PubMed · 8805528
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E N Baker, T L Blundell, E Dodson, G Dodson, G L Gilliland, J L Sussman, M Vijayan. 1996-02-15. Diffraction data deposition.. https://doi.org/10.1016/s0969-2126(96)00024-x
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A protocol for complete isotopic labeling of iso-1-cytochrome c from the eukaryote Saccharomyces cerevisiae is reported. Assignments are reported for the vast majority of the 15N amide resonances in both oxidized and reduced states. 15N heteronuclear relaxation experiments were collected to study the picosecond-nanosecond backbone dynamics of this protein. Relaxation rates were computed and fit to spectral density functions by a model-free analysis. Backbone amides in the overlapping loop B/C region are the most flexible on the picosecond-nanosecond time scale in both forms of the protein. The results show that, on average, the protein backbone is slightly more dynamic in the oxidized than the reduced state, though not significantly so. Exchange terms, which suggest significant motion on a time scale at least an order of magnitude slower than the overall correlation time of 5.2 ns, were required for only two residues in the reduced state and 27 residues in the oxidized state. When analyzed on a per-residue basis, the lower order parameters found in the oxidized state were scattered throughout the protein, with a few continuous segments found in loop C and the C-terminal helix, suggesting greater flexibility of these regions in the oxidized state. The results provide dynamic interpretations for previously presented structural and functional data, including redox-dependent changes that occur in the protein. The way is now paved for extensive dynamic analysis of variant cytochromes c.
Spectroscopic and chemical modification studies of modified flavins bound to old yellow enzyme have led to predictions about the flavin environment of this enzyme. These studies analyzed solvent accessibility and hydrogen bonding patterns of particular flavin atoms, in addition to suggesting amino acid residues that are in close proximity to those atoms. Here, these studies are evaluated in the light of the crystal structure of old yellow enzyme to reveal that the spectroscopic and modified flavin results are generally consistent with the crystal structure. This highlights the fact that these are useful methods for studying flavin binding site structure. Although several of the inferred properties of the flavin environment are not consistent with the crystal structure, these discrepancies occurred in cases where an incorrect choice was made from among multiple plausible explanations for an experimental result. We conclude that modified flavin studies are powerful probes of flavin environment; however, it is risky to specify details of interactions, especially because of uncertainties due to induced charge delocalization in the flavin.
This article describes new methods for X-ray crystallographic refinement and nuclear magnetic resonance (NMR) structure determination that are available in the recent release of the X-PLOR software, X-PLOR 98.0. The major new features of the X-PLOR 98.0 software are: (i) the introduction of maximum likelihood methods (Pannu and Read, Acta Crystallogr 1996;A52:659-668) for X-ray crystallographic refinement with structure factor amplitude, intensity and phase probability targets, (ii) the addition of the Andersen thermal coupling method for temperature control during simulated annealing refinements, (iii) a new utility function for converting reflection data in to the X-PLOR format, (iv) validated scripts and performance enhancements for structure determination from NMR distance restraints using torsion angle dynamics, (v) fast code for direct nuclear Oberhauser effect (NOE) refinement using matrix doubling and gaussian quadratures, (vi) methodologies for using ambiguous restraint information to perform automated iterative peak assignment and structure determination (Nilges et al., J Mol Biol 1997;269: 408-422). Additional developments in methodology for refining crystal structures from poor initial models include the implementation of a fast adaptive bulk solvent scattering correction and an energy minimization routine that makes use of second derivative information. Trial crystallographic refinements with an energy minimization protocol that includes these enhancements indicate significantly improved convergence. The quality of the resulting models appears comparable to models obtained from refinement protocols that incorporate torsion angle dynamics. Test applications of the new energy minimizer to NMR structure refinement with using NOE calculations also show improved convergence, leading to more optimized final models.