Showing papers on "Circular dichroism published in 1996"
01 Jan 1996
TL;DR: This chapter discusses Circular Dichroism Using Synchrotron Radiation: From Ultraviolet to Xrays, and the applications to Conformational Analysis of Biomolecules.
Abstract: Remembrance of Things Past: A Career in Chiroptical Research (JT Yang) Theory of Circular Dichroism of Proteins (RW Woody) Determination of Protein Secondary Structure (SY Venyaminov, JT Yang) Aromatic and Cystine Sidechain Circular Dichroism in Proteins (RW Woody, AK Dunker) Stoppedflow Circular Dichroism (K Kuwajima) Circular Dichroism of Collagen and Related Polypeptides (RS Bhatnagar, CA Gough) CD Spectroscopy and the Helixcoil Transition in Peptides and Polypeptides (NR Kallenbach et al) The GBPIiGBP Sheet=Coil Transition of Polypeptides: As Determined by Circular Dichroism (L Tilstra, WL Mattice) Turns (A Perczel, M Hollosi) Differentiation between Transmembrane Helices and Peripheral Helices by the Deconvolution of Circular Dichroism Spectra of Membrane Proteins (GD Fasman) Theories of Circular Dichroism for Nucleic Acids (D Keller) Determination of the Conformation of Nucleic Acids by Electronic CD (WC Johnson, Jr) Circular Dichroism of Proteinnucleic Acid Interactions (DM Gray) Carbohydrates (ES Stevens) Chaperones (GD Fasman) Vibrational Circular Dichroism: Applications to Conformational Analysis of Biomolecules (TA Keiderling) Circular Dichroism Using Synchrotron Radiation: From Ultraviolet to Xrays (JC Sutherland) Circular Dichroism Instrumentation (WC Johnson, Jr) Vibrational Raman Optical Activity of Biomolecules (LD Barron et al) 252 illustrations Index
1,631 citations
TL;DR: The β-hexapeptide (H-β-HVal-β -HAla-Leu)2-2-OH (2) was obtained from the component Lβ-amino acids by conventional peptide synthesis, including fragment coupling as discussed by the authors.
Abstract: The β-hexapeptide (H-β-HVal-β-HAla-β-HLeu)2-OH (2) was prepared from the component L-β-amino acids by conventional peptide synthesis, including fragment coupling. A cyclo-β-tri- and a cyclo-β-hexapeptide were also prepared. The β-amino acids were obtained from α-amino acids by Arndt-Eistert homologation. All reactions leading to the β-peptides occur smoothly and in high yields. The β-peptides were characterized by their CD and NMR spectra (COSY, ROESY, TOCSY, and NOE-restricted modelling), and by an X-ray crystal-structure analysis. β-Sheet-type structures (in the solid state) and a compact, left-handed or (M) 31 helix of 5-A pitch (in solution) were discovered. Comparison with the analogous secondary structures of α-peptides shows fundamental differences, the most surprising one at this point being the greater stability of β-peptide helices. There are structural relationships of β-peptides with oligomers of β-hydroxyalkanoic acids, and dissimilarities between the two classes of compounds are a demonstration of the power of H-bonding. The β-hexapeptide 2 is stable to cleavage by pepsin at pH 2 in H2O for at least 60 h at 37°, while the corresponding α-peptide H-(Val-Ala-Leu)2-OH is cleaved instantaneously under these conditions. The implication of the described results are discussed.
642 citations
TL;DR: This review compares various methods of obtaining structural information from CD data and the advantages and pitfalls of each technique are detailed.
603 citations
TL;DR: It is shown that p21 and NH2-terminal fragments that are active as Cdk inhibitors lack stable secondary or tertiary structure in the free solution state and the flexibility, or disorder, of free p21 is associated with binding diversity and insights are offered into the role for structural disorder in mediating binding specificity in biological systems.
