Use of essential and molecular dynamics to study gamma B-crystallin unfolding after non-enzymic post-translational modifications

Essential and Molecular Dynamics (ED/MD) have been used to model the conformational changes of a protein implicated in a conformational disease-cataract, the largest cause of blindness in the world-after non-enzymic post-translational modification. Cyanate modification did not significantly alter flexibility, while the Schiff's base adduct produced a more flexible N-terminal domain, and intra-secondary structure regions, than either the cyanate adduct or the native structure. Glycation also increased linker flexibility and disrupted the charge network. A number of post-translational adducts showed structural disruption around Cys15 and increased linker flexibility; this may be important in subsequent protein aggregation. Our modelling results are in accord with experimental evidence, and show that ED/MD is a useful tool in modelling conformational changes in proteins implicated in disease processes. (C) 2003 Published by Elsevier Ltd.

A novel Bayesian approach to drug design with simple and complex enzymes: Use with lens aldehyde dehydrogenase and the glyoxalase pathway

Purpose: Acquiring details of kinetic parameters of enzymes is crucial to biochemical understanding, drug development, and clinical diagnosis in ocular diseases. The correct design of an experiment is critical to collecting data suitable for analysis, modelling and deriving the correct information. As classical design methods are not targeted to the more complex kinetics being frequently studied, attention is needed to estimate parameters of such models with low variance. Methods: We have developed Bayesian utility functions to minimise kinetic parameter variance involving differentiation of model expressions and matrix inversion. These have been applied to the simple kinetics of the enzymes in the glyoxalase pathway (of importance in posttranslational modification of proteins in cataract), and the complex kinetics of lens aldehyde dehydrogenase (also of relevance to cataract). Results: Our successful application of Bayesian statistics has allowed us to identify a set of rules for designing optimum kinetic experiments iteratively. Most importantly, the distribution of points in the range is critical; it is not simply a matter of even or multiple increases. At least 60 % must be below the KM (or plural if more than one dissociation constant) and 40% above. This choice halves the variance found using a simple even spread across the range.With both the glyoxalase system and lens aldehyde dehydrogenase we have significantly improved the variance of kinetic parameter estimation while reducing the number and costs of experiments. Conclusions: We have developed an optimal and iterative method for selecting features of design such as substrate range, number of measurements and choice of intermediate points. Our novel approach minimises parameter error and costs, and maximises experimental efficiency. It is applicable to many areas of ocular drug design, including receptor-ligand binding and immunoglobulin binding, and should be an important tool in ocular drug discovery.