Estimation Of Secondary Structure Content Research Articles

In Situ Protein Secondary Structure Determination in Ice: Raman Spectroscopy-Based Process Analytical Tool for Frozen Storage of Biopharmaceuticals

A Raman spectroscopy-based method for in situ monitoring of secondary structural composition of proteins during frozen and thawed storage was developed. A set of reference proteins with different α-helix and β-sheet compositions was used for calibration and validation in a chemometric approach. Reference secondary structures were quantified with circular dichroism spectroscopy in the liquid state. Partial least squares regression models were established that enable estimation of secondary structure content from Raman spectra. Quantitative secondary structure determination in ice was accomplished for the first time and correlation with existing (qualitative) protein structural data from the frozen state was achieved. The method can be used in the presence of common stabilizing agents and is applicable in an industrial freezer setup. Raman spectroscopy represents a powerful, noninvasive, and flexibly applicable tool for protein stability monitoring during frozen storage.

CAPITO—a web server-based analysis and plotting tool for circular dichroism data

Circular dichroism (CD) spectroscopy is one of the most versatile tools to study protein folding and to validate the proper fold of purified proteins. Here, we aim to provide a readily accessible, user-friendly and platform-independent tool capable of analysing multiple CD datasets of virtually any format and returning results as high-quality graphical output to the user. CAPITO (CD Anaylsis and Plotting Tool) is a novel web server-based tool for analysing and plotting CD data. It allows reliable estimation of secondary structure content utilizing different approaches. CAPITO accepts multiple CD datasets and, hence, is well suited for a wide application range such as the analysis of temperature or pH-dependent (un)folding and the comparison of mutants. http://capito.nmr.fli-leibniz.de. cwiede@fli-leibniz.de or mago@fli-leibniz.de Supplementary data are available at Bioinformatics online.

Structural and Functional Characterization of Recombinant Matrilin-3 A-domain and Implications for Human Genetic Bone Diseases

Mutations in matrilin-3 result in multiple epiphyseal dysplasia, which is characterized by delayed and irregular bone growth and early onset osteoarthritis. The majority of disease-causing mutations are located within the beta-sheet of the single A-domain of matrilin-3, suggesting that they disrupt the structure and/or function of this important domain. Indeed, the expression of mutant matrilin-3 results in its intracellular retention within the rough endoplasmic reticulum of cells, where it elicits an unfolded protein response. To understand the folding characteristics of the matrilin-3 A-domain we determined its structure using CD, analytical ultracentrifugation, and dual polarization interferometry. This study defined novel structural features of the matrilin-3 A-domain and identified a conformational change induced by the presence or the absence of Zn(2+). In the presence of Zn(2+) the A-domain adopts a more stable "tighter" conformation. However, after the removal of Zn(2+) a potential structural rearrangement of the metal ion-dependent adhesion site motif occurs, which leads to a more "relaxed" conformation. Finally, to characterize the interactions of the matrilin-3 A-domain we performed binding studies on a BIAcore using type II and IX collagen and cartilage oligomeric matrix protein. We were able to demonstrate that it binds to type II and IX collagen and cartilage oligomeric matrix protein in a Zn(2+)-dependent manner. Furthermore, we have also determined that the matrilin-3 A-domain appears to bind exclusively to the COL3 domain of type IX collagen and that this binding is abolished in the presence of a disease causing mutation in type IX collagen.

Estimation of protein secondary structure content directly from NMR spectra using an improved empirical correlation with averaged chemical shift

We have recently shown that the averaged chemical shift (ACS) of a nucleus in the protein backbone correlates well empirically to its secondary structure content (SSC). This allows the estimation of SSC directly from the NMR spectrum without the time intensive process of chemical shift assignment. Here, we present an empirical correlation that accounts both for contributions to the relevant protein and chemical shift databases made subsequent to the original analysis, and for missing or inconsistently referenced resonances. Our results affirm that this method provides a significant tool for initial structural prediction from NMR data prior to complete chemical shift assignment.

Secondary structure of the mammalian 70-kilodalton heat shock cognate protein analyzed by circular dichroism spectroscopy and secondary structure prediction

Heat shock proteins are rapidly synthesized when cells are exposed to stressful agents that cause protein damage. The 70-kDa heat shock induced proteins and their closely related constitutively expressed cognate proteins bind to unfolded and aberrant polypeptides and to hydrophilic peptides. The structural features of the 70-kDa heat shock proteins that confer the ability to associate with diverse polypeptides are unknown. In this study, we have used circular dichroism (CD) spectroscopy and secondary structure prediction to analyze the secondary structure of the mammalian 70-kDa heat shock cognate protein (hsc 70). The far-ultraviolet CD spectrum of hsc 70 indicates a large fraction of alpha-helix in the protein and resembles the spectra one obtains from proteins of the alpha/beta structural class. Analysis of the CD spectra with deconvolution methods yielded estimates of secondary structure content. The results indicate about 40% alpha-helix and 20% aperiodic structure within hsc 70 and between 16-41% beta-sheet and 21-0% beta-turn. The Garnier-Osguthorpe-Robson method of secondary structure prediction was applied to the rat hsc 70 amino acid sequence. The predicted estimates of alpha-helix and aperiodic structure closely matched the values derived from the CD analysis, whereas the predicted estimates of beta-sheet and beta-turn were midway between the CD-derived values. Present evidence suggests that the polypeptide ligand binding domain of the 70-kDa heat shock protein resides within the C-terminal 160 amino acids [Milarski, K. L., & Morimoto, R. I. (1989) J. Cell Biol. 109, 1947-1962].(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of methionine oxidation and deletion of amino-terminal residues on the conformation of parathyroid hormone. Circular dichroism studies.

Circular dichroism (CD) studies of parathyroid hormone (PTH), its oxidized forms, and some fragments of the hormone are described. The CD spectrum of native PTH (84 amino acids) and the active fragment, 1-34 PTH, suggests that most of the secondary structure resides in the amino-terminal segment of this hormone. Oxidation of the methionine residue at position 18 has a small impact on secondary structure, whereas oxidation of the methionine at position 8 produces substantial changes. Oxidation of both methionines produces secondary structure changes that are greater than the sum of those seen upon oxidation of the individual methionines. The CD spectrum for the 3-34 fragment of PTH is identical to that of the 1-34 fragment, and that of the 7-34 fragment is only slightly different. The spectra of the 13-34 and 19-34 fragments are markedly altered from that of the 1-34 peptide, and those of the 9-84 and 19-84 fragments of native PTH are significantly different from the intact hormone. Computer-assisted estimates of secondary structure content, and difference spectra, were utilized to evaluate the secondary structure content of the peptides. These results suggest that residues 6-12 are important in formation of helical secondary structure and that a reverse turn may be important for the folding of PTH into a conformation with high affinity for receptors. Residues 1 and 2 appear to make no contribution to the secondary structure and may be directly involved in activation of receptors.