Secondary Structure of Proteins Through Circular Dichroism Spectroscopy

Johnson, W. Curtis

doi:10.1146/annurev.bb.17.060188.001045

Cited by 553 publications

(354 citation statements)

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“…3A) and a shoulder expanding from 220 to 230 nm. Such spectrum resembles, except for the shoulder, that of a random coil structure (17,18). The spectrum was invariable in the pH range 2.0 -8.5 (data not shown).…”

Section: Circular Dichroism Spectroscopymentioning

confidence: 83%

Human p8 Is a HMG-I/Y-like Protein with DNA Binding Activity Enhanced by Phosphorylation

Encinar¹,

Mallo²,

Mizyrycki³

et al. 2001

Journal of Biological Chemistry

117

147

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We have studied the biochemical features, the conformational preferences in solution, and the DNA binding properties of human p8 (hp8), a nucleoprotein whose expression is affected during acute pancreatitis. Biochemical studies show that hp8 has properties of the high mobility group proteins, HMG-I/Y. Structural studies have been carried out by using circular dichroism (near-and far-ultraviolet), Fourier transform infrared, and NMR spectroscopies. All the biophysical probes indicate that hp8 is monomeric (up to 1 mM concentration) and partially unfolded in solution. The protein seems to bind DNA weakly, as shown by electrophoretic gel shift studies. On the other hand, hp8 is a substrate for protein kinase A (PKA). The phosphorylated hp8 (PKAhp8) has a higher content of secondary structure than the nonphosphorylated protein, as concluded by Fourier transform infrared studies. PKAhp8 binds DNA strongly, as shown by the changes in circular dichroism spectra, and gel shift analysis. Thus, although there is not a high sequence homology with HMG

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Section: Circular Dichroism Spectroscopymentioning

confidence: 83%

Human p8 Is a HMG-I/Y-like Protein with DNA Binding Activity Enhanced by Phosphorylation

Encinar¹,

Mallo²,

Mizyrycki³

et al. 2001

Journal of Biological Chemistry

117

147

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“…Qualitative changes in the CD spectra of the modified samples are clearly indicated, pointing to changes in the protein secondary structure. However, a reliable quantitative estimation of these changes would need CD spectra down to wavelengths of about 185-190 nm [29]. Our calculations using spectra in the restricted wavelength range 207 -250 nm point to a decrease in a-helical structure of about 5% at 92% succinylation, without significant changes in fl-sheet.…”

Section: Spectroscopic Studiesmentioning

confidence: 87%

Changes of the oligomeric structure of legumin from pea (Pisum sativum L.) after succinylation

Schwenke

Zirwer

Gast

et al. 1990

European Journal of Biochemistry

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The influence of various levels of succinylation on the structure of the legumin from pea seed has been studied by the techniques of sedimentation velocity, viscometry, fluorescence and circular dichroism spectroscopy, as well as dynamic light scattering. The protein dissociates gradually into the 3s subunit forming a 7s intermediate. At a level of 75 -80% succinylation, sudden unfolding of the protein occurs characterized by drastic changes in viscometric and spectroscopic properties. The fluorescence spectra point to the formation of a novel organized structure at a moderate degree of modification before the molecular unfolding takes place.The succinylated subunit was shown to have a sedimentation coefficient of 3.2S, a diffusion coefficient of 5.03 x cm2 . s p l a Stokes' radius of 4.24 nm, a partial specific volume of 0.703 ml/g, an intrinsic viscosity of 0.13 dl/g, a molar mass of 52.2 kDa and a frictional ratio of 1.74.Legumin is one of the main storage proteins in the seeds of pea (Pisum sativum L.). It belongs to the group of the socalled l l S globulins, having sedimentation coefficients between lls and 14S, and molecular masses between 300 kDa and 400 kDa [l]. These proteins possess an oligomeric structure characterized by an arrangement of six subunits (3s components) [2 -41. The molecular mass of pea legumin was reported by different authors to be in the range 330 -410 kDa [l, 5, 61. By means of small-angle X-ray scattering, a value of 359 f 25 kDa was obtained [7]. Disulphide-bonded polypeptide pairs with molecular mass 54 kDa constitute most of the 3s subunit of the legumin [8]. In addition, polypeptide pairs varying in molecular mass over 35-58 kDa have been observed which associate in various ways to give rise to different molecular forms of legumin IS].Due to very similar quaternary structures [9, lo], the 11s plant proteins show an analogous behaviour dissociating into subunits [l 11. Therefore, the decay of oligomeric structure by an increase of negative charge caused by succinylation effects similar changes in the structure of different 11s proteins. Although there are specific differences in the dissociation behaviour of the peanut [12], sunflower seed [13] and rape seed [14] 11s globulins, a step-by-step decay of the native structure, and a marked change in conformation at a critical step of succinylation, were observed in each case.The present paper deals with the effect of succinylation on the oligomeric structure of the 11s globulin (legumin) from pea. Structural changes of the protein were followed using ultracentrifugation, viscometry, circular dichroism and fluorescence spectroscopy. The physicochemical properties of the dissociation products, including molecular mass and shape, were investigated by a combination of hydrodynamic methods, including dynamic light scattering. MATERIALS AND METHODSLegumin was isolated and purified according to Gueguen et al. [15]. Lyophilized preparations containing small amounts of aggregated material were purified additionally by gel filtration on a ...

