Estimation of the number of α‐helical and β‐strand segments in proteins using circular dichroism spectroscopy

Sreerama, Narasimha; Venyaminov, Sergei Yu.; Woody, Robert W.

doi:10.1110/ps.8.2.370

Cited by 687 publications

(474 citation statements)

References 34 publications

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“…The important features of the KIS landscape lie in its kinetic connectivity between adjacent states as well as in the inclusion of trajectories originating from the misfolded β-hairpin-like intermediates for the determination of the rates involved. These intermediates may be probed by optical techniques such as nanosecond (39) CD spectroscopy, which enables the measurement of the contribution of the α-helix, β-strand, and random-coil in the ensemble (40).…”

Section: Resultsmentioning

confidence: 99%

Dominance of misfolded intermediates in the dynamics of α-helix folding

Lin

Shorokhov

Zewail

2014

Proc. Natl. Acad. Sci. U.S.A.

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Helices are the "hydrogen atoms" of biomolecular complexity; the DNA/RNA double hairpin and protein α-helix ubiquitously form the building blocks of life's constituents at the nanometer scale. Nevertheless, the formation processes of these structures, especially the dynamical pathways and rates, remain challenging to predict and control. Here, we present a general analytical method for constructing dynamical free-energy landscapes of helices. Such landscapes contain information about the thermodynamic stabilities of the possible macromolecular conformations, as well as about the dynamic connectivity, thus enabling the visualization and computation of folding pathways and timescales. We elucidate the methodology using the folding of polyalanine, and demonstrate that its α-helix folding kinetics is dominated by misfolded intermediates. At the physiological temperature of T = 298 K and midfolding time t = 250 ns, the fraction of structures in the native-state (α-helical) basin equals 22%, which is in good agreement with time-resolved experiments and massively distributed, ensemble-convergent molecular-dynamics simulations. We discuss the prominent role of β-strandlike intermediates in flight toward the native fold, and in relation to the primary conformational change precipitating aggregation in some neurodegenerative diseases.protein folding | misfolding intermediates I f there is one signature characteristic of biological macromolecules, it is the presence of helices. The double helix is the dominant feature of DNA and RNA, and, in proteins, the α-helix is the most ubiquitous structural motif. The formation and decay of α-helices play a pivotal role in protein folding (and misfolding). Indeed, even for proteins containing little native helical structure, helix formation, concomitant with hydrophobic collapse, seems to be a universal precursor of the folding process (1), much as the nonnative conversion of helical (or disordered) to β-strand-rich structures is the precursor to protein aggregation diseases such as Alzheimer's (2). Although the thermodynamics of such transformations was well understood beginning with the 1950s, the dynamical pathways and rates involved have remained an area of active research. Thus, relatively little was known about the associated kinetics until the end of the 1990s (3). With the advent of temperature-jump (T-jump) experimental techniques, the rates of the helix−coil transitions became readily measurable (4-6), whereas the progress in temporal resolution spanning both the nanosecond (7) and picosecond (8) regimes elucidated the early steps of folding/unfolding. Importantly, studies of the elementary steps accompanying such processes can now uncover the nature of kinetic-intermediate states, as well as their characteristic lifetimes (8, 9).Here, we introduce a statistical mechanical method of constructing dynamical free-energy landscapes of helices. The dynamical landscape allows folding pathways and rates to be visualized and computed in terms of elementary structural steps. For a r...

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Section: Resultsmentioning

confidence: 99%

Dominance of misfolded intermediates in the dynamics of α-helix folding

Lin

Shorokhov

Zewail

2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Circular dichroism is an excellent method to analyze the conformation of proteins and peptides (Greenfield et al, 1996). The far-UV spectrum of CD generally reflects secondary structures of protein (Sreerama et al, 1999;Johnson, 1999). Various empirical methods and analyses have been developed for quantitative estimation of the secondary structure content of protein (Bohm et al, 1992;Sreerama et al, 2000;Fink, 1995).…”

Section: Discussionmentioning

confidence: 99%

“…CD spectra were recorded in an average of 10 scans between 190 and 260 nm, with a 1.0 nm bandwidth, a scanning rate of 10 nm/min, a wavelength step of 0.2 nm and a time constant of 2 s. CD band intensities were expressed as molar ellipticities, [Â] M, in degrees cm 2 dmol −1 . The extents of secondary structures of 3D8 scFv were determined from the ellipticities using K2d software (Sreerama et al, 2000(Sreerama et al, , 1999Johnson et al, 1999;Bohm et al, 1992). Fluorescence emission spectra were obtained using a spectrofluorometer Japan) to analyze tertiary structures of recovered 3D8 scFv.…”

