Protein crystallographic methods

Extensive biochemical and spectroscopic studies have been undertaken on hCP in order to investigate the nature of the copper centers and their role in structure-function relationships. However, the protein is very susceptible to aggregation, proteolysis, loss of copper, and other chemical degradations and requires careful preparation and handling in these circumstances it is difficult to review all the literature objectively and comprehensively. A three-dimensional crystal structure of hCP has been reported at a nominal resolution of 3.1A [7], but this resolution has been extended to just beyond 3.0 A. This chapter will summarize some of the more important biochemical and spectroscopic studies of the protein. It will then focus on the structural results recently obtained by X-ray crystallographic methods and attempt to explain putative functions of the protein in terms of its molecular structure. 

Protein-based methods rely upon the structural information extracted from the X-ray crystallographic and/or homology protein structures. These also include docking techniques for the exploration of possible binding modes of a ligand to a given transporter protein. 

A X-ray crystallographic method for detecting the transient accumulation of intermediates in enzyme catalysis, protein folding, ligand-binding interactions, and other processes involving macromolecules. The approach is premised on the well documented retention of substantial reactivity of biological macromolecules, even in the crystalline state. 

Sharma and Reed, 1976)]. In proteins the coordination number 4 is most common, where the zinc ion is typically coordinated in tetrahedral or distorted tetrahedral fashion. The coordination polyhedron of structural zinc is dominated by cysteine thiolates, and the metal ion is typically sequestered from solvent by its molecular environment the coordination polyhedron of catalytic zinc is dominated by histidine ligands, and the metal ion is exposed to bulk solvent and typically binds a solvent molecule (Vallee and Auld, 1990). The inner-sphere coordination number of catalytic zinc may increase to 5 during the course of enzymatic turnover, and several five-coordinate zinc enzyme—substrate, enzyme product, and enzyme-inhibitor complexes have been studied by high-resolution X-ray crystallographic methods (reviewed by Matthews, 1988 Christianson and Lipscomb, 1989). The coordination polyhedron of zinc in five coordinate examples may tend toward either trigonal bipyramid or octahedral-minus-one geometry. 

At present two three-dimensional structures are available, one determined by the 2D-NMR for solution protein (Cd,MT) and the other using the conventional crystallographic method . These are in conflict and major discrepancies exist between the two structures. In addition, in the recent crystallographic refinement where metal-sulphur clusters were also refined, metal-sulphur bond distances are obtained which are in serious error with respect to the EXAFS determined distances despite the claimed accuracy. For example, the Cd—S distances show considerable variation from 2.5 A the average terminal ligand distance is 3.2 A while the bridging... 

The first serious attempt at interpreting the protein hydration by crystallographic methods was nude 1978 on rubvedoxin, a molecule consisting of only 54 amino adds [835]. The refinement by conventional (unconstrained) least squares methods included a total of 127 water sites and converged at an R-factor of 12.7% with 1.2 A resolution data. The water oxygen atoms were added to the model only if their temperature factors were <50 A2 and their electron density was >0.3 e/A3, i.e., corresponding to the electron density expected for liquid water, 0.34 e/A3. 

The data describing hydration in the nucleic acids are more limited than that of the proteins. In the RNA series, two dinucleoside phosphates [536, 538], one trinucleoside diphosphate [864] and one tetradecamer [666] have been studied by crystallographic methods in the DNA series, many more oligonucleotides were investigated. The oligonucleotide sequences are such that the molecules are self-com-plementary and therefore can readily form double helices (Thble 24.2). This implies that the duplexes exhibit inherent twofold symmetry which, however, only coincides with a crystallographic twofold axis in a few exceptional cases. 

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