[27] Circular dichroism and optical rotatory dispersion of proteins and polypeptides

Resistin, a small cysteine rich protein secreted by adipocytes, has been proposed to be a link between obesity and type II diabetes by modulating the insulin signaling pathway and thus inducing insulin resistance. Resistin protein, with 11 cysteine residues, was not significantly homologous at the amino acid level to any other known cysteine rich proteins. Resistin cDNA derived from human subcutaneous adipose tissue was expressed in Escherichia coli as an N-terminal six-His-tag fusion protein.The overexpressed recombinant resistin was purified to homogeneity from inclusion bodies, after solubilization in 8 M urea, using a metal affinity column. While MALDI-TOF mass spectrometric analysis of the purified protein generated a single peak corresponding to the estimated size of 11.3 kDa, the protein exhibited a concentration-dependent oligomerization which is evident from size exclusion chromatography. The oligomeric structure was SDS-insensitive but -mercaptoethanol-sensitive, pointing to the importance of disulfide linkages in resistin oligomerization. Estimation of free cysteine residues using the NBD-Cl assay revealed a concentration-and time-dependent increase in the extent of formation of disulfide linkages. The presence of intermolecular disulfide bond(s), crucial in maintaining the global conformation of resistin, was further evident from fluorescence emission spectra. Circular dichroism spectra revealed that recombinant resistin has a tendency to reversibly convert from R-helical to -sheet structure as a direct function of protein concentration. Our novel observations on the biophysical and biochemical features of human resistin, particularly those shared with prion proteins, may have a bearing on its likely physiological function.

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“…Spectra were signal averaged for four accumulations and baseline corrected by subtracting the spectra of the respective blank (22).…”

Section: Methodsmentioning

confidence: 99%

Human Recombinant Resistin Protein Displays a Tendency To Aggregate by Forming Intermolecular Disulfide Linkages

et al. 2003

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“…The solution was placed in a quartz cell with a path length of 0.2 cm. The mean residue molar ellipticity of each polymer was calculated on the basis of the measured apparent ellipticity by following the literature-reported formulas: Ellipticity ([θ] in cm 2 deg dmol − 1 ) = (millidegrees×mean residue weight)/(path length in millimetres×concentration of polypeptide in mg ml − 1 ) 39,40 . For the temperature-dependent experiments, the temperature of the sample chamber (where the quartz cell was placed) was controlled by a water bath (from 4 °C to 70 °C).…”

Section: Synthesis Of Pvblg-x (mentioning

confidence: 99%

Ionic polypeptides with unusual helical stability

et al. 2011

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Water-soluble peptides that adopt stable helical conformations are attractive motifs because of their importance in basic science and their broad utility in medicine and biotechnology. Incorporating charged amino-acid residues to improve peptide solubility, however, usually leads to reduced helical stability because of increased side-chain charge repulsion, reduced side-chain hydrophobicity and the disruption of intramolecular hydrogen bonding. Here, we show that water-soluble, ultra-stable α-helical polypeptides can be produced by elongating charge-containing amino-acid side chains to position the charges distally from the polypeptide backbone. The strategy has been successfully applied to the design and synthesis of watersoluble polypeptides bearing long, charged side chains and various functional moieties that possess unusual helical stability against changing environmental conditions, including changes in the pH and temperature and the presence of denaturing reagents.

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“…In addition to the far-UV CD spectra, which monitor a protein's secondary structural content, we also measured the near-UV (250-350 nm) CD spectra, which can be used to monitor a protein's tertiary structure in the vicinity of the aromatic amino acid residues Tyr, Trp, and Phe (Adler et al, 1973). The near-UV CD spectrum can be sensitive to small changes in tertiary structure due to protein-protein interactions and/or changes in solvent conditions.…”

Section: Spectra Analysis Of Mammentioning

confidence: 99%

Mutagenesis, biochemical, and biophysical characterization of Mycoplasma arthritidis-derived mitogen

Li¹,

Zhao²,

Guo³

et al. 2007

Molecular Immunology

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Mycoplasma arthritidis -derived mitogen (MAM) is a superantigen (SAg) that can activate large fractions of T cells bearing particular TCR Vβ elements. Here we report the mutagenesis, biochemical and biophysical studies on the dimerization of MAM in solution. Our studies showed that although MAM mainly exists as a monomer in solution, a small percentage of MAM molecules form homodimer at high protein concentration, regardless of the presence of Zn 2+ . A distinct peak corresponding to a MAM homodimer was detected in the presence of EDTA, using both chemical cross-linking and analytical ultracentrifugation methods. Further mutagenesis studies revealed that single mutation of residues at the interface of the crystallographic dimer of MAM does not significantly affect the dimerization of MAM in solution. Circular dichroism (CD) analysis indicated that addition of Zn 2+ does not induce conformational changes of MAM from its apo-state. Thermal denaturation experiments indicated that addition of Zn 2+ to MAM solution resulted in a decrease of melting point (T m ), whereas addition of EDTA did not affect the T m of MAM. These results imply that there is no defined Zn 2+ -binding site on MAM.

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[27] Circular dichroism and optical rotatory dispersion of proteins and polypeptides

Cited by 591 publications

References 228 publications

Human Recombinant Resistin Protein Displays a Tendency To Aggregate by Forming Intermolecular Disulfide Linkages

Human Recombinant Resistin Protein Displays a Tendency To Aggregate by Forming Intermolecular Disulfide Linkages

Ionic polypeptides with unusual helical stability

Mutagenesis, biochemical, and biophysical characterization of Mycoplasma arthritidis-derived mitogen

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