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“…6a). The secondary structure contents were calculated using VARSLC1 (20) with the CD data in the range of 184 -260 nm for each peptide with the exception of S09 (which has very low solubility) and summarized in Table II. Although not obvious in the CD spectra, each peptide forms more or less sheet structures.…”

Section: Biosynthesis Purification and Secondary Structures Of Recomentioning

confidence: 99%

Biosynthesis, Purification, and Substrate Specificity of Severe Acute Respiratory Syndrome Coronavirus 3C-like Proteinase

Fan

Wei

Qian

et al. 2004

Journal of Biological Chemistry

330

422

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The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. In order to understand the active form and the substrate specificity of the enzyme, we have cloned, expressed, and purified SARS 3C-like proteinase. Analytic gel filtration shows a mixture of monomer and dimer at a protein concentration of 4 mg/ml and mostly monomer at 0.2 mg/ml, which correspond to the concentration used in the enzyme assays. The linear decrease of the enzymatic-specific activity with the decrease of enzyme concentration revealed that only the dimeric form is active and the dimeric interface could be targeted for structural-based drug design against SARS 3C-like proteinase. By using a high pressure liquid chromatography assay, SARS 3C-like proteinase was shown to cut the 11 peptides covering all of the 11 cleavage sites on the viral polyprotein with different efficiency. The two peptides corresponding to the two self-cleavage sites are the two with highest cleavage efficiency, whereas peptides with non-canonical residues at P2 or P1 positions react slower. The P2 position of the substrates seems to favor large hydrophobic residues. Secondary structure studies for the peptide substrates revealed that substrates with more ␤-sheetlike structure tend to react fast. This study provides a basic understanding of the enzyme catalysis and a full substrate specificity spectrum for SARS 3C-like proteinase, which are helpful for structural-based inhibitor design against SARS and other coronavirus.The outbreak of a severe atypical pneumonia in early 2003 has caused 8422 cases and 916 related deaths. The World Health Organization has designated the illness as severe acute respiratory syndrome (SARS).1 A novel form of coronavirus has been identified as the major cause of SARS (1, 2). The genome of SARS coronavirus has been sequenced within a short period of time after confirmation of the virus (3, 4). Currently, 23 genome sequences of different variations of SARS coronavirus have been released at the NCBI web site (www.ncbi.nlm.nih. gov/). Coronaviruses are members of positive-stranded RNA viruses featuring the largest viral RNA genomes up to date. The SARS coronavirus replicase gene encompasses two overlapping translation products, polyproteins 1a (ϳ450 kDa) and 1ab (ϳ750 kDa), which are conserved both in length and amino acid sequence to other coronavirus replicase proteins. Polyproteins 1a and 1ab are cleaved by the internally encoded 3C-like proteinase to release functional proteins necessary for virus replication. The SARS 3C-like proteinase is fully conserved among all of the released SARS coronavirus genome sequences and is highly homologous with other coronavirus 3C-like proteinase.Two crystal structures of coronavirus 3C-like proteinase from transmissible gastroenteritis virus (TGEV) (5) and human coronavirus (hCoV) 229E have been solved (6). The structure of coronavirus 3C-like proteinase contains three domains. The first two domains form ...

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Secondary Structure of Proteins Through Circular Dichroism Spectroscopy

Cited by 553 publications

References 0 publications

Human p8 Is a HMG-I/Y-like Protein with DNA Binding Activity Enhanced by Phosphorylation

Human p8 Is a HMG-I/Y-like Protein with DNA Binding Activity Enhanced by Phosphorylation

Changes of the oligomeric structure of legumin from pea (Pisum sativum L.) after succinylation

Biosynthesis, Purification, and Substrate Specificity of Severe Acute Respiratory Syndrome Coronavirus 3C-like Proteinase

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