RESUMEN
Circular dichroism (CD) spectra at variable temperatures have been recorded for human cytosolic sialidase NEU2 in buffered water solutions and in the presence of divalent cations. The results show the prevalence of ß-strands together with a considerable amount of α-helical structure, while in the solid state, from both previous X-ray diffraction analysis and our CD data on film samples, the content of ß-strands is higher. In solution, a significant change in CD spectra occurs with an increase in temperature, related to a decrease in α-helix content and a slight increase in ß-strand content. In the same range of temperatures, the enzymatic activity decreases. Although the overall structure of the protein appears to be particularly stable, molecular dynamics simulations performed at various temperatures evidence local conformational changes possibly relevant for explaining the relative lability of enzymatic activity.
Asunto(s)
Neuraminidasa/metabolismo , Dicroismo Circular , Simulación por Computador , Cristalización , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Neuraminidasa/química , Neuraminidasa/genética , Conformación Proteica , Espectrofotometría InfrarrojaRESUMEN
The nπ* and ππ* transitions of polypeptides mix significantly with very high energy transitions in the poly(Pro)II conformation, as evidenced by the strongly nonconservative CD spectrum in the 170-250 nm region. Because of this, the exciton model, the standard quantum mechanical model for predicting absorption and CD spectra of polypeptides, gives poor results for the poly(Pro) II (P(II)) conformation, although it works well for the α-helix, ß-sheet, and ß-turns. The exciton theory has been extended to include the effects of mixing of discrete peptide transitions near 200 nm with the large number of uncharacterized transitions in the deep ultraviolet. These latter transitions dominate the polarizability, and their mixing with the discrete transitions can be described via bond and lone-pair polarizability tensors, derivable by ab initio methods. This extended exciton method gives a good description of the CD spectrum of (Ala)(n) oligomers in the P(II) conformation. For this conformation, the polarizability contributions lead to a strong negative band near 200 nm that dominates the calculated and observed CD spectrum. The model does not give a good description of the CD of (Pro)(n) oligomers, probably because of conformational heterogeneity or nonadditive contributions of the Pro side chains. The model improves the calculated CD spectra of α-helical (Ala)(n) oligomers. Although the high-energy transitions make only a small net contribution to the CD of the α-helix in the 200 nm region, they enhance the negative exciton band at â¼205 nm and largely cancel the negative exciton band near 175 nm, substantially improving agreement with experiment.
Asunto(s)
Dicroismo Circular , Péptidos/química , Proteínas/química , Rayos UltravioletaRESUMEN
The poly(Pro)II (P(II)) conformation is increasingly recognized as an important element in peptide and protein conformation. Circular dichroism (CD) is one of the most useful methods for detecting and characterizing P(II). Although the standard exciton-based model for predicting peptide CD spectra works well for alpha-helices and beta-sheets, it fails to reproduce the P(II) CD spectrum because it does not account for mixing of the n pi* and pi pi* transitions with transitions in the deep UV, which is significant for the P(II) conformation. In this work, the exciton model is extended to include this mixing, using ab initio-derived bond polarizability tensors to calculate the contributions of the high-energy transitions. The strong negative 195-nm and weaker positive 220-nm CD bands of P(II) are reproduced for (Ala)(n) conformers in the P(II) region of the Ramachandran map. For the canonical P(II) conformation from fiber diffraction of poly(Pro)II (-77, +146), the results are poor, but conformations with less negative phi (approximately -60 degrees) and more positive psi (> or = 160 degrees) give spectra showing the P(II) characteristics. The CD of (Pro)(n) is not reproduced by the calculations, probably because variations in (phi,Psi), ring puckering, and cis-trans isomerism are not included in the model The extended model also gives improved results for alpha-helical polypeptides, leading to increased amplitude for the 205-nm band and decreased amplitude for a negative band predicted near 180 nm.
