Structural analysis of proteins

Direct protein sequencing, by the methods discussed above (Sects. 5.1.5 and 5.1.6), is the preferred route to primary structure. It is now widely accepted that an indirect approach, via the sequence of cloned cDNA is also appropriate, and possibly easier. It does need to be established, however, that the protein primary structure is not affected by tissue specific mRNA processing (splicing or editing) or by post-translational modification such as N- or C-terminal processing or protein splicing. 

Secondary structure is most easily assessed by spectroscopic methods, in particular circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). These techniques can be used to determine the content of a-helices, /3-sheets and, with 

X-ray crystallography and NMR are, of course, the indispensable sources of information about protein tertiary structure at high resolution (Rhodes 1993 McRee 1993  

Information about the domain structure of proteins can be derived from the results of limited proteolysis, since linker regions between domains are often particularly susceptible to protease cleavage. It is advisable to try several proteases with differing specificities, and to follow the kinetics of cleavage by SDS-PAGE. The accumulation of an intermediate is suggestive, but no more than this, of the existence of a stable domain. 

The stoichiometry and molar masses of component subunits in a multi-subunit protein can usually be elucidated from the results of SDS-PAGE and measurements of native molar mass. This does not, however, give any topological information about 

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Applications of Molecular Dynamics for Structural Analysis of Proteins and Peptides... 

Swiderek, K. M. Alpert, A. J. Heckendorf, A. Nugent, K. Patterson, S. D. Structural analysis of proteins and peptides in the presence of detergents Tricks of the trade. ABRF News 1997. 

Gorman, J.J., Corino, G.L., and Mitchell, S.J. (1987) Fluorescent labeling of cysteinyl residues. Application to extensive primary structure analysis of protein on a microscale. Eur. J. Biochem. 168, 169-179. 

Volume III Methods for Structural Analysis of Protein Pharmaceuticals W. Jiskoot, D J.A. Crommelin... 

Like most other approaches to structural analysis of proteins, however, DSC is most valuable when it is not the only source of structural information concerning a given protein. DSC can provide evidence concerning the number of domains, or "cooperative unfolding units" into which a protein is divided, the stabilities of the individual domains. 

H is particularly important in NMR experiments because of its high sensitivity and natural abundance. For macromolecules, 1H NMR spectra can become quite complicated. Even a small protein has hundreds of 1H atoms, typically resulting in a one-dimensional NMR spectrum too complex for analysis. Structural analysis of proteins became possible with the advent of two-dimensional NMR techniques (Fig. 3). These methods allow measurement of distance-dependent coupling of nuclear spins in nearby atoms through space (the nuclear Overhauser effect (NOE), in a method dubbed NOESY) or the coupling of nuclear spins in atoms connected by covalent bonds (total correlation spectroscopy, or TOCSY). 

The 13C chemical shift contour map for the Cp carbon of the L-alanine residue in peptides and polypeptides was made as a function of the dihedral angles(, W) by using the experimental data. Also, the corresponding calculated map was made by using the ab initio coupled Hartree-Fock method with the gauge included atomic orbitals(GIAO-CHF). From these results, it was found that the calculated map explains the chemical shift behavior of the a-helix and p-sheet forms in poly(L-alanine) and some proteins. This suggests that the calculated map is applicable to the structural analysis of proteins with complicated structure. 

From these results, it was found that the calculated map explains well the chemical shift behavior of the a-helix and p-sheet forms in poly(L-Ala) and some proteins. This suggests that the calculated map is applicable to the structural analysis of proteins with complicated structure. 

Ikeya, T., Terauchi, T., Guntert, P. and Kainosho, M. (2006). Evaluation of stereo-array isotope labeling (SAIL) patterns for automated structural analysis of proteins with CYANA. Magn. Reson. Chem. 44, S152-S157. 

Choice of Basis Sets for the Secondary Structure Analysis of Proteins... 

A variety of experimental setups were developed for structure analysis of proteins, based on the excitation of the tryptophan moiety (the most brightly fluorescent proteogenic amino acid residue), that produces an intrinsic fluorescence emission of a folded protein. Tryptophane residues excited at wavelength values around 280-290 nm emit at a characteristic wavelength range (330-350 nm) thus reporting on how much this residue is buried within the protein. Techniques such... 

Attenuated Total Reflectance FTIR (ATR-FTIRf) is a method that has been applied by a number of workers for the study of protein conformation. ATR has been used for monitoring adsorption of proteins or blood components to sur ces (1,2), and for the structural analysis of proteins dried onto an IRE (thin film) (3,4). It has also been used for exploring the effects of solution conditions on the structure of proteins irreversibly adsorbed to an IRE (5,6,7), and has been shown to be usefel for studying the secondary structure and ligand binding properties of membrane proteins (8,9). 

Takakuwa, T. Kurosu, Y. Sakayanagi, N. Kaneuchi, F. Takeuchi, N. Wada, A. Senda, M. Direct combination of a high performance liquid chromatograph and a circular dichroism spectrometer for separation and structural analysis of proteins. J. Liq. Chromatogr. 1987,10, 2759-2769. 

C. Structural Analysis of Protein-Ligand Complexes in Drug Design Examples from Immunophilin Research... 

B. R. Brooks, in Supercomputer Research in Chemistry and Chemical Engineering, ACS Symposium Series 353. K. F. Jensen, and D. G. Truhlar, Eds., American Chemical Society, Washington, D.C., 1987, pp. 123-145. Applications of Molecular Dynamics for Structural Analysis of Proteins and Peptides. R. H. Reid, C. A. Hooper, and B. R. Brooks, Biopolymers, 28, 525 (1989). Computer Simulations of a Tumor Surface Octapeptide Epitope. 

Griffin P R, Coffman J A, Hood L E, et al. (1991). Structural analysis of proteins by capillary HPLC electrospray tandem mass spectrometry. Int. J. Mass Spectrom. 111 131-149. 

Ault-Riche D, Atkinson B, Kumble K D (2005). Methods for Structural analysis of Protein Pharmaceuticals. AAPS Press, Arlington, V.A. pp 545-571. 

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