlations between subunits provide information about the mechanism of action of the protein. The mechanical motions driven by the different modes provide variable binding surfaces and variable sized cavities in the interior to enable accommodation of a broad range of protein substrates. These modes of motion could be used to manipulate the substrate's conformations.

Tryptophan synthase (TRPS), with linearly arrayed subunits alphabetabetaalpha, catalyzes the last two reactions in the biosynthesis of L-tryptophan. The two reactions take place in the respective alpha- and beta-subunits of the enzyme, and the intermediate product, indole, is transferred from the alpha- to the beta-site through a 25 A long hydrophobic tunnel. The occurrence of a unique ligand-mediated long-range cooperativity for substrate channeling, and a quest to understand the mechanism of allosteric control and coordination in metabolic cycles, have motivated many experimental studies on the structure and catalytic activity of the TRPS alpha2beta2 complex and its mutants. The dynamics of these complexes are analyzed here using a simple but rigorous theoretical approach, the Gaussian network model. Both wild-type and mutant structures, in the unliganded and various liganded forms, are considered. The substrate binding site in the beta-subunit is found to be closely coupled to a group of hinge residues (beta77-beta89 and beta376-beta379) near the beta-beta interface. These residues simultaneously control the anticorrelated motion of the two beta-subunits, and the opening or closing of the hydrophobic tunnel. The latter process is achieved by the large amplitude fluctuations of the so-called COMM domain in the same subunit. Intersubunit communications are strengthened in the presence of external aldimines bound to the beta-site. The motions of the COMM core residues are coordinated with those of the alpha-beta hinge residues beta174-beta179 on the interfacial helix betaH6 at the entrance of the hydrophobic tunnel. And the motions of betaH6 are coupled, via helix betaH1 and alphaL6, to those of the loop alphaL2 that includes the alpha-subunit catalytically active residue Asp60. Overall, our analysis sheds light on the molecular machinery underlying subunit communication, and identifies the residues playing a key role in the cooperative transmission of conformational motions across the two reaction sites.

Recently, we developed a simple analytical model based on local residue packing densities and the distribution of tertiary contacts for describing the conformational fluctuations of proteins in their folded state. This so-called Gaussian network model (GNM) is applied here to the interpretation of experimental hydrogen exchange (HX) behavior of proteins in their native state or under weakly denaturing conditions. Calculations are performed for five proteins: bovine pancreatic trypsin inhibitor, cytochrome c, plastocyanin, staphylococcal nuclease, and ribonuclease H. The results are significant in two respects. First, a good agreement is reached between calculated fluctuations and experimental measurements of HX despite the simplicity of the model and within computational times 2 or 3 orders of magnitude faster than earlier, more complex simulations. Second, the success of a theory, based on the coupled conformational fluctuations of residues near the native state, to satisfactorily describe the native-state HX behavior indicates the significant contribution of local, but cooperative, fluctuations to protein conformational dynamics. The correlation between the HX data and the unfolding kinetics of individual residues further suggests that local conformational susceptibilities as revealed by the GNM approach may have implications relevant to the global dynamics of proteins.

A low-resolution model is used together with recently developed knowledge-based potentials for exploring the dynamics of proteins. Configurations are generated using a Monte Carlo/Metropolis scheme combined with a singular value decomposition technique (SVD). The approach is shown to characterize the cooperative motions in good detail, at least 1 order of magnitude faster than atomic simulations. Trajectories are partitioned into modes, and the slowest ones are analyzed to elucidate the dominant mechanism of collective motions. Calculations performed for bacteriophage T4 lysozyme, a two-domain enzyme, demonstrate that the structural elements within each domain are subject to strongly coupled motions, whereas the motions of the two domains with respect to each other are strongly anticorrelated. This type of motion, evidenced by the synchronous fluctuations of the domain centroids by up to +/-4.0 A in opposite directions, is comparable to the movements observed by recent spin-labeling experiments in solution. The potential of mean force governing these fluctuations is shown to be anharmonic. The beta-sheet region at the N-terminal domain and the helix E in the C-terminal domain are identified as regions important for mediating cooperative motions and, in particular, for the opening and closing of the active-site cleft between the domains. Residues Leu66-Phe67 in the central helix C stop the propagation of correlated motions between the domains. There is a correlation between the groups involved in highly cooperative motions revealed by simulations and the highly protected regions during unfolding measured by pulsed H/D exchange and 2-D NMR.

