Current Protein and Peptide Science - Volume 1, Issue 3, 2000

The present knowledge on the stereochemical mechanism of action of glucose (or xylose) isomerase, one of the highest tonnage industrial enzymes, is summarized. First we deal shortly with experimental methods applied to study the structure and function of this enzyme enzyme kinetics, protein engineering, X-ray crystallography, nuclear magnetic and electron paramagnetic resonance spectroscopy. Computational methods like homology modeling, molecular orbital, molecular dynamics and continuum electrostatic methods are also shortly treated. We discuss mostly those results and their contribution to the elucidation of the mechanism of action that have been published in the last decade. Structural characteristics of free xylose isomerase as well as its complexes with various ligands are depicted. This information provides a tool for the study of structural details of the enzyme mechanism. We present a general mechanism where the first step is ring opening, which is followed by the extension of the substrate to an open-chain conformation, a proton shuttle with the participation of a structural water molecule and the rate-determining hydride shift. The role of metal ions in the catalytic process is discussed in detail. Finally we present main trends in efforts of engineering the enzyme and delineate the prospective future lines. The review is completed by an extended bibliography with over100 citations.

Advances in molecular biology may mean that almost any protein sequence can be synthesised, but perhaps this has served to highlight the inadequacy of theoretical work. For a given protein fold, it is probably not possible to reliably predict an ideal sequence. We identify and survey several aspects of the problem. Firstly, it is not clear what is the best way to score a sequence-structure pair. Secondly, there is no consensus as to what the score function should represent (free energy or some abstract measure of sequence-structure compatibility). Finally, the number of possible sequences is astronomical and searching this space poses a daunting optimisation problem. These problems are discussed in the light of recent experimental successes.

Efforts to use computers in predicting the secondary structure of proteins based only on primary structure information started over a quarter century ago (1-3). Although the results were encouraging initially, the accuracy of the pioneering methods generally did not attain the level required for using predictions of secondary structures reliably in modelling the three-dimensional topology of proteins. During the last decade, however, the introduction of new computational techniques as well as the use of multiple sequence information has lead to a dramatic increase in the success rate of prediction methods, such that successful 3D modelling based on predicted secondary structure has become feasible (e.g., Ref 4). This review is aimed at presenting an overview of the scale of the secondary structure prediction problem and associated pitfalls, as well as the history of the development of computational prediction methods. As recent successful strategies for secondary structure prediction all rely on multiple sequence information, some methods for accurate protein multiple sequence alignments will also be described. While the main focus is on prediction methods for globular proteins, also the prediction of trans-membrane segments within membrane proteins will be briefly summarised. Finally, an integrated iterative approach tying secondary structure prediction and multiple alignment will be introduced (5).

Because synthetic short peptides bearing critical binding residues, can chemically mimic the folded antigenic determinants on proteins, short synthetic peptides can generate antibodies that react with cognate sequences in intact folded proteins. According to this mimotope theory, we produced site-specific antibodies by immunization with short peptides which overlapped each other and covered the entire protein, and used them for domain mapping of influenza virus RNA polymerase (antibody-scanning method). We also used a tagged-epitope and its monoclonal antibodies for topology mapping of clathrin light chains in clathrin triskelions by electron microscopy. Both methods using specific epitopes in combination with their antibodies enable us to determine the domains of interesting proteins systematically without the need to generate monoclonal antibodies or mutant proteins.

Protein folding and assembly in the cell requires the assistance of molecular chaperones. These components prevent off pathway folding reactions that lead to aggregation. They are also critical factors in organismal stress physiology, protecting cells against heat shock and providing thermotolerance. Among this important protein family are chaperonins. They form large cylindrical double ring complexes with a central cavity where protein binding and folding takes place in an ATP-dependent manner. Recently, components functionally related to the eubacterial and organellar chaperonins have been found in the cytosol of archaebacteria and of eukaryotic cells. Based on their sequences and structural features, they have been classified as group II chaperonins, to distinguish them from the group I chaperonins occuring in bacteria. Of particular interest in the group II family is the eukaryotic CCT complex, whose function in protein folding and assembly has been demonstrated mainly for the cytoskeletal proteins tubulin and actin. Together with the Hsp70 chaperone system, it can be considered as an essential helper factor to facilitate the folding of native proteins in the eukaryotic cytosol. Recent structural data have opened the path to a molecular understanding of group II chaperonins and have helped to define their role in cellular protein folding.