Methods for Protein Structure Determination

Most of our structural information comes from x-ray crystallographic analysis of protein crystals and from the use of nuclear magnetic resonance spectroscopy in solution. Each of these techniques has advantages and limitations which makes them suitable for a complementary range of problems. The first protein structure determined at a sufficient resolution to trace the path of the polypeptide chain was that of myoglobin in 1960. Since that time many thousands of structures corresponding to hundreds of different proteins have been determined. The coordinates of the atoms in many protein and nucleic acid structures are available from the Protein Data Bank, which may be accessed via the Internet or World Wide Web (http //www.pdb.bnl.gov). 

Question What generalizations or folding rules can be drawn from the known data base of experimentally determined protein structures  

Most electrically charged groups are on the surface of the molecule, interacting with water. Exceptions to this rule are often catalytically important residues in enzymes, which may well be partially stabilized by specific polar interactions within a hydrophobic portion of the molecule. 

Most nonpolar (e g., hydrocarbon) groups are in the interior of the molecule, thus avoiding thermodynamically unfavorable contact with water. Exceptions to this may function as specific binding sites on the surface of the molecule for other proteins or ligands. 

Maximal hydrogen bonding occurs within the molecule. 

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V Kanelis, JD Forman-Kay, LE Kay (2001) Multidimensional NMR methods for protein structure determination, IUBMB Life 52 (6) 291-302... 

W. Braun, Q. Rev. Biophys., 19, 115 (1987). Distance Geometry and Related Methods for Protein Structure Determination from NMR Data. 

Edman, Pehr Victor (1916-1977), a pioneer in the field of peptide and protein chemistry. Already for his Ph.D he had isolated and purified angiotensin. In 1947, he accepted an associate professorship at the University of Lund (Sweden), and in 1957 Edman came to Melbourne as the first John Holt director of research at St. Vincent s School of Medical Research. In 1972, he moved to Germany, having been appointed Professor at the Max-Planck-Institute for Biochemistry at Martinsried. He is the inventor of an important method for protein structure determination, named the Edman degradation [F. J. Morgan, Edman, Pehr Victor (1916-1977), Austr. Diet. Biograph. 1996, Volume 14, pp. 78-79]. 

It has been recognized that many of the time-consuming interactive tasks carried out by an expert during the process of spectral analysis could be done more efficiently by automated computational systems [6]. Over the past few years, this potential has been realized to some degree. Today automated methods for NMR structure determination are playing a more and more prominent role and can be expected to largely supersede the conventional manual approaches to solving three-dimensional protein structures in solution. 

Petoukhov, M. V., and Svergun, D. I. (2003). New methods for domain structure determination of proteins from solution scattering data.J. Appl. Crystallogr. 36, 540-544. 

The molecular replacement method is often used for protein structure determination. It involves determining the orientation and the position of the known structure (or a portion of the structure) with respect to the crystallographic axes three angles describe the orientation... 

For the determination of the electron density map of a molecule the amplitudes and phases of the waves scattered by a single crystal are required for a number of Bragg reflections. Common X-ray techniques yield the product (Ah vh) (Ah 1waves scattered by the electrons of the molecule into the Bragg reflection H. Thus, the scattering amplitude Ah is obtained, but the phase information is lost. The solution of this phase problem for protein structure determination is based on Perutz and Kendrew s isomorphous replacement method (108-111). In this procedure Bragg reflections have to be measured at least three times, first on a crystal of native molecules, and then on two crystals, in which reference scatterers (for example Hg atoms) have been substituted at well-defined positions. From the difference of the measured intensities one can calculate the relative phases without ambiguity. 

The molecular replacement method used for protein structure determination (50,51) involves determining the orientation and the position in the unit cell of a known structure such as that of a homologous protein that has previously been determined or the same protein in a different unit cell (a polymorph). For the rotation function the Patterson map is systematically laid down upon itself in all possible orientations (Fig. 23). Six parameters that define the position and orientation of the protein in the unit cell are found from maxima in a function that describes the extent of overlap between the two placements of the Patterson function. This function will reveal the relative orientations of protein molecules in the unit cell. The rotation function is thus a computational tool used to assess the agreement or degree of coincidence of two Patterson functions, one from a model and the other from the diffraction pattern. 

Chemists rely on diffraction methods for the structural determination of molecular solids (i.e. solids composed of discrete molecules), non-molecular solids (e.g. ionic materials) and, to a lesser extent, gaseous molecules. As the technique has been developed, its range of applications has expanded to include polymers, proteins and other macromolecules. The most commonly applied techniques are single crystal and powder X-ray diffraction. Electron diffraction is important for the structural elucidation of molecules in the gas phase and for the study of solid surfaces. Neutron diffraction is used for the accurate location of light atoms (e.g. H, D or Li), or if one needs to distinguish between atoms of similar atomic numbers, e.g. C and N, or Ni and Cu. 

The chemical nature of coenzymes has been established for a much longer time than that of the enzymes themselves. The explanation is that all enzymes are proteins and, as indicated before, the methods for their structural determination (sequential analysis) have been developed only very recently. Coenzymes, on the other hand, are low molecular weight compounds which can be studied by the methods of organic chemistry. 

D. Schneidman-Duhovny, A. Rossi, A. Avila-Sakar, S.J. Kim, J. Velazquez-Muriel, P. Strop, H. Liang, K. A. Krukenberg, M. Liao, H. M. Kim, S. Sobhanifar, V. Dotsch, A. Rajpal, J. Pons, D. A. Agard, Y. Cheng, and A. Sali, Bioinformatics, 28, 3282-3289 (2012). A Method for Integrative Structure Determination of Protein-Protein Complexes. 

Electrostatic stabilization, 181, 195,225-228 Empirical valence bond model, see Valence bond model, empirical Energy minimization methods, 114-117 computer programs for, 128-132 convergence of, 115 local vr. overall minima, 116-117 use in protein structure determination,... 

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