Abstract: The cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1/Sdi1, important for p53-dependent cell cycle control, mediates G1/S arrest through inhibition of Cdks and possibly through inhibition of DNA replication. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2 terminus. We show, using proteolytic mapping, circular dichroism spectropolarimetry, and nuclear magnetic resonance spectroscopy, that p21 and NH2-terminal fragments that are active as Cdk inhibitors lack stable secondary or tertiary structure in the free solution state. In sharp contrast to the disordered free state, however, the p21 NH2 terminus adopts an ordered stable conformation when bound to Cdk2, as shown directly by NMR spectroscopy. We have, thus, identified a striking disorder-order transition for p21 upon binding to one of its biological targets, Cdk2. This structural transition has profound implications in light of the ability of p21 to bind and inhibit a diverse family of cyclin-Cdk complexes, including cyclin A-Cdk2, cyclin E-Cdk2, and cyclin D-Cdk4. Our findings suggest that the flexibility, or disorder, of free p21 is associated with binding diversity and offer insights into the role for structural disorder in mediating binding specificity in biological systems. Further, these observations challenge the generally accepted view of proteins that stable secondary and tertiary structure are prerequisites for biological activity and suggest that a broader view of protein structure should be considered in the context of structure-activity relationships.
570 citations
TL;DR: The observation of the fast kinetics of helix melting in a small 21-residue alanine-based peptide demonstrates that secondary structure formation is fast enough to be a key event at early times in the protein-folding process and that helices are capable of forming before long range tertiary contacts are made.
Abstract: The helix is a common secondary structural motif found in proteins, and the mechanism of helix-coil interconversion is key to understanding the protein-folding problem. We report the observation of the fast kinetics (nanosecond to millisecond) of helix melting in a small 21-residue alanine-based peptide. The unfolding reaction is initiated using a laser-induced temperature jump and probed using time-resolved infrared spectroscopy. The model peptide exhibits fast unfolding kinetics with a time constant of 160 +/- 60 ns at 28 degrees C in response to a laser-induced temperature jump of 18 degrees C which is completed within 20 ns. Using the unfolding time and the measured helix-coil equilibrium constant of the model peptide, a folding rate constant of approximately 6 x 10(7) s-1 (t1/2 = 16 ns) can be inferred for the helix formation reaction at 28 degrees C. These results demonstrate that secondary structure formation is fast enough to be a key event at early times in the protein-folding process and that helices are capable of forming before long range tertiary contacts are made.
557 citations
TL;DR: In this article, the authors investigated the relationship between nitroxide side-chain mobility and protein structure by spin-labeling mutants of T4 lysozyme with a sulfhydryl-specific reagent.
Abstract: Thirty single cysteine substitution mutants of T4 lysozyme have been prepared and spin-labeled with a sulfhydryl-specific nitroxide reagent in order to systematically investigate the relationship between nitroxide side-chain mobility and protein structure. The perturbation caused by replacement of a native residue with a nitroxide amino acid was assessed from the resulting changes in biological activity, circular dichroism, and free energy of folding. The nitroxide produced context-dependent changes in stability and activity similar to those observed for substitution with natural amino acids at the same site but had little effect on the circular dichroism spectra. At solvent-exposed sites, the structural perturbation appears to be small at the level of the backbone fold. Nitroxide side-chain mobility faithfully reflects the protein tertiary fold at all sites investigated. The primary determinants of nitroxide side-chain mobility are tertiary interactions and backbone dynamics. Tertiary interactions constrain the side-chain mobility to an extent closely correlated with the degree of interaction. At interhelical loop sites, the side chains have a high mobility, consistent with high crystallographic thermal factors. On the exposed surfaces of alpha-helices, the side-chain mobility is not restricted by interactions with nearest neighbor side chains but appears to be determined by backbone dynamics. An unexpected result is a striking difference between the mobility of residues near the C- and N-termini of helices. These results provide the foundation for another dimension of information in site-directed spin-labeling experiments that can be interpreted in terms of the protein tertiary fold, its equilibrium dynamics and time-dependent conformational changes.
538 citations
TL;DR: In this article, the enantiomers of a racemic photoresponsive material represent two distinct states that can be modulated with irradiation at a single wavelength by changing the handedness of the light.
Abstract: The enantiomers of a racemic photoresponsive material represent two distinct states that can be modulated with irradiation at a single wavelength by changing the handedness ofthe light. Dynamic control over molecular chirality was obtained by the interconversion of enantiomers of helically shaped molecules with either left or right circular polarized light (CPL). Photoresolution of the bistable compound as a dopant in a nematic liquid crystalline phase by CPL irradiation led to a chiral mesoscopic phase. The chiral information inherent to CPL is therefore transmitted to the bistable molecule, followed by amplification and macroscopic expression of the chirality.
