Content secondary-structure

These sources of error in estimating secondary structure content from CD and FTIR spectra suggest that the estimates, especially for amyloid fibrils, should be considered qualitative. 

There is a correlation between the backbone conformations which commonly flank disulfides and the frequency with which disulfides occur in the different types of overall protein structure (see Section III,A for explanation of structure types), although it is unclear which preference is the cause and which the effect. There are very few disulfides in the antiparallel helical bundle proteins and none in proteins based on pure parallel /3 sheet (except for active-site disulfides such as in glutathione reductase). Antiparallel /3 sheet, mixed /8 sheet, and the miscellaneous a proteins have a half-cystine content of 0-5%. Small proteins with low secondary-structure content often have up to 15-20% half-cystine. Figure 52 shows the structure of insulin, one of the small proteins in which disulfides appear to play a major role in the organization and stability of the overall structure. 

Table 5. Secondary-structure content of the wild type and foiu" mutant enzymes...

Name RMSB Energy Secondary Structure Content... 

Table 1. Energies (in kcal/mol) of the 10 lowest energy decoys obtained in the basin hopping simulations of the HIV accessory protein. The table shows the backbone RMS deviation to the NMR structure and secondary structure content. The first row designates the secondary structure content of the NMR structure.

P17G <7> (<7> alters distribution of multiple conformations, lowered secondary structure content, poorer affinity to substrates, reduced catalytic efficiency [52]) [52]... 

The protein concentration should be as high as possible, consistent with the absorbance in the cell not exceeding 1.0 to 1.5, and must be accurately determined by UV absorbance. Values of mean residue ellipticity and secondary structure content results are both affected by uncertainties in protein concentration. 

In conclusion, the analysis of spectra properly recorded to 185 nm, or lower where possible, can give useful estimates of secondary structure content, but the content of turns and of P-structure should be interpreted with caution. Fourier transform infrared spectroscopy (FTIR) provides better estimates of the latter. When using the results of far-UV CD determination to characterize reproducibility of folding for different samples, it is important first to compare the spectra visually and to look for possible trends or factors that may explain small differences, rather than to rely solely on comparison of derived secondary structure contents. 

Manning, M.C. 1989. Underlying assumptions in the estimation of secondary structure content in proteins by circular dichroism spectroscopy—a critical review. J. Pharmacol. Biomed. Anal. 7 110.3-1119. 

A good account of the practice offar-UV CD spectroscopy and a description and assessment of the determination of protein secondary structure content. 

Conformational changes are detected in the near-UV CD spectrum if they affect the conformation or environment of side chain aromatic residues. On the other hand, they affect the far-UV CD spectrum only if substantial changes in secondary structure content occur. Larger amounts of proteins are required for near-UV CD than for far-UV CD spectroscopy given that absorptions in the former are at least an order of magnitude smaller. Because the pH-dependent conformational changes in BmPBP involve aromatic side chains, possibly tryptophan, they were also mirrored in fluorescence spectroscopy. This is fortuitous given that fluorescence requires smaller amounts of protein, which can consequently be obtained from natural sources. It is important to point out that for such experiments, however, proteins must be scrupulously pure and devoid of other chromophores. 

Table 10. Secondary structure content (%) in penicillolysin, apoenzyme and...

Both approaches are empirical. They depend on comparing unknown spectra with spectra which represent presumably known structures. They give relatively accurate percentages of helix, P-sheet, reverse turn, and unfolded structure, but quantitate only the average secondary structure content [108]. The relative success of these spectroscopic methods gives confidence that more detailed information about specific vibrational characteristics of peptides and proteins will provide valuable and useful contributions to the study of these problems. The developments... 

