Protein secondary structure and circular dichroism: A practical guide

Johnson, W. Curtis

doi:10.1002/prot.340070302

Cited by 962 publications

(755 citation statements)

References 22 publications

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“…If dissolved in 30% aqueous TFE at pH 7.0, RNase acquires a slightly enhanced content of a-helix with respect to that of the protein in buffer alone. In fact, the intensity of the negative ellipticity in the 200-250-nm region of RNase in aqueous TFE is greater than that of the protein in buffer only, and shows characteristics typical of helical polypeptides with minima of ellipticity near 208 and 222 nm (Greenfield & Fasman, 1969;Johnson, 1990). The enhanced content of helicity is much more evident if the protein is dissolved in 50% TFE (Fig.…”

Section: Measurementsmentioning

confidence: 92%

“…Between 200 and 250 nm, the CD spectrum of RNase Thl is very similar to that of the intact protein, indicating that the secondary structure of the protein is unchanged after fission of the peptide bond Asn 34-Leu 35. On the other hand, the CD spectrum of RNase Th2, with two peptide bonds broken, is largely changed with respect to that of the intact protein and displays features most typical of a random-coil polypeptide (Greenfield & Fasman, 1969;Johnson, 1990).…”

Section: Nicked Rnasementioning

confidence: 99%

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Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

et al. 1997

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We have examined the proteolysis of bovine pancreatic ribonuclease A (RNase) by thermolysin when dissolved in aqueous buffer, pH 7.0, in the presence of 50% (v/v) trifluoroethanol (TFE). Under these solvent conditions, RNase acquires a conformational state characterized by an enhanced content of secondary structure (helix) and reduced tertiary structure, as given by CD measurements. It was found that the TFE-resistant thermolysin, despite its broad substrate specificity, selectively cleaves the 124-residue chain of RNase in its TFE state (20-42"C, 6-24 h) at peptide bond Asn 34-Leu 35, followed by a slower cleavage at peptide bond Thr 45-Phe 46. In the absence of TFE, native RNase is resistant to proteolysis by thermolysin. Two nicked RNase species, resulting from cleavages at one or two peptide bonds and thus constituted by two (1-34 and 35-124) (RNase Thl) or three (1-34, 35-45 and 46-124) (RNase Th2) fragments linked covalently by the four disulfide bonds of the protein, were isolated to homogeneity by chromatography and characterized. CD measurements provided evidence that RNase Thl maintains the overall conformational features of the native protein, but shows a reduced thermal stability with respect to that of the intact species (-ATm 16 "C); RNase Th2 instead is fully unfolded at room temperature. That the structure of RNase Thl is closely similar to that of the intact protein was confirmed unambiguously by two-dimensional NMR measurements. Structural differences between the two protein species are located only at the level of the chain segment 30-41, i.e., at residues nearby the cleaved Asn 34-Leu 35 peptide bond. RNase Thl retained about 20% of the catalytic activity of the native enzyme, whereas RNase Th2 was inactive. The 3 1-39 segment of the polypeptide chain in native RNase forms an exposed and highly flexible loop, whereas the 41-48 region forms a &strand secondary structure containing active site residues. Thus, the conformational, stability, and functional properties of nicked RNase Thl and Th2 are in line with the concept that proteins appear to tolerate extensive structural variations only at their flexible or loose parts exposed to solvent. We discuss the conformational features of RNase in its TFE-state that likely dictate the selective proteolysis phenomenon by thermolysin.Keywords: CD; limited proteolysis; NMR; ribonuclease A; thermolysin; trifluoroethanol Short-chain alcohols, such as methanol, ethanol, propanol, chloroethanol, and, in particular, trifluoroethanol, have been used frequently to induce a-helical conformations in otherwise randomly coiled or partially structured polypeptides (Toniolo et al., 1975;

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Section: Measurementsmentioning

confidence: 92%

Section: Nicked Rnasementioning

confidence: 99%

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

et al. 1997

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“…4, left panel) shows a strong negative ellipticity centered at 217 nm. This CD band is characteristic of &sheet conformation (Greenfield & Fasman, 1969;Johnson, 1990;Waterhous & Johnson, 1994). At 0.13 m M , where…”

Section: Conformational Analysismentioning

confidence: 99%

A folding pathway for βpep‐4 peptide 33mer: From unfolded monomers and β‐sheet sandwich dimers to well‐structured tetramers