365 citations
345 citations
01 Jan 1996
TL;DR: Several methods of CD analysis of proteins can provide estimates of α helix, β sheet, β turn, and unordered form, which are presumed to have the same structure in the crystalline state and in aqueous solution.
Abstract: Circular dichroism (CD) is one of the most sensitive physical techniques for determining structures and monitoring structural changes of biomolecules. It can directly interpret the changes of protein secondary structure, even though the method is empirical. The far-ultraviolet (far-UV) CD specra (below 250 nm) of proteins are extremely sensitive toward protein structure, and the near-UV spectra reflect the contributions of aromatic side chains, disulfide bonds, and induced CD bands of prosthetic groups. Together these measurements provide information about the overall structure of a protein molecule as well as its local conformation around the aromatic and prosthetic groups and disulfide linkages. The ease of CD measurements is attractive, but CD, unlike two other powerful techniques—x-ray diffraction of protein crystals and NMR for protein solutions—cannot determine the three-dimensional structure of a protein. In this chapter we will discuss several methods of CD analysis of proteins, which can provide estimates of α helix, β sheet, β turn, and unordered form. These empirical methods utilize a set of reference proteins of known structure from x-ray diffraction studies. Thus, proteins are presumed to have the same structure in the crystalline state and in aqueous solution.
302 citations
TL;DR: The intrinsic capacity of Pro to disrupt beta-sheets was further reflected in a survey of porins where Pro was found to be selectively excluded from the core of membrane-spanning beta-sheet barrels, and an observation that argues against the doctrine that Pro is the most potent alpha-helix breaker as established in aqueous media is argued.
Abstract: Proline is established as a potent breaker of both alpha-helical and beta-sheet structures in soluble (globular) proteins. Thus, the frequent occurrence of the Pro residue in the putative transmembrane helices of integral membrane proteins, particularly transport proteins, presents a structural dilemma. We propose that this phenomenon results from the fact that the structural propensity of a given amino acid may be altered to conform to changes imposed by molecular environment. To test this hypothesis on proline, we synthesized model peptides of generic sequence H2N-(Ser-LyS)2-Ala- Leu-Z-Ala-Leu-Z-Trp-Ala-Leu-Z-(Lys-Ser)3-OH (Z = Ala and/or Pro). Peptide conformations were analyzed by circular dichroism spectroscopy in aqueous buffer, SDS, lysophosphatidylglycerol micelles, and organic solvents (methanol, trifluoroethanol, and 2-propanol). The helical propensity of Pro was found to be greatly enhanced in the membrane-mimetic environments of both lipid micelles and organic solvents. Proline was found to stabilize the alpha-helical conformation relative to Ala at elevated temperatures in 2-propanol, an observation that argues against the doctrine that Pro is the most potent alpha-helix breaker as established in aqueous media. Parallel studies in deoxycholate micelles of the temperature-induced conformational transitions of the single-spanning membrane bacteriophage IKe major coat protein, in which the Pro-containing wild type was compared with Pro30 --> Ala mutant, Pro was found to protect the helix, but disrupt the beta-sheet structure as effectively as it does to model peptides in water. The intrinsic capacity of Pro to disrupt beta-sheets was further reflected in a survey of porins where Pro was found to be selectively excluded from the core of membrane-spanning beta-sheet barrels. The overall data provide a rationale for predicting and understanding the structural consequences when Pro occurs in the context of a membrane.
278 citations
TL;DR: It is proposed that this unique ability of the peptides to induce nonbilayer structures in phosphatidylcholine model membranes is due to the presence of two tryptophans at both sides of the membrane/water interface, which prevent the peptide from aggregating when the mismatch is increased.
Abstract: We have investigated the effect of several hydrophobic polypeptides on the phase behavior of diacylphosphatidylcholines with different acyl chain length. The polypeptides are uncharged and consist of a sequence with variable length of alternating leucine and alanine, flanked on both sides by two tryptophans, and with the N- and C-termini blocked. First it was demonstrated by circular dichroism measurements that these peptides adopt an α-helical conformation with a transmembrane orientation in bilayers of dimyristoylphosphatidylcholine. Subsequent 31P NMR measurements showed that the peptides can affect lipid organization depending on the difference in hydrophobic length between the peptide and the lipid bilayer in the liquid-crystalline phase. When a 17 amino acid residue long peptide (WALP17) was incorporated in a 1/10 molar ratio of peptide to lipid, a bilayer was maintained in saturated phospholipids containing acyl chains of 12 and 14 C atoms, an isotropic phase was formed at 16 C atoms, and an invert...
