Dramatic Shape Changes Occur as Cytochrome c Folds.

Extensive experimental studies on the folding of cytochrome c (Cyt c) make this small protein an ideal target for atomic detailed simulations for the purposes of quantitatively characterizing the structural transitions and the associated time scales for folding to the native state from an ensemble of unfolded states. We use previously generated atomically detailed folding trajectories by the stochastic difference equation in length to calculate the time-dependent changes in the small-angle X-ray scattering (SAXS) profiles. Excellent agreement is obtained between experiments and simulations for the time-dependent SAXS spectra, allowing us to identify the structures of the folding intermediates, which shows that Cyt c reaches the native state by a sequential folding mechanism. Using the ensembles of structures along the folding pathways, we show that compaction and the sphericity of Cyt c change dramatically from the prolate ellipsoid shape in the unfolded state to the spherical native state. Our data, which are in unprecedented quantitative agreement with all aspects of time-resolved SAXS experiments, show that hydrophobic collapse and amide group protection coincide on the 100 microseconds time scale, which is in accordance with ultrafast hydrogen/deuterium exchange studies. Based on these results, we propose that compaction of polypeptide chains, accompanied by dramatic shape changes, is a universal characteristic of globular proteins, regardless of the underlying folding mechanism.

Sugar-binding sites on the surface of the carbohydrate-binding module of CBH I from Trichoderma reesei.

Molecular dynamics simulations were carried out for a system consisting of the carbohydrate-binding module (CBM) of the cellulase CBH I from Trichoderma reesei (Hypocrea jecorina) in a concentrated solution of ß-D-glucopyranose, to determine whether there is any tendency for the sugar molecules to bind to the CBM. In spite of the general tendency of glucose to behave as an osmolyte, a marked tendency for the sugar molecules to bind to the protein was observed. However, the glucose molecules tended to bind only to specific sites on the protein. As expected, the hydrophobic face of the sugar molecules, comprising the axial H1, H3, and H5 aliphatic protons, tended to adhere to the flat faces of the three tyrosine side chains on the planar binding surface of the CBM. However, a significant tendency to bind to a groove-like feature on the upper surface of the CBM was also observed. These results would not be inconsistent with a model of the mechanism for this globular domain in which the cellodextrin chain being removed from the surface of crystalline cellulose passes over the upper surface of the CBM, presumably then available for hydrolysis in the active site tunnel of this processive cellulase.

Structure and axis curvature in two dA6 x dT6 DNA oligonucleotides: comparison of molecular dynamics simulations with results from crystallography and NMR spectroscopy.

Molecular dynamics (MD) simulations have been performed on the A6 containing DNA dodecamers d(GGCAAAAAACGG) solved by NMR and d(CGCAAAAAAGCG) solved by crystallography. The experimental structures differ in the direction of axis bending and in other small but important aspects relevant to the DNA curvature problem. Five nanosecond MD simulations of each sequence have been performed, beginning with both the NMR and crystal forms as well as canonical B-form DNA. The results show that all simulations converge to a common form in close proximity to the observed NMR structure, indicating that the structure obtained in the crystal is likely a strained form due to packing effects. A-tracts in the MD model are essentially straight. The origin of axis curvature is found at pyrimidine-purine steps in the flanking sequences.

Structural preference for changes in the direction of the Ca2+-induced transition: a study of the regulatory domain of skeletal troponin-C.

The determinants for specificity in the Ca(2+)-dependent response of the regulatory N-terminal domain of skeletal troponin-C are a combination of intrinsic and induced properties. We characterized computationally the intrinsic propensity of this domain for structural changes similar to those observed experimentally in the Ca(2+)-induced transition. The preference for such changes was assessed by comparing the structural effect of the harmonic and quasiharmonic vibrations specific for each Ca(2+) occupancy with crystallographic data. Results show that only the Ca(2+)-saturated form of the protein features a slow vibrational motion preparatory for the transition. From the characteristics of this mode, we identified a molecular mechanism for transition, by which residues 42-51 of helix B and of the adjacent linker move toward helices (A, D), and bind to the surface used by the protein to interact with troponin-I. By obstructing the access of the target to hydrophobic residues important in the formation of the complex, helix B and the adjacent linker act as an autoinhibitory structural element. Specific properties of the methionines at the interaction surface were found to favor the binding of the autoinhibitory region. Located over hydrophobic residues critical for binding, the methionines are easily displaceable to increase the accessibility of these residues to molecular encounter.

Molecular dynamics simulation studies of induced fit and conformational capture in U1A-RNA binding: do molecular substates code for specificity?

Molecular dynamics (MD) simulations on stem loop 2 of U1 small nuclear RNA and a construct of the U1A protein were carried out to obtain predictions of the structures for the unbound forms in solution and to elucidate dynamical aspects of induced fit upon binding. A crystal structure of the complex between the U1A protein and stem loop 2 RNA and an NMR structure for the uncomplexed form of the U1A protein are available from Oubridge et al. (Nature, 1994, Vol. 372, pp. 432-438) and Avis et al. (Journal of Molecular Biology, 1996, Vol. 257, pp. 398-411), respectively. As a consequence, U1A-RNA binding is a particularly attractive case for investigations of induced fit in protein-nucleic acid complexation. When combined with the available structural data, the results from simulations indicate that structural adaptation of U1A protein and RNA define distinct mechanisms for induced fit. For the protein, the calculations indicate that induced fit upon binding involves a non-native thermodynamic substate in which the structure is preorganized for binding. In contrast, induced fit of the RNA involves a distortion of the native structure in solution to an unstable form. However, the RNA solution structures predicted from simulation show evidence that structures in which groups of bases are favorably oriented for binding the U1A protein are thermally accessible. These results, which quantify with computational modeling recent proposals on induced fit and conformational capture by Leuillot and Varani (Biochemistry, 2001, Vol. 40, pp. 7947-7956) and by Williamson (Nature Structural Biology, 2000, Vol. 7, pp. 834-837) suggest an important role for intrinsic molecular architecture and substates other than the native form in the specificity of protein-RNA interactions.