Asunto(s)
Péptidos/química , Dicroismo Circular , Estructura Secundaria de ProteínaRESUMEN
The visible (alpha) and near-UV (beta) CD bands of rhodopsin have been studied extensively, but their source(s) have never been definitively established. Do they result from the intrinsic chirality of the polyene chromophore of the protonated Schiff base of retinal (retPSB) or from the coupling of the transitions of this chromophore with those of protein groups? We have calculated the contributions of these two mechanisms to the CD of rhodopsin. The intrinsic CD of the retPSB chromophore was calculated using time-dependent density functional theory (TDDFT) and, for comparison, the semiempirical ZINDO method. First-order perturbation theory was used to calculate the effects of coupling of the retPSB transitions with the pi pi* transitions of the aromatic chromophores and the pi pi* and n pi* transitions of the peptide groups in rhodopsin. Calculations were performed for eight structures based upon the two molecules in the asymmetric unit of four crystal structures. The most reliable results were obtained from TDDFT calculations on the structure of Okada et al. (J. Mol. Biol. 2004, 342, 571), PDB 1U19. Averaging over the two molecules in the asymmetric unit, the intrinsic rotational strengths are 0.62 +/- 0.00 DBM (Debye-Bohr magneton) and 0.90 +/- 0.03 DBM for the alpha- and beta-bands, respectively. The contributions from coupling with protein groups are, respectively, -0.32 +/- 0.05 and -0.01 +/- 0.03 DBM. Our results show that the visible/near-UV CD bands of rhodopsin are determined by the intrinsic chirality of the retPSB chromophore and that the contributions of coupling with the protein are significantly smaller for the alpha-band and negligible for the beta-band.
Asunto(s)
Dicroismo Circular/métodos , Rodopsina/química , Animales , Bovinos , Modelos Químicos , Modelos Moleculares , Conformación Proteica , Teoría Cuántica , Espectrofotometría Ultravioleta/métodos , EstereoisomerismoRESUMEN
A computational model was developed to examine the phototriggered folding of a caged protein, a protein modified with an organic photolabile cross-linker. Molecular dynamics simulations of the modified 36-residue fragment of subdomain B of chicken villin head piece with a photolabile linker were performed, starting from both the caged and the uncaged structures. Construction of a free-energy landscape, based on principal components as well as on radius of gyration versus root-mean-square deviation, and circular dichroism calculations were employed to characterize folding behavior and structures. The folded structures observed in the molecular dynamics trajectories were found to be similar to that of the wild-type protein, in agreement with the published experimental results. The free-energy landscapes of the modified and wild-type proteins have similar topology, suggesting common thermodynamic/kinetic behavior. The existence of small differences in the free-energy surface of the modified protein from that of the native protein, however, indicates subtle differences in the folding behavior.
Asunto(s)
Modelos Moleculares , Proteínas de Neurofilamentos/química , Fragmentos de Péptidos/química , Pliegue de Proteína , Animales , Pollos , Dicroismo Circular , Reactivos de Enlaces Cruzados/efectos de la radiación , Cinética , Modelos Teóricos , Fotoquímica , TermodinámicaRESUMEN
Circular dichroism spectra of proteins are sensitive to protein secondary structure. The CD spectra of alpha-rich proteins are similar to those of model alpha-helices, but beta-rich proteins exhibit CD spectra that are reminiscent of CD spectra of either model beta-sheets or unordered polypeptides. The existence of these two types of CD spectra for beta-rich proteins form the basis for their classification as betaI- and betaII-proteins. Although the conformation of beta-sheets is largely responsible for the CD spectra of betaI-proteins, the source of betaII-protein CD, which resembles that of unordered polypeptides, is not completely understood. The CD spectra of unordered polypeptides are similar to that of the poly(Pro)II helix, and the poly(Pro)II-type (P2) structure forms a significant fraction of the unordered conformation in globular proteins. We have compared the beta-sheet and P2 structure contents in beta-rich proteins to understand the origin of betaII-protein CD. We find that betaII-proteins have a ratio of P2 to beta-sheet content greater than 0.4, whereas for betaI-proteins this ratio is less than 0.4. The beta-sheet content in betaI-proteins is generally higher than that in betaII-proteins. The origin of two classes of CD spectra for beta-rich proteins appears to lie in their relative beta-sheet and P2 structure contents.
