GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM RABBIT MUSCLE

The rabbit muscle is one of the most frequently used sources for glyceraldehyde-3-phosphate dehydrogenase. This chapter describes the assay method and properties of the enzyme, glyceraldehyde-3-phosphate dehydrogenase, isolated from the rabbit muscle. Classical isolation procedures involve the high solubility of the nicotinamide adenine dinucleotide kinase (NAD+)-containing enzyme in (NH4)2SO4 solutions; several recrystallization steps from concentrated (NH4)2SO4 solutions at pH values above 8.0 are also involved. The steps involved in the purification procedure of the enzyme are (1) the extraction of the rabbit muscle, (2) ammonium sulfate fractionation, and (3) chromatography. The preparation of holoenzyme, apoenzyme, and tetra (3-phosphoglyceroyl) enzyme are also discussed in the chapter. The specific activity of the enzyme prepared is about 185 units per milligram of protein. The enzyme migrates as a single band in polyacrylamide gel electrophoresis and has a molecular weight of 145,000. It contains 3.8–4.0 highly reactive cysteine residues, as determined with dithionitrobenzoate, and can bind 4.0 molecules of NAD+ or nicotinamide adenine dinucleotide dehydrogenase (NADH) per enzyme molecule

Orientation restraints in molecular dynamics simulations using time and ensemble averaging

In this article we present methodology for simulating protein dynamics while imposing restraints derived from NMR measurements on partially ordered molecules. Such measurements may include residual dipolar couplings and chemical-shift anisotropies. We define a restraint potential for use in molecular dynamics and energy minimization. The presented potential is consistent with the simultaneously optimized molecular order tensor. Restraining can be performed with time and ensemble averaging. We performed a large number of molecular dynamics simulations of the histidine containing phosphocarrier protein with restraints on backbone N-H vector orientations derived from residual dipolar couplings. From these simulations it is evident that the use of time-or ensemble-averaged restraints is essential to leave the fluctuations of the restrained vectors unaffected. Without averaging the fluctuations of the restrained vectors are reduced significantly. This also has the effect of decreasing the apparent molecular order-parameter tensor. (C) 2003 Elsevier Inc. All rights reserved

MOLECULAR-DYNAMICS SIMULATION TECHNIQUES FOR DETERMINATION OF MOLECULAR-STRUCTURES FROM NUCLEAR-MAGNETIC-RESONANCE DATA

This chapter discusses the molecular dynamics (MD) simulation techniques for determination of molecular structures from nuclear magnetic resonance (NMR) data. This chapter presents with a short description of the MD technique and explain how the force field can be modified in order to bring or keep protons close together when a NOE between them has been measured. The combined use of distance geometry calculations and distance bounds driven dynamics gives insight into the accuracy of structure determinations by NMR. Refinement by restrained molecular dynamics improves the quality of the structures in terms of energetics. It is important in this respect to discriminate between uncertainties in the structure determination and thermal fluctuations. Computer simulation of at least the high-frequency modes of a molecule's thermal motions by MD and calculation of the experimentally observed parameters by proper averaging over computed trajectories can be used to support a dynamic model, in which more than one “dynamic conformation” may have to be included to explain all experimental results

TWO-DIMENSIONAL H-1-NMR STUDIES ON THE LAC REPRESSOR DNA-BINDING DOMAIN - FURTHER RESONANCE ASSIGNMENTS AND IDENTIFICATION OF NUCLEAR OVERHAUSER ENHANCEMENTS

The assignments of several 1H lines in the spectrum of the lac repressor DNA‐binding domain (or “headpiece”) are documented. These new assignments complement those obtained in a previous study [E.R.P. Zuiderweg, R. Kaptein, and K. Wüthrich (1983) Eur. J. Biochem. 137 , 279] and were made from pure absorption phase two‐dimensional nuclear Overhauser enhancement (2D NOE) spectra and 1H 2D double‐quantum spectra of this protein. Based on the complete set of resonance assignments, a large number of interresidue NOEs are identified; these are documented and their relation with distance constraints is discussed. The identification of these NOEs is a prerequiste for the determination of the spatial structure of this protein, for which no crystallographic data are available