TL;DR: In the aggregated phases, a splitting of the excited state into two exciton levels occurs as mentioned in this paper, and the exciton level is split into two levels of exciton activation.
Abstract: Conformationally disordered poly{3,4-di[(S)-2-methylbutoxy]thiophene} is prepd via monomer polymn by anhyd FeCl3 Optically active ordered polymer is prepd by cooling of the polymer to -30 Deg In the aggregated phases a splitting of the excited state into two exciton levels occurs
TL;DR: The present results suggest that the folding reaction of β-lactoglobulin follows a non-hierarchical mechanism, in which non-native α-helical structures play important roles.
Abstract: It is generally assumed that folding intermediates contain partially formed native-like secondary structures. However, if we consider the fact that the conformational stability of the intermediate state is simpler than that of the native state, it would be expected that the secondary structures in a folding intermediate would not necessarily be similar to those of the native state. β-Lactoglobulin is a predominantly β-sheet protein, although it has a markedly high intrinsic preference for α-helical structure. We have studied the refolding kinetics of bovine β-lactoglobulin using stopped-flow circular dichroism and find that a partly α-helical intermediate accumulates transiently before formation of the native β-sheets. The present results suggest that the folding reaction of β-lactoglobulin follows a non-hierarchical mechanism, in which non-native α-helical structures play important roles.
TL;DR: In this article, it was suggested that silicates be considered for therapeutic use to delay the onset of plaques and tangles in Alzheimer's disease, which was also reversed by the addition of silicates.
01 Jan 1996
TL;DR: The central theoretical parameter of rotational strength will be defined and the mechanisms by which electronic transitions contribute to CD will be discussed qualitatively, after which the methods by which CD is calculated will be described.
Abstract: In this chapter, the basic phenomenon of circular dichroism (CD) will be described. The central theoretical parameter of rotational strength will then be defined. The mechanisms by which electronic transitions contribute to CD, i.e., acquire rotational strength, will then be discussed qualitatively, after which the methods by which CD is calculated will be described. The most important group in the electronic spectroscopy of proteins, the peptide group, will then be discussed. Finally, theoretical studies of the principal types of peptide secondary structure will be surveyed. The reader should note that aromatic and disulfide groups are not discussed in this chapter, but are covered in a separate chapter (Woody and Dunker, Chapter 4), along with experimental studies of these important protein chromophores.
TL;DR: The conditions at which the alcohol-induced molten globule exists could be similar to those existing near negatively charged membrane surfaces and might explain how the molten globules state can be achieved under physiological conditions.
Abstract: Methanol-induced conformational transitions in cytochrome c have been studied by near- and far-UV circular dichroism, Trp fluorescence, microcalorimetry, and diffusion measurements. The existence of at least two cooperative stages of transition has been shown. At the first stage, the native protein is transformed into an intermediate which has only traces of tertiary structure, but has a native-like secondary structure content and is relatively compact; i.e., it has properties of the molten globule state. On the second stage, the alcohol-induced molten globule is transformed into a more helical state, typical of proteins at high alcohol concentrations. The conditions at which the alcohol-induced molten globule exists (moderately low pH and moderately low dielectric constant) could be similar to those existing near negatively charged membrane surfaces. Consequently, these results might explain how the molten globule state can be achieved under physiological conditions.
TL;DR: In this paper, a distance dependency of the porphyrin coupling was investigated in the range between 10 and 50 A. Over short interchromophoric distances, significant changes in the conformational distribution introduced by the bulky porphrin chromophores were observed.
Abstract: The present study (see ref 1) delineates the scope and limitations of porphyrin chromophores for structural studies by the exciton coupled circular dichroic (CD) method. A distance dependency of the porphyrin coupling was investigated in the range between 10 and 50 A. Over short interchromophoric distances, significant changes in the conformational distribution introduced by the bulky porphyrin chromophores were observed. Over longer distances, the porphyrins showed ca. 10-fold sensitivity increase over commonly used chromophores, and an effective direction for the interacting porphyrin transition moments was assigned by comparison. Porphyrins at the termini of dimeric steroids and brevetoxin B exhibited exciton coupling over interchromophoric distances up to 50 A. These results represent the porphyrins as promising reporter chromophores for extending the exciton coupled CD method to structural studies of biopolymers.
