Kim Vilbour Andersen

AUTOMATED AND SEMIAUTOMATED ANALYSIS OF HOMO- AND HETERONUCLEAR MULTIDIMENSIONAL NUCLEAR MAGNETIC RESONANCE SPECTRA OF PROTEINS : THE PROGRAM PRONTO

Publisher Summary This chapter describes the programs MNMR and Pronto. It describes the program as it has been developed for the analysis of both homo- and heteronuclear protein NMR data in two-, three-, and four-dimensional spectra. In Pronto there is no facility that fully automated for the analysis of multidimensional heteronuclear spectra. This is because the analytical tools provided in the program have proved so efficient and because the users of the program typically are specialists. The ultimate goal of making computer programs for the analysis of protein NMR spectra is to create a system that does any step in the analysis automatically. Such a program has so far not been made, although a number of approaches that automate fragments of the analysis have been reported. The strategy for the development of Pronto has been primarily to create a software program that combines automation and user-controlled analysis. The strategy for analyzing NMR data sets is a straightforward process that can relatively easily be described schematically and also translated into a computer program.

A nuclear magnetic resonance study of the hydrogen-exchange behaviour of lysozyme in crystals and solution

Amide hydrogen/deuterium exchange behaviour has been studied for all of the peptide amides of hen lysozyme by means of two-dimensional n.m.r. spectroscopy. The amides have been grouped into four categories on the basis of their rates of exchange in solution at pH 4.2 and 7.5. The distribution of the amides into the different categories has been examined in the light of the crystallographic structural information, considering the type of secondary structure, the nature of hydrogen bonding and the distance from the protein surface. None of these features was found to determine uniquely the pattern of hydrogen exchange rates within the protein. The exchange behaviour of the individual amides could, however, in general be rationalized by a combination of these features. Hydrogen exchange was also monitored in both tetragonal and triclinic crystals of lysozyme, by allowing exchange to take place in the crystals prior to dissolution and recording of n.m.r. spectra under conditions where further exchange was minimized. This enabled direct comparison to be made of the exchange behaviour in the crystals and solution. A reduction in exchange rate was observed in the crystalline state relative to solution for a substantial number of amides and distinct differences between exchange in the different crystals could be observed. These differences between the solution and the different crystal states do not, however, correlate in a simple manner with proximity to intermolecular contacts in the crystals. However, the existence of these contacts, which are on the surface of the protein molecule, have a profound effect on the exchange of amides in the interior of the protein. The results indicate that the spectrum of fluctuations giving rise to hydrogen exchange may be significantly altered by the intermolecular interactions present within the crystalline state.

The three-dimensional structure of acyl-coenzyme A binding protein from bovine liver: structural refinement using heteronuclear multidimensional NMR spectroscopy.

SummaryThe 3D structure of bovine recombinant acyl-coenzyme A binding protein has been determined using multidimensional heteronuclear magnetic resonance spectroscopy in a study that combines investigations of 15N-labeled and unlabeled protein. The present structure determination is a refinement of the structure previously determined (Andersen, K.V. and Poulsen, F.M. (1992) J. Mol. Biol., 226, 1131–1141). It is based on 1096 distance restraints and 124 dihedral angle restraints of which 69 are for ϕ-angles and 8 for chiral centers and 47 for prochiral centers. The new experimental input for the structure determination has provided an increase of 263 distance restraints, 5 ϕ-angle restraints, and 32 χ-angle restraints in 2 chiral centers, and 31 prochiral centers restraining an additional 23 χ1, 8 χ2, and 1 χ3 angles. The increase of 300 distance and dihedral angle restraints representing an additional 30% of input parameters for the structure determination has been shown to be in agreement with the first structure. A set of 29 structures has been calculated and each of the structures has been compared to a mean structure to give an atomic root mean square deviation of 0.44±0.12 Å (1 Å is 0.1 nm) for the backbone atoms C, Cα, and N in the four α-helices A1, residues 4–15, A2, residues 21–36, A3, residues 51–62 and A4, residues 65–84. The loop-region of residues Gly45-Lys50 could not be defined by the restraints obtained by NMR.The program PRONTO has been used for the spectrum analysis, assignment of the individual nuclear Overhauser effects, the integration of the cross peaks, and the measurement of the coupling constants. The programs DIANA, X-PLOR and INSIGHT have been used in the structure calculations and evaluations.

Outline of a Computer Program for the Analysis of Protein NMR Spectra

It was realized early on in the work process preceeding the determination of three-dimensional structures of proteins in solution by 1H NMR spectroscopy that the very large amount of data in the analysis required the use of computers, primarily as computational devices for ‘number-crunching’; however, the potential of the computer as a bookkeeper and an automation device was also anticipated. The result has been the development of a large number of computer programs, that can assist the analysis of protein NMR data at all levels, from Fourier transformation, automated cross peak identification, amino acid spin system recognition, and sequential and stereospecific assignments.1–10 The development in this field has been promising. In particular, recent analyses of three- and four-dimensional spectra have shown that the larger dispersion of these favors automation.10 However, the full automation of the analysis of protein two-dimensional NMR data involving complete assignment of both NOESY, TOCSY and COSY spectra, full integration of peak volumes, and accurate determination of coupling constants has not yet been achieved. The major reason for this is partly the complexity of the problem, partly the coincidental overlap of resonances, and partly the line broadening of nuclei in certain regions of proteins.