Protein structure by NMR

In some ways it is easier to obtain protein structures by NMR spectroscopy. Proteins need not be crystallized, just purified and dissolved. Would it be desirable to use NMR to learn about the structure of the active site of an enzyme, to design an inhibitor Why or why not ... 

A major bottleneck in solving protein structures by NMR is the highly peakpicking and assignment of chemical shifts and NOEs. The strategy of the assignment process and stmcture calculation can be found in an excellent review [30]. In general, for a -labeled protein, a series of double/triple resonance experiments are... 

Determination of protein structure by nmr. Proton nmr is the most appropriate tool for this purpose because of the large number of hydrogen atoms present in proteins and the high natur abundance of the H isotope C and N are generally of too low an abundance to be of use in th context unless extra atoms of these isotopes have been purposely incorporated into the protein during their biosynthesis. All the H nuclei in a protein can be observed by pmr except those of the hydrogen atoms of -NH-, -NH2, -OH... 

NMRCORE, and OLDERADO are programs that were initially developed for use by scientists solving protein structures by NMR spectroscopy. These programs aid in the superposition and clustering of protein structures. OLDERADO is a combination of the NMRCLUST and NMRCORE methods. The methodologies employed in OLDERADO are discussed below as separate entities devoted to a common task. 

Einally, structural properties that depend directly neither on the data nor on the energy parameters can be checked by comparing the structures to statistics derived from a database of solved protein structures. PROCHECK-NMR and WHAT IE [94] use, e.g., statistics on backbone and side chain dihedral angles and on hydrogen bonds. PROSA [95] uses potentials of mean force derived from distributions of amino acid-amino acid distances. 

The first step in determination of a structure by NMR spectroscopy involves assignment of individual proton resonances. Development of high-field spectrometers and the use of a second dimension (2D-NMR) along with isotopic substitution (11) and sophisticated pulse sequences (12) make it possible to almost completely assign the proton spectrum of proteins of about 15 kD molecular weight (13—17). Some 2D-pulse sequences commonly used in the study of macromolecules are shown in Figure 1. 

Clore GM, Gronenborn AM. Determining the structures of large proteins and protein complexes by NMR. Trends Biotechnol 1998 16 22-34. 

Traditionally, the structural characterization of designed proteins is carried out by CD spectroscopy, which unfortunately provides only limited structural information at the atomic level. As the understanding of protein design develops more proteins appear that have well-defined structures and the determination of their solution structures by NMR spectroscopy is clearly the main tool for elucidating structure-function relationships. Key information is obtained simply from the ID spectrum (Fig. 7). 

W. F. van Gunsteren, R. Kaptein, and E. R. P. Zuiderweg, in Proceedings of the NATO/ CECAM Workshop on Nucleic Acid Conformation and Dynamics, W. K. Olsen, Ed., Centre de Calcul Atomique et Moleculaire, Orsay, 1984, pp. 79-82. Use of Molecular Dynamics Computer Simulations When Determining Protein Structure by 2D-NMR. 

Fig. 5. The complex of cytochrome/and plastocyanin as determined by paramagnetic NMR (PDB Accession Code 2PCF). The solution structure of Spinacia oleracea plastocyanin was determined by NMR, while cytochrome f was modeled from the previous crystal structure of the soluble domain of Brassica rapa cytochrome / (PDB Code ICTM), with only the contacts between the two proteins determined by NMR. The distance shown is between the heme Fe atom in cytochrome / and the eN of the His-87 copper ligand in plastocyanin.

Structures of Blue Copper Proteins Solved by NMR Spectroscopy... 

Luchette, P. A., Prosser, R. S. and Sanders, C. R. (2002) Oxygen as a paramagnetic probe of membrane protein structure by cysteine mutagenesis and F-19 NMR spectroscopy. Journal of the American Chemical Society, 124(8), 1778-1781. 

Modern Techniques in Protein NMR, ed. N. R. Krishna and L. J. Berliner, Kluwer Academic/Plenum PubL, New York, N.Y., 1998 G. M. Clore and A. M, Gronenbom, Determining Structures of Large Proteins and Protein Complexes by NMR , p. 3 K. H. Gardner and L. E. Kay, Multidimensional H-Based NMR Methods for Resonance Assignment, Structure Determination, and the Study of Protein Dynamics , p. 27... 

ME Girvin and RH Fillingame (1995) Determination of local protein structure by spin label difference 2D NMR the region neighboring Asp61 of subunit c of the F Fq ATP sythase. Biochemistry 34 1635-1645... 

Many of the experimental approaches to study protein structure by solid-state MAS NMR have been devised relatively recently. Not surprisingly, the number of biophysical applications using such methods is still limited. Nevertheless, we will present in Chapter 3 different areas of research where MAS-based solid-state NMR has already been applied and which may pave the way for future applications. We also refer the interested reader to... 

The quality of the three-dimensional structure of proteins determined by nmr is influenced by many factors. The factors are reviewed by focusing on structure calculation problems. High-performance computing implementations are introduced to improve the conformational sampling of the calculations. The quality and improvements of the nmr-derived structural information are also discussed. 

Fourier transform mass spectrometry was devised by Comisarow and Marshall in 1974 at the University of British Columbia. This method uses Fourier transformation, which is a mathematical way to convert a set of data involving a time domain into a set of frequencies. The mathematical concept of Fourier transformation is important and has been used in analyses of the protein structure by mass spectrometry, X-ray crystallography, and nuclear magnetic resonance (NMR). 

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