TL;DR: In this article, low-temperature optical absorption, circular dichroism, magnetic circular dichroidism, and sulfur K-edge X-ray absorption spectra have been measured for the green "blue" copper center (type 1) in Achromobacter cycloclastes nitrite reductase.
Abstract: Low-temperature optical absorption, circular dichroism, magnetic circular dichroism, and sulfur K-edge X-ray absorption spectra have been measured for the green “blue” copper center (type 1) in Achromobacter cycloclastes nitrite reductase. Combined with density functional calculations, the results of these spectroscopies have been used to define the extremely “perturbed” electronic structure of this site relative to that of the prototypical “classic” site found in plastocyanin. Experimentally calibrated density functional calculations have been further used to determine the specific geometric distortions which generate the perturbed electronic structure. These studies indicate that the principal electronic structure changes in nitrite reductase, relative to plastocyanin, are a rotation of the Cu dx2-y2 half-filled, highest occupied molecular orbital (HOMO) and an increase in the ligand field strength at the Cu center. The HOMO rotation increases the pseudo-σ interaction and decreases the π interaction of ...
TL;DR: In this paper, the triple-helical propensities of template-assembled collagen-based polypeptides were investigated using circular dichroism, ultraviolet absorbance, optical rotation, and nuclear magnetic resonance measurements.
Abstract: Template-assembled collagen-based polypeptides KTA-[Gly-(Gly-Pro-Hyp)n-NH2]3 (n = 1, 3, 5, 6; KTA is cis,cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylic acid, also known as the Kemp triacid) and acetyl-terminated single-chain collagen-based analogs Ac-(Gly-Pro-Hyp)n-NH2 (n = 1, 3, 5, 6, 9) were synthesized by solid phase segment condensation methods. The triple-helical propensities of these collagen analogs were investigated using circular dichroism, ultraviolet absorbance, optical rotation, and nuclear magnetic resonance measurements. The acetyl analogs, Ac-(Gly-Pro-Hyp)n-NH2 (n = 6, 9), assume a stable triple-helical conformation in H2O (0.2 mg/mL) at room temperature. By contrast, Ac-(Gly-Pro-Hyp)5-NH2 adopts a triple-helical conformation in H2O only below 18 °C at a concentration of 0.2 mg/mL. For the template-assembled collagen analogs, results show that KTA-[Gly-(Gly-Pro-Hyp)n-NH2]3 (n = 5, 6) peptides form triple-helical structures which have melting temperatures above 70 °C in H2O. These meltin...
TL;DR: The third helix of antennapedia homeodomain pAntp-(43-58) can translocate through cell membrane and has been used as an intracellular vehicle for delivering peptides and oligonucleotides and lipid polymorphism in different ways depending on the mixtures of acidic lipids.
Abstract: The third helix of antennapedia homeodomain pAntp-(43-58) can translocate through cell membrane and has been used as an intracellular vehicle for delivering peptides and oligonucleotides. The conformational and associative behaviour of two peptidic vectors pAntp-(43-58) and [Pro50] pAntp-(43-58) has been analyzed by different biophysical methods. pAntp-(43-58) adopts an amphipathic helical structure in 30% (by vol.) hexafluoroisopropanol, in perfluoro-tert-butanol and in the presence of SDS micelles. CD spectra indicate that the conformation of [Pro50]pAntp-(43-58) in contrast to pAntp-(43-58) is independent of the media used. 1H-NMR spectroscopy in SDS micelles or in perfluoro-tert-butanol allows detection of aggregated peptides probably in a ribbon 2(7) type conformation. These conformations became the predominant structure when Gln50 was replaced by Pro50. Interproton-distance restraints derived from NOE measurements have been classified in two groups corresponding to two types of structures: alpha-helix and essentially extended structures. Consecutive CH alpha (i)/ CH alpha (i + 1) NOEs are only compatible with aggregates. Simulated annealing calculation of dimeric structure agrees with phi and psi angles in the beta-sheet and gamma-turn regions. Fluorescence spectroscopy analysis has shown that the indole groups of both peptides penetrate into SDS micelles; both peptides also induce the formation of micelles at very low concentration of SDS (20 microM). Similar interaction was observed with reverse-phase micelles made of bis(2-ethyhexyl) sodium sulfosuccinate and small unilamellar vesicles (SUV) made of a mixture of phosphatidylcholine/phosphatidylserine. 31P-NMR of vesicles (SUV and large unilamellar vesicles) indicated that the addition of pAntp analogues did not affect the size of phosphatidylcholine/phosphatidylserine vesicles. The addition of pAntp analogues to lipidic dispersions modulates lipid polymorphism in different ways depending on the mixtures of acidic lipids.