Structural feasibility and conformational requirements for the sequence 5'-d-GGTACIAGTACC-3' to adopt a hairpin loop with I6 and A7 in the loop are studied. It is shown that a hairpin loop containing only two nucleotides can readily be formed without any unusual torsional angles. Stacking is continued on the 5'-side of the loop, with the I6 stacked upon C5. The base A7, on the 3'-side of the loop, can either be partially stacked with I6 or stick outside without stacking. Loop closure can be achieved for both syn and anti conformations of the glycosidic torsions for G8 while maintaining the normal Watson-Crick base pairing with the opposite C5. All torsional angles in the stem fall within the standard B-family of DNA helical structures. The phosphodiesters of the loop have trans,trans conformations. Loop formation might require the torsion about the C4'-C5' bond of G8 to be trans as opposed to the gauche+ observed in B-DNA. These results are discussed in relation to melting temperature studies [Howard et al. (1991) Biochemistry (preceding paper in this issue)] that suggest the formation of very stable hairpin structures for this sequence.

A new method is presented to examine the complete range of folded topologies accessible in the compact state of globular proteins. The procedure is to generate all conformations, with volume exclusion, upon a lattice in a space restricted to the individual protein's known compact conformational space. Using one lattice point per residue, we find 10(2)-10(4) possible compact conformations for the five small globular proteins studied. Subsequently, these conformations are evaluated in terms of residue-specific, pairwise contact energies that favor nonbonded, hydrophobic interactions. Native structures for the five proteins are always found within the best 2% of all conformers generated. This novel method is simple and general and can be used to determine a small group of most favorable overall arrangements for the folding of specific amino acid sequences within a restricted space.

By using conformational free energy calculations, we have studied the sequence dependence of flexibility and its anisotropy along various conformational variables of DNA base pairs. The results show the AT base step to be very flexible along the twist coordinate. On the other hand, homonucleotide steps, GG(CC) and AA(TT), are among the most rigid sequences. For the roll motion that would correspond to a bend, the TA step is most flexible, while the GG(CC) step is least flexible. The flexibility of roll is quite anisotropic; the ratio of fluctuations toward the major and minor grooves is the largest for the GC step and the smallest for the AA(TT) and CG steps. Propeller twisting of base pairs is quite flexible, especially of A.T base pairs; propeller twist can reach 19 degrees by thermal fluctuation. We discuss the effect of electrostatic parameters, comparison with available experimental results, and biological relevance of these results.

Prokaryotic promoters have been extensively studied to relate sequence features to promoter function. Here we examine the relationship between double-helix stability and promoter activity. The double-helix stability is evaluated from sequence data by free energy computation, based on reported values of dinucleotide free energies for strand separation. For a collection of 168 promoters, we find that within a 500-nucleotide span around the transcription initiation site the -10 region is the least stable. There is no correlation between the free energies and the rates of RNA polymerase-promoter open complex formation measured for 25 promoters. We also compare the free energies of 121 promoter mutations across the -35 and -10 consensus regions with the free energies of the corresponding wild-type sequences. These pairwise mutant-wild-type comparisons provide a particularly good test since the examined sequences differ only in one nucleotide so that all other sequence-dependent effects remain the same. About 80% of the mutations in the -10 region that show increased/reduced promoter activity are less/more stable than the wild types. The observed high free energy peak and the mutation data strongly support the conjecture that the instability, or melting properties, of the -10 region plays a significant role in promoter function.

Conformational analysis of DNA shows that the origin of the B-form double helix can be attributed in large part to the atomic charge pattern in the base pairs. The charge patterns favor specific helical stacking of the base pairs. Base pairs alone--without backbones--have a strong tendency to form helix, indicating that the backbones play a rather passive role in determining the basic helical structure of DNA. It is mainly the electrostatic interactions determined by the charge pattern on base pairs that stabilize a particular helical conformation. The charge pattern in the base pairs appears to be responsible for much of the sequence dependence of DNA conformation, rather than steric clashes.