Identifying Protein Structures

Growing a protein crystal is a tedious and often not successful process. 

If a protein crystal could be grown, it is usually small and fragile and contains solvents and impurities that barely can be separated. 

If a good crystal can be obtained, the crystal structure does usually not reflect the shape of the protein in its natural environment. 

If a diffraction pattern can be obtained, it contains information only about the intensity of the diffracted wave the phase information cannot be experimentally determined and must be estimated by other means (usually referred to as the phase problem). 

The selection of points from which intensities are collected is limited, leading to a limited resolution, which reduces the differentiation between atoms. 

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Interest centers on the extent to which the reported crystal structures of individual glycosylascs (a) arc able to identify an active-site residue which, by nature and spatial disposition, might serve to protonate a bound enolic substrate or wrong glycosyl fluoride anomer and (b) are able to identify protein structural features that, could dictate the direction of approach of acceptor cosubstratcs to the reaction center. 

Fig, 10.25 The six environment categories used by the 3D profiles method. (Figure adapted from Bowie j U, R Liith. and D Eisenberg 1991. A Method to Identify Protein Sequences That Fold into a Known Three-Dunensinnal Structure. Science 253 164-170.)... 

Ithough knowledge-based potentials are most popular, it is also possible to use other types potential function. Some of these are more firmly rooted in the fundamental physics of iteratomic interactions whereas others do not necessarily have any physical interpretation all but are able to discriminate the correct fold from decoy structures. These decoy ructures are generated so as to satisfy the basic principles of protein structure such as a ose-packed, hydrophobic core [Park and Levitt 1996]. The fold library is also clearly nportant in threading. For practical purposes the library should obviously not be too irge, but it should be as representative of the different protein folds as possible. To erive a fold database one would typically first use a relatively fast sequence comparison lethod in conjunction with cluster analysis to identify families of homologues, which are ssumed to have the same fold. A sequence identity threshold of about 30% is commonly... 

The techniques described thus far cope well with samples up to 10 kDa. Molecular mass determinations on peptides can be used to identify modifications occurring after the protein has been assembled according to its DNA code (post-translation), to map a protein structure, or simply to confirm the composition of a peptide. For samples with molecular masses in excess of 10 kDa, the sensitivity of FAB is quite low, and such analyses are far from routine. Two new developments have extended the scope of mass spectrometry even further to the analysis of peptides and proteins of high mass. 

A. Identifying Known Protein Structures Related to the Target Sequence... 

JU Bowie, R Liithy, D Eisenberg. A method to identify protein sequences that fold into a known three-dimensional structure. Science 253 164-170, 1991. 

Similar residues in the cores of protein structures especially hydrophobic residues at the same positions, are responsible for common folds of homologous proteins. Certain sequence profiles of conserved residue successions have been identified which give rise to a common fold of protein domains. They are organized in the smart database (simple modular architecture research tool) http //smait.embl-heidelberg.de. 

A new chapter on the primary structure of proteins, which provides coverage of both classic and newly emerging proteomic and genomic methods for identifying proteins. A new section on the appHcation of mass spectrometry to the analysis of protein structure has been added, including comments on the identification of covalent modifications. 

If cellular redox state, determined by the glutathione status of the heart, plays a role in the modulation of ion transporter activity in cardiac tissue, it is important to identify possible mechanisms by which these effects are mediated. Protein S-,thiolation is a process that was originally used to describe the formation of adducts of proteins with low molecular thiols such as glutathione (Miller etal., 1990). In view of the significant alterations of cardiac glutathione status (GSH and GSSG) and ion-transporter activity during oxidant stress, the process of S-thiolation may be responsible for modifications of protein structure and function. 

Methods that utilize structural data of the target, generally identified by protein crystallography, to look for molecules that complement the binding site through favorable protein-ligand interactions (protein structure-based VS or SBVS). 

Because protein ROA spectra contain bands characteristic of loops and turns in addition to bands characteristic of secondary structure, they should provide information on the overall three-dimensional solution structure. We are developing a pattern recognition program, based on principal component analysis (PCA), to identify protein folds from ROA spectral band patterns (Blanch etal., 2002b). The method is similar to one developed for the determination of the structure of proteins from VCD (Pancoska etal., 1991) and UVCD (Venyaminov and Yang, 1996) spectra, but is expected to provide enhanced discrimination between different structural types since protein ROA spectra contain many more structure-sensitive bands than do either VCD or UVCD. From the ROA spectral data, the PCA program calculates a set of subspectra that serve as basis functions, the algebraic combination of which with appropriate expansion coefficients can be used to reconstruct any member of the... 

Because of this length dependence, IR and VCD of -turns may provide a means of discriminating and detecting them in a protein structure. Short Aib peptide results provide examples of type III /3-turn VCD (Yasui et al., 1986b), while cyclic peptides have been used to study type I and II turns, which are implied to have unique VCD band shapes (Wyssbrod and Diem, 1992 Xie et al., 1995). We have identified distinct turn modes... 

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