Protein flexibility and enzymatic catalysis.

The dynamic nature of protein structures has been recognized, established, and accepted as an intrinsic fundamental property with major consequences to their function. Nowadays, proteins are considered as networks of continuous motions, which reflect local flexibility and a propensity for global structural plasticity. Protein-protein and protein-small ligand interactions, signal transduction and assembly of macromolecular machines, allosteric regulation and thermal enzymatic adaptation are processes which require structural flexibility. In general, enzymes represent an attractive class among proteins in the study of protein flexibility and they can be used as model systems for understanding the implications of protein fluctuations to biological function. Flexibility of the active site is considered as a requirement for reduction of free energy barrier and acceleration of the enzymatic reaction while there is growing evidence which concerns the connection between flexibility and substrate turnover rate. Moreover, the role of conformational flexibility has been well established in connection with the accessibility of the active site, the binding of substrates and ligands, and release of products, stabilization and trapping of intermediates, orientation of the substrate into the binding cleft, adjustment of the reaction environment, etc.

Multidimensional molecular replacement.

A method is described which attempts to simultaneously and independently determine the positional and orientational parameters of all molecules present in the asymmetric unit of a target crystal structure. This is achieved through a reverse Monte Carlo optimization of a suitable statistic (such as the R factor or the linear correlation coefficient between the observed and calculated amplitudes of the structure factors) in the 6n-dimensional space defined by the rotational and translational parameters of the n search models. Results from the application of this stochastic method - obtained with a space-group-general computer program which has been developed for this purpose - indicate that with present-day computing capabilities the method may be applied successfully to molecular-replacement problems for which the target crystal structure contains up to three molecules per asymmetric unit. It is also shown that the method may be useful in cases where the assumption of topological segregation of the self- and cross-vectors in the Patterson function is violated (as may happen, for example, in closely packed crystal structures).

Side-chain conformations in 4-alpha-helical bundles.

The distribution of the chi(1), chi(2) dihedral angles in a dataset consisting of 12 unrelated 4-alpha-helical bundle proteins was determined and qualitatively compared with that observed in globular proteins. The analysis suggests that the 4-alpha-helical bundle motif could occasionally impose steric constraints on side chains: (i) the side-chain conformations are limited to only a subset of the conformations observed in globular proteins and for some amino acids they are sterically more constrained than those in helical regions of globular proteins; (ii) aspartic acid and asparagine occasionally adopt rotamers that have not been previously reported for globular or helical proteins; (iii) some rotamers of tyrosine and isoleucine are predominantly or exclusively associated with hydrophobic core positions (a, d); (iv) mutations in the hydrophobic core occur preferentially between residue types which among other physicochemical properties also share a predominant rotamer.

Crystallization and diffraction to ultrahigh resolution (0.8 A) of a designed variant of the Rop protein.

The Rop protein is the paradigm of a highly regular four-alpha-helix bundle and as such has been subject to numerous structural and mutagenesis studies. Crystals of a designed Rop variant which establishes a continuous heptad pattern through the bend region have been obtained by a combination of vapour-diffusion and seeding techniques. The crystals diffract to ultrahigh (0.8 A) resolution using synchrotron radiation and cryogenic conditions.

On the distribution of the bulk-solvent correction parameters.

The distribution of the bulk-solvent correction parameters (B(sol), k(sol)) (as determined with an exponential scaling algorithm based on Babinet's principle) for 219 crystal structures deposited in the Protein Data Bank is presented. The distribution shows that (i) the range of values observed is far wider than the usually cited parameter range, (ii) the observed k(sol) values do not correlate with their assumed physical meaning and (iii) the two parameters are not independent and a reasonable agreement with the experimental data can be obtained through the application of a simple exponential function. These findings are interpreted in terms of the inability of the currently used algorithms to uncouple the values of the two parameters during macromolecular refinement.

A stochastic approach to molecular replacement.