Table 10.1 summarizes neural network applications for protein structure prediction. Protein secondary structure prediction is often used as the first step toward understanding and predicting tertiary structure because secondary structure elements constitute the building blocks of the folding units. An estimated 90% or so of the residues in most proteins are involved in three classes of secondary structures, the a-helices, p-strands or reverse turns. Related to the secondary structure prediction are also the prediction of solvent accessibility, transmembrane helices, and secondary structure content (10.2). Neural networks have also been applied to protein tertiary structure prediction, such as the prediction of the backbones or side-chain packing, and to structural class prediction (10.3). 

Muskal, S. M. Kim, S.-H. (1992). Predicting protein secondary structure content. A tandem neural network approach. J Mol Biol 225,713-27. 

The nature of the unfolded state in denaturant and how it relates to the denatured state under native conditions in the bilayer is a major issue in all denaturation experiments. Thermodynamic arguments from the two-stage model suggest that the relevant denatured state has lost its tertiary structure and maintained the transmembrane helix secondary structure. As noted above, CD spectra on thermally denatured bacteriorhodopsin suggest that the denatured protein maintains most of its helical secondary structure. The extent to which tertiary structure is disrupted is unclear, however. It is possible that some stable interhelical interactions are maintained even at high temperature. The helical secondary structure content is also maintained in SDS micelles, and near-UV circular dichroism (CD) spectra suggest substantial loss or... 

Far UV circular dichroism spectra contain information about secondary structure content in proteins and peptides. Small peptides are almost invariably unstructured in aqueous solution, but can adopt regular secondary structure upon binding to a protein. In general, the secondary structure of a protein is unlikely to change dramatically upon binding to a peptide. However, as shown in this work, the binding of a target peptide to a mutated protein with an altered structure (compared with the wildtype) may partially restore the "native" structure of the protein. 

FSD spectra are frequently curve-fit to obtain an estimate of the secondary structure content of the protein being examined. This is justifiable because, in theory, Fourier self-deconvolution should not affect the relative areas of component bands. In practice however, it was found that this assumption is not valid. The relative areas of bands at the edges of the amide I region are increased by FSD. Therefore the following procedure was used for structural analysis. 

Finally, the secondary structure content was evaluated by calculating the relative areas of all component bands. Such quantification of structural content is based on the assumption that the extinction coefficients for all structural types are similar. The band assignments used have been adapted from other studies (13, 16, 17). 

Although a qualitative visual comparison of second-derivative spectra can be useful to assess the influence of additives on protein structure during lyophilization, a quantitative comparison is often also desirable. For research on lyophilization-induced structural transitions two approaches can be employed. Occasionally, there is a need to know the secondary structural content. Then, the relative band areas can be determined with curve fitting (see [ 11,50-52,54]). For example, the percentage of intermolecular P sheet can be used to calculate the percentage of aggregated protein in dried samples [11,14]. 

Combined SAXS/Circular dichroism beamline. Biological macromolecules, such as proteins, carbohydrates and nucleic acids, are composed of many optically active or chiral units that exhibit large Circular Dichroism (CD) signals. CD spectroscopy has therefore been used extensively in the study of proteins, where asymmetric carbon atoms in their amino acid backbone give rise to a CD spectrum. The shape of the spectrum depends on the protein s secondary structure content and allows the proportions of helix, beta structure, turns and random to be determined. 

CD is an excellent method for determining the secondary structure content of proteins in their native state, but it is limited by the fact that much of the information is located at wavelengths (below 200 nm) where the light output from conventional Xenon lamps diminishes markedly. In contrast, the flux obtained from synchrotron light remains high at these wavelengths. Also, the inherent polarisation of synchrotron radiation makes it the ideal light source for CD experiments. 

Gene finding is difficult and, as may be expected, not all approaches give the desired results. This has given rise to papers describing methods that do not work. The NCRNASCAN paper by Rivas and Eddy [43] is a good example. Contrary to the authors expectations, real RNAs do not generally have any more secondary-structure content than random sequence [43],... 

Figure 4.7 CD spectra of polypeptides in pure conformations as indicated (left). These form a typical basis set for the deduction of secondary structure content in a protein sample of interest by mathematical analysis of the recorded CD spectrum (right).

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