Mayo

Ilyina

1998

Protein Science

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It was recently reported that a de novo designed peptide 33mer, Ppep-4, can form well-structured P-sheet sandwich tetramers (Ilyina E, Roongta V, Mayo KH, 1997b, Biochemistry 36:5245-5250). For insight into the pathway of Ppep-4 folding, the present study investigates the concentration dependence of Ppep-4 self-association by using 'H-NMR pulsedfield gradient (PFG)-NMR diffusion measurements, and circular dichroism. Downfield chemically shifted a H resonances, found to arise only from the well-structured Ppep-4 tetramer state, yield the fraction of tetramer within the oligomer equilibrium distribution. PFG-NMR-derived diffusion coefficients, D, provide a means for deriving the contribution of monomer and other oligomer states to this distribution. These data indicate that tetramer is the highest oligomer state formed, and that inclusion of monomer and dimer states in the oligomer distribution is sufficient to explain the concentration dependence of D values for /.?pep-4. Equilibrium constants calculated from these distributions [2.5 X lo5 M" for M-D and 1.2 X lo4 M-' for D-T at 31 3 K] decrease only slightly, if at all, with decreasing temperature indicating a hydrophobically mediated, entropy-driven association/folding process. Conformational analyses using NMR and CD provide a picture where "random coil" monomers associate to form molten globule-like P-sheet sandwich dimers that further associate and fold as well-structured tetramers. /.?pep-4 folding is thermodynamically linked to self-association. As with folding of singlechain polypeptides, the final folding step to well-structured tetramer ppep-4 is rate limiting.

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“…Far-UV (190-260 nm) circular dichroism (CD) spectra were measured with a Jasco J-715 spectrometer [32]. The CD experiment was performed at 0.5 nm intervals, in a 0.1 cm quartz cuvette, at 20°C.…”

Section: Circular Dichroism Spectrometrymentioning

confidence: 99%

“…CD is useful for the determination of proteins' secondary structure, as it allows the researcher to visually verify the diVerential manner in which a protein absorbs left-and right-hand circular polarized lights [32]. To examine the compositions of the secondary structures of the BubR1 constructs, we obtained the CD spectra of constructs 1-286 and 1-320.…”

Section: Circular Dichroism For Bubr1 Constructsmentioning

confidence: 99%

Identification and purification of a soluble region of BubR1: A critical component of the mitotic checkpoint complex

Yoon

Kang

Kim

et al. 2005

Protein Expression and Purification

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The mitotic checkpoint complex (MCC) ensures the Wdelity of chromosomal segregation, by delaying the onset of anaphase until all sister chromatids have been properly attached to the mitotic spindle. In essence, this MCC-induced delay is achieved via the inhibition of the anaphase-promoting complex (APC). Among the components of the MCC, BubR1 plays two major roles in the functions of the mitotic checkpoint. First, BubR1 is able to inhibit APC activity, either by itself or as a component of the MCC, by sequestering a APC coactivator, known as Cdc20. Second, BubR1 activates mitotic checkpoint signaling cascades by binding to the centromere-associated protein E, a microtubule motor protein. Obtaining highly soluble BubR1 is a prerequisite for the study of its structure. BubR1 is a multi-domain protein, which includes a KEN box motif, a mad3-like region, a Bub3 binding domain, and a kinase domain. We obtained a soluble BubR1 construct using a three-step expression strategy. First, we obtained two constructs from BLAST sequence homology searches, both of which were expressed abundantly in the inclusion bodies. We then adjusted the lengths of the two constructs by secondary structure prediction, thereby generating partially soluble constructs. Third, we optimized the solubility of the two constructs by either chopping or adding a few residues at the C-terminus. Finally, we obtained a highly soluble BubR1 construct via the Escherichia coli expression system, which allowed for a yield of 10.8 mg/L culture. This report may provide insight into the design of highly soluble constructs of insoluble multi-domain proteins.  2005 Elsevier Inc. All rights reserved.Keywords: BubR1; Structure; Anaphase-promoting complex In the eukaryotic cell cycle, mitosis consists of six sequential phases: prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis. During metaphase, two sister chromatids align in the equatorial plane of the dividing cell, while mitotic spindles attach themselves to the kinetochores in the centromeres of the sister chromatids. The mitotic spindle-attached sister chromatids are then separated, and pulled into each spindle pole, thereby completing chromosomal duplication. The mitotic checkpoint (also called the spindle assembly checkpoint) ensures the Wdelity of this chromosomal segregation, by delaying the onset of anaphase until all sister chromatids have been properly attached to the mitotic spindle [1]. Bub3 [5], and BubR1 [6,7] are some characteristic mitotic checkpoint proteins. These proteins associate with the unattached kinetochores and are involved in the delay in the metaphase/ anaphase transition [4,5,[7][8][9]. In essence, this delay occurs due to inhibition of the anaphase-promoting * Corresponding author. Fax: +82 2 741 7947.

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Protein secondary structure and circular dichroism: A practical guide

Cited by 962 publications

References 22 publications

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

A folding pathway for βpep‐4 peptide 33mer: From unfolded monomers and β‐sheet sandwich dimers to well‐structured tetramers

Identification and purification of a soluble region of BubR1: A critical component of the mitotic checkpoint complex

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