Section: Circular Dichroism and Fluorescence Spectroscopymentioning

confidence: 99%

Optimized stability retention of a monoclonal antibody in the PLGA nanoparticles

Son

Lee

Joung

et al. 2009

International Journal of Pharmaceutics

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“…CD spectra in the far-UV range were smoothed using the JASCO noise reduction routine. The CDPro program suite (34), a modified version of three methods (SELCON3 (35), CONTIN/LL locally linearized approximation (38) of CONTIN (39), and CDSSTR (40)), was used to calculate HET-C2 secondary structure from far-UV CD spectra. Tertiary structure class was assessed using the CDPro CLUSTER program (37) (see supplemental material for details).…”

Section: Methodsmentioning

confidence: 99%

Structural Determination and Tryptophan Fluorescence of Heterokaryon Incompatibility C2 Protein (HET-C2), a Fungal Glycolipid Transfer Protein (GLTP), Provide Novel Insights into Glycolipid Specificity and Membrane Interaction by the GLTP Fold

Kenoth

Simanshu

Kamlekar

et al. 2010

Journal of Biological Chemistry

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HET-C2 is a fungal protein that transfers glycosphingolipids between membranes and has limited sequence homology with human glycolipid transfer protein (GLTP). The human GLTP fold is unique among lipid binding/transfer proteins, defining the GLTP superfamily. Herein, GLTP fold formation by HET-C2, its glycolipid transfer specificity, and the functional role(s) of its two Trp residues have been investigated. X-ray diffraction (1.9 Å ) revealed a GLTP fold with all key sugar headgroup recognition residues (Asp 66 , Asn 70 , Lys 73 , Trp 109 , and His 147 ) conserved and properly oriented for glycolipid binding. Far-UV CD showed secondary structure dominated by ␣-helices and a cooperative thermal unfolding transition of 49°C, features consistent with a GLTP fold. Environmentally induced optical activity of Trp/Tyr/Phe (2:4:12) detected by near-UV CD was unaffected by membranes containing glycolipid but was slightly altered by membranes lacking glycolipid. Trp fluorescence was maximal at ϳ355 nm and accessible to aqueous quenchers, indicating free exposure to the aqueous milieu and consistent with surface localization of the two Trps. Interaction with membranes lacking glycolipid triggered significant decreases in Trp emission intensity but lesser than decreases induced by membranes containing glycolipid. Binding of glycolipid (confirmed by electrospray injection mass spectrometry) resulted in a blue-shifted emission wavelength maximum (ϳ6 nm) permitting determination of binding affinities. The unique positioning of Trp 208 at the HET-C2 C terminus revealed membrane-induced conformational changes that precede glycolipid uptake, whereas key differences in residues of the sugar headgroup recognition center accounted for altered glycolipid specificity and suggested evolutionary adaptation for the simpler glycosphingolipid compositions of filamentous fungi.Self/nonself recognition is a universally important process, encompassing intercellular interactions ranging from vertebrate immune responses to somatic chimera formation in protists, filamentous fungi, sponges, ascidians, and tunicates. Self/ nonself discrimination becomes critical for filamentous fungi during hyphal fusion, which enables the exchange of cytoplasm and nuclei during the assimilative growth phase (1-4). Nonself recognition triggers a postfusion, programmed cell death process known as vegetative incompatibility, which leads to heterokaryon death. The benefits of vegetative incompatibility include prevention of transmission of deleterious cytoplasmic elements (i.e. viruses) as well as restriction of plundering by parasitic genotypes.het genes are known to play a major role in vegetative incompatibility processes that occur in Neurospora crassa and Podospora anserina. het genes exhibit extensive polymorphism and generally encode proteins carrying a HET domain (1-4). Originally, this domain was linked to the het-c2 gene of P. anserina, where it was shown to encode a protein similar in size and with limited sequence homology to mammalian glycolipid transfer...

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Estimation of the number of α‐helical and β‐strand segments in proteins using circular dichroism spectroscopy

Cited by 687 publications

References 34 publications

Dominance of misfolded intermediates in the dynamics of α-helix folding

Dominance of misfolded intermediates in the dynamics of α-helix folding

Optimized stability retention of a monoclonal antibody in the PLGA nanoparticles

Structural Determination and Tryptophan Fluorescence of Heterokaryon Incompatibility C2 Protein (HET-C2), a Fungal Glycolipid Transfer Protein (GLTP), Provide Novel Insights into Glycolipid Specificity and Membrane Interaction by the GLTP Fold

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