TL;DR: The results suggest that in vivo lipid binding by apoA-1 may be mediated via the molten globular apolipoprotein state in plasma under near-physiological conditions.
Abstract: Apolipoprotein A-1 (apoA-1) in complex with high-density lipoprotein is critically involved in the transport and metabolism of cholesterol and in the pathogenesis of atherosclerosis. We reexamined the thermal unfolding of lipid-free apoA-1 in low-salt solution at pH approximately 7, by using differential scanning calorimetry and circular dichroism. At protein concentrations <5 mg/ml, thermal unfolding of apoA-1 is resolved as an extended peak (25 degrees C-90 degrees C) that can be largely accounted for by a single reversible non-two-state transition with midpoint Tm 57 +/- 1 degree C, calorimetric enthalpy deltaH(Tm)= 200 +/- 20 kcal/mol (1 kcal = 4.18 kJ), van't Hoff enthalpy deltaHv(Tm) approximately 32.5 kcal/mol, and cooperativity deltaHv(Tm)/deltaH(Tm) approximately 0.16. The enthalpy deltaH(Tm) can be accounted for by melting of the alpha-helical structure that is inferred by CD to constitute approximately 60% of apoA-1 amino acids. Farand near-UV CD spectra reveal noncoincident melting of the secondary and tertiary structural elements and indicate a well-defined secondary structure but a largely melted tertiary structure for apoA-1 at approximately 37 degrees C and pH 7. This suggests a molten globular-like state for lipid-free apoA-1 under near-physiological conditions. Our results suggest that in vivo lipid binding by apoA-1 may be mediated via the molten globular apolipoprotein state in plasma.
TL;DR: Methanol-induced conformational transitions of cytochrome c(cyt c) at acidic pH values were investigated with a combined use of far and near-UV CD, fluorescence, NMR spectroscopy and small-angle X-ray scattering showing that the I(M) state has no specific tertiary structure but has a secondary structural content and tryptophan environment similar to those in the native state.
TL;DR: The contribution of specific packing to the stability of the sperm whale apomyoglobin intermediate is studied by urea denaturation monitored by circular dichroism and fluorescence and proposed to be stabilized by both partially formed native-like tertiary, and non-specific hydrophobic interactions forming a subdomain folding intermediate.
Abstract: The contribution of specific packing to the stability of the sperm whale apomyoglobin intermediate has been studied by urea denaturation monitored by circular dichroism and fluorescence. Mutations disrupting native packing sites within the subdomain formed by the A, G and H helices destabilize the intermediate, in contrast to the conclusion drawn from earlier studies of pH-induced unfolding. Based on these results, the intermediate is proposed to be stabilized by both partially formed native-like tertiary, and non-specific hydrophobic interactions forming a subdomain folding intermediate. The results help to explain how the intermediate acquires its structure and stability.
TL;DR: The combination of infrared spectroscopy and hydrogen-deuterium exchange has great potential in characterization of even subtle structural differences in proteins induced by naturally occurring point mutations and/or site-directed mutagenesis.
Abstract: Structural differences between two genetic variants of bovine beta-lactoglobulins (type A and B) in aqueous solutions were characterized using Fourier transform infrared and circular dichroism spectroscopies. To probe differences in structural dynamics, the effects hydrogen-deuterium exchange were also compared for the two proteins. The infrared spectra recorded in H2O solution for the two proteins were nearly identical in the conformationlly sensitive amide I region. The only exceptions were small differences at the band ascribed to a high-wavenumber beta-sheet component near 1693 cm-1 and the band assigned to turns at 1684 cm-1. In contrast, when the proteins were prepared in D2O solution, marked spectral differences were observed at all regions ascribed to beta-sheet and turn structures. These differences are consistent with the structural differences of the two variants at amino acid residues 64 and 118, which are located at a turn and a beta-sheet structure, respectively, as revealed by X-ray crystallographic studies [Monaco et al. (1987) J. Mol. Biol. 197, 695-706]. The circular dichroism spectra for the two proteins were essentially identical, both before and after hydrogen-deuterium exchange. Therefore, hydrogen-deuterium exchange did not alter the proteins' secondary structure. The enhancement of the amide I spectral difference upon hydrogen-deuterium exchange was ascribed to the differences in the structural mobility of the two proteins. Since the rate of exchange was greater for variant A, it was concluded that this variant has greater structural mobility than variant B. These findings indicate that the combination of infrared spectroscopy and hydrogen-deuterium exchange has great potential in characterization of even subtle structural differences in proteins induced by naturally occurring point mutations and/or site-directed mutagenesis.