The classical approach to the problem of placing n copies of a search model in the asymmetric unit of a target crystal structure is to divide this 6n-dimensional optimization problem into a succession of three-dimensional searches (rotation-function followed by translation-function searches for each of the models). Here, it is shown that a structure-determination method based on a reverse Monte Carlo minimization of a suitably chosen statistic in the 6n-dimensional space defined by the rotational and translational parameters of the n molecules is both feasible and practical, at least for small n. Because all parameters of all molecules are determined simultaneously, this algorithm is expected to improve the signal-to-noise ratio in difficult cases involving high crystallographic/non-crystallographic symmetry in tightly packed crystal forms. Preliminary results from the application of this method (obtained with a space-group general computer program which has been developed for this purpose) are presented.

Crystallization of type I chloramphenicol acetyltransferase: an approach based on the concept of ionic strength reducers.

Chloramphenicol acetyltransferase (CAT) is responsible for bacterial resistance to chloramphenicol. It catalyzes inactivation of the antibiotic by acetyl-group transfer from acetyl CoA to one or both hydroxyl groups of chloramphenicol. Type I CAT possesses some unique properties which are not observed in other CAT variants. Type I CAT overexpressed in Escherichia coli was purified and crystals with a resolution limit of 2.22 A have been obtained using a novel procedure which is based on the concept of 'ionic strength reducers'. The crystals have the symmetry of space group P1 and unit-cell parameters a = 96.46, b = 113.86, c = 114.21 A, alpha = 119.9, beta = 94.1, gamma = 98.6 degrees. These dimensions are consistent with four to six trimers per unit cell, corresponding to a solvent fraction ranging from 65 to 47%.

Protein plasticity to the extreme: changing the topology of a 4-alpha-helical bundle with a single amino acid substitution.

BACKGROUND: Conventional wisdom has it that two proteins sharing 98.4% sequence identity have nearly identical three-dimensional structures. Here we provide a counter-example to this statement by showing that a single amino acid substitution can change the topology of a homodimeric 4-alpha-helical bundle protein. RESULTS: We have determined the high-resolution crystal structure of a 4-alpha-helical protein with a single alanine to proline mutation in the turn region, and show that this single amino acid substitution leads to a complete reorganisation of the whole molecule. The protein is converted from the canonical left-handed all-antiparallel form, to a right-handed mixed parallel and antiparallel bundle, which to the best of our knowledge and belief represents a novel topological motif for this class of proteins. CONCLUSIONS: The results suggest a possible new mechanism for the creation and evolution of topological motifs, show the importance of loop regions in determining the allowable folding pathways, and illustrate the malleability of protein structures.

Meaningful refinement of polyalanine models using rigid-body simulated annealing: application to the structure determination of the A31P Rop mutant.

Conventional refinement methods, when applied to even correctly positioned polyalanine models of a target structure, result in a systematic distortion of the molecular geometry and to a concomitant increase in the mean phase difference from the correct phase set. Here, it is shown that iterative rigid-body simulated-annealing refinement of polyalanine models employing successively fewer residues per rigid body (down to one alanine residue per body) at a very high initial temperature (of the order of T0 = 10000 K) and with the geometric energy terms switched on, not only preserves the geometry of the model but can also converge to an essentially correct polyalanine trace of the target structure, even when the starting model deviates systematically and significantly from the sought structure. As an example of the application of the method, details are presented of the structure determination of the Ala31Pro mutant of the Rop protein, where an initial roughly positioned polyalanine model (giving an average phase difference of 78.2 degrees from the final phase set) was successfully refined against a 1.8 A resolution native data set, leading to an essentially correct model of the main chain with an average displacement of its atomic positions from the final model of 0.275 A. The phases calculated from this refined polyalanine model had an average difference of 43.8 degrees from the final phase set (corresponding to a mean figure of merit of 0.72) and gave a readily interpretable electron-density map.

Low-resolution structural characterization of the arginine repressor/activator from Bacillus subtilis: a combined X-ray crystallographic and electron microscopical approach.

Attempts to determine the X-ray crystal structure of the intact homohexameric arginine repressor/activator from B. subtilis have so far been unsuccessful. The major problem appears to be the lack of an isomorphous heavy-atom derivative with a manageable number of substitution sites. Here it is shown how electron microscopy of thin three-dimensional crystals, the same as those used for the X-ray crystallographic studies, made it possible (i) to obtain experimental support for some conclusions drawn on the basis of X-ray data alone, (ii) to determine the low-resolution distribution of electron density in several different crystallographic projections, and (iii) to obtain a tentative low-resolution model of the whole hexamer.