Determination of protein conformation

Techniques and methods for the study of protein adsorption have been well reviewed 4). It is now generally recognized that it is not necessarily the type and amount of protein present at the surface which is most important, but rather the orientation and conformational state of those proteins. At present it is virtually impossible to predict the specific conformation of an adsorbed protein at a particular interface. The techniques used in the determination of protein conformation in solution or in the solid state do not usually apply to adsorbed proteins. Hence, the difference between adsorbed and bulk solution protein conformation has to be inferred indirectly. 

T. F. Havel and K. Wiithrich, J. Mol Biol., 182, 281 (1985). An Evaluation of the Combined Use of Nuclear Magnetic Resonance and Distance Geometry for the Determination of Protein Conformations in Solution. 

Schwehm, J. M., and Stites, W. E. (1998). Application of automated methods for determination of protein conformational stability. Methods Enzymol. 295, 150-170. 

Although hydrogen bonds do not contribute to stability they are a major determinant of protein conformation. The necessity to form hydrogen bonds accounts for the or-helices and /8-strands that abound in protein structures. 

Ragg, E., Tagliavini, F., Malesani, P., Monticelli, L., Bugiani, O., Forloni, G., and Salmona, M. (1999). Determination of solution conformations of PrP106-126, a neurotoxic fragment of prion protein, by 1H NMR and restrained molecular dynamics. Eur.J. Biochem. 266, 1192-1201. 

In de novo three-dimensional structure determinations of proteins in solution by NMR spectroscopy, the key conformational data are upper distance limits derived from nuclear Overhauser effects (NOEs) [11, 14]. In order to extract distance constraints from a NOESY spectrum, its cross peaks have to be assigned, i.e. the pairs of hydrogen atoms that give rise to cross peaks have to be identified. The basis for the NOESY assignment... 

Protein-Ligand Interactions Exchange processes and determination of ligand conformation and protein-ligand contacts, 238, 657 nuclear magnetic studies of protein-peptide complexes,... 

The stericaily permitted conformations for various di- and tripeptides are described using mathematical and computer methods. The effects of variations in the size and shape of the side-chain groups on the allowed conformations are assessed. Other factors which are investigated are the effects of possible variations in the geometry of the planar amide backbone and in the van der Waal s contact distances between atoms on the stericaily permitted backbone conformations. The evaluation of the steric restrictions emphasizes their Important role as a determinant in protein conformation. 

Hydrophobic forces The hydrophobic effect is the name given to those forces that cause nonpolar molecules to minimize their contact with water. This is clearly seen with amphipathic molecules such as lipids and detergents which form micelles in aqueous solution (see Topic El). Proteins, too, find a conformation in which their nonpolar side chains are largely out of contact with the aqueous solvent, and thus hydrophobic forces are an important determinant of protein structure, folding and stability. In proteins, the effects of hydrophobic forces are often termed hydrophobic bonding, to indicate the specific nature of protein folding under the influence of the hydrophobic effect. 

Chemical modifications of proteins have been carried out for a long time prior to any interest in the understanding of the chemical basis of the process. Early studies were motivated by the interest in quantitative determination of proteins and amino acids that conform its structure [104]. Intramolecular reactions occur naturally in posttranslational modifications such as disulfide bonding, glycosylation, or terminal residue cyclization. These modifications are relevant in structure-function relationships. They can produce conformational changes in order to switch between... 

Mchaourab, H.S. and Perozo, E. (2000) Determination of proteins folds and conformational dynamics using spin-labeling ESR spectroscopy, in Berliner, L, Eaton, S., and Eaton, G. (eds.),, Magnetic Resonance in Biology, V. 18, Kluwer Academic Publishers. Dordrecht, pp. 185-248. 

At this juncture, it is useful to discuss the experimental methods that are of value in studying and separating the various kinds of interactions in macromolecular systems. A variety of experimental methods have been applied to the determination of protein structure and conformation in solution, and these have been summarized by Kauzmann (1959). In the discussion which follows, emphasis is placed on those methods which have so far been of most use in studies of proteins in nonaqueous solvents, and these remarks should be considered as supplementary to the Kauzmann summary. 

The absorbance of the aromatic amino acids can be used for rapid and reliable determinations of protein concentration, provided that its amino acid composition is known (see Sect. 5.1.2.3). Spectrophotometric determination of nucleic add concentrations requires information not only about base composition but also about conformation, i.e., whether the nucleic acid is single- or double-stranded (see-Sect. 5.2.1). 

Of all the mutant calmodulins studied, the B4K mutant has the most altered a-helical secondary structure, based on the CD signal at 222nm, (Fig. 2A). Values obtained from current samples of 62K and B2Q mutants show somewhat weaker far UV-CD than previously reported, (4), although the absolute values for a given mutant are critically dependent upon determination of protein concentration. When the WFF peptide is added, the CD (222nm) increases for all the proteins, as shown in Fig. 2B. For WT-protein this increase in intensity is interpreted as deriving mainly from the bound peptide adopting an a-helical conformation (2). 

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