TL;DR: Simplified partially folded models, based on the crystal structure of hen lysozyme, support a working model for the intermediate, whose structure may be correlated with that of the kinetic intermediate found in the refolding pathway studied by Dobson and coworkers.
TL;DR: These in vitro results correlate well with the in vivo observed partial inhibition of bacteriophage λ growth in a DnaJΔ144-200 mutant background, and show that the Zn(II) ions help to stabilize DnJ's tertiary structure.
TL;DR: Post-translational modification, possibly lysine monomethylation, appears to be the single most important stabilizing factor that distinguishes the native hyperthermophile protein from small mesophile proteins.
TL;DR: Peptides corresponding to the cytoplasmic tails of the α and β subunits of the integrin receptor αβ were synthesized and used to characterize their interaction with cations and with one another.
TL;DR: The binding of monoclonal antibody Fab fragments dramatically increased the solubility of both gp41 constructs and it is proposed that gp41 folds into its membrane fusion‐active conformation, when expressed alone.
Abstract: The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein is composed of a soluble glycopolypeptide gp120 and a transmembrane glycopolypeptide gp41. These subunits form non-covalently linked oligomers on the surface of infected cells, virions and cells transfected with the complete env gene. Two length variants of the extracellular domain of gp41 (aa 21-166 and aa 39-166), that both lack the N-terminal fusion peptide and the C-terminal membrane anchor and cytoplasmic domain, have been expressed in insect cells to yield soluble oligomeric gp41 proteins. Oligomerization was confirmed by chemical cross-linking and gel filtration. Electron microscopy and circular dichroism measurements indicate a rod-like molecule with a high alpha-helical content and a high melting temperature (78 degrees C). The binding of monoclonal antibody Fab fragments dramatically increased the solubility of both gp41 constructs. We propose that gp41 folds into its membrane fusion-active conformation, when expressed alone.
TL;DR: It is demonstrated that the intramolecular disulfide bonds of protegrins are required for their antiparallel beta-sheet conformation in membrane-mimetic environments and for their potent antimicrobial activity in media containing NaCl concentrations comparable to those found in serum and extracellular fluids.
Abstract: Protegrins are 2-kDa antimicrobial peptides that contain 16–18 amino acid residues and two intramolecular disulfide bonds. We studied the contribution of these disulfide bonds to the bactericidal activity of protegrins in physiological concentrations of NaCl by comparing protegrin PG-1 with variants that lacked one or both cysteine disulfides. Whereas the bactericidal and liposome-lytic properties of protegrin PG-1 were enhanced by adding 100 mM NaCl to the phosphate-buffered medium, NaCl addition strongly inhibited the effects of its linearized, disulfide-free variant, [A6, A8, A13, A15]protegrin-1. Whereas protegrin PG-1 manifested β-sheet structure by CD (circular dichroism) and ATR-FTIR (attenuated-total-reflectance-Fourier-transform-infrared) spectroscopy in buffer or membrane-mimetic environments, [A6, A8, A13, A15]protegrin-1 manifested disordered structure in phosphate buffer and α-helical characteristics in membrane-mimetic environments. Both single-disulfide protegrin variants, [A8, A13]protegrin-1 and [A6, A15]protegrin-1, assumed β-sheet conformations with liposomes that simulated bacterial membranes, and both retained substantial bactericidal activity when 100 mM NaCl was present. These findings demonstrate that the intramolecular disulfide bonds of protegrins are required for their antiparallel β-sheet conformation in membrane-mimetic environments and for their potent antimicrobial activity in media containing NaCl concentrations comparable to those found in serum and extracellular fluids.