Asunto(s)
Estructura Secundaria de Proteína , Proteínas/química , Dicroismo CircularRESUMEN
Analysis of circular dichroism spectra of proteins provides information about protein secondary structure. Analytical methods developed for such an analysis use structures and spectra of a set of reference proteins. The reference protein sets currently in use include soluble proteins with a wide range of secondary structures, and perform quite well in analyzing CD spectra of soluble proteins. The utility of soluble protein reference sets in analyzing membrane protein CD spectra, however, has been questioned in a recent study that found current reference protein sets to be inadequate for analyzing membrane proteins. We have examined the performance of reference protein sets available in the CDPro software package for analyzing CD spectra of 13 membrane proteins with available crystal structures. Our results indicate that the reference protein sets currently available for CD analysis perform reasonably well in analyzing membrane protein CD spectra, with performance indices comparable to those for soluble proteins. Soluble + membrane protein reference sets, which were constructed by combining membrane proteins with soluble protein reference sets, gave improved performance in both soluble and membrane protein CD analysis.
Asunto(s)
Proteínas Bacterianas/química , Dicroismo Circular , Proteínas de la Membrana/química , Estructura Secundaria de Proteína , Halobacterium salinarum/química , Estándares de Referencia , Rhodobacter capsulatus/química , Rhodobacter sphaeroides/química , Rhodopseudomonas/química , Solubilidad , Especificidad de la EspecieRESUMEN
The gene 5 protein (g5p) of the Ff virus contains five Tyr, individual mutants of which have now all been characterized by CD spectroscopy. The protein has a dominant tyrosyl 229-nm L(a) CD band that is shown to be approximately the sum of the five individual Tyr contributions. Tyr41 is particularly important in contributing to the high cooperativity with which the g5p binds to ssDNA, and Y41F and Y41H mutants are known to differ in dimer-dimer packing interactions in crystal structures. We compared the solution structures and binding properties of the Y41F and Y41H mutants using CD spectroscopy. Secondary structures of the mutants were similar by CD analyses and close to those derived from the crystal structures. However, there were significant differences in the binding properties of the two mutant proteins. The Y41H protein had an especially low binding affinity and perturbed the spectrum of poly[d(A)] in 2 mM Na(+) much less than did Y41F and the wild-type gene 5 proteins. Moreover, a change in the Tyr 229 nm band, assigned to the perturbation of Tyr34 at the dimer-dimer interface, was absent in titrations with the Y41H mutant under low salt conditions. In contrast, titrations with the Y41H mutant in 50 mM Na(+) exhibited typical CD changes of both the nucleic acid and the Tyr 229-nm band. Thus, protein-protein and g5p-ssDNA interactions appeared to be mutually influenced by ionic strength, indicative of correlated changes in the ssDNA binding and cooperativity loops of the protein or of indirect structural constraints.
Asunto(s)
Proteínas de Unión al ADN , Tirosina/química , Proteínas Virales/química , Sustitución de Aminoácidos , Dicroismo Circular , ADN de Cadena Simple , Dimerización , Escherichia coli , Mutación , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Virales/genéticaRESUMEN
Recent experimental and theoretical advances in understanding the electronic excited states of simple amides are reviewed. Polarized reflection spectroscopy of single crystals of N-acetylglycine shows that the direction of the first pipi* (NV(1)) transition dipole moment of a secondary amide differs by approximately 15 degrees from that of a primary amide. Ab initio calculations on simple amides support this conclusion. Ab initio studies of di- and tri-amides demonstrate that several inter-amide charge-transfer (CT) transitions occur in the 150-175-nm region, between the NV(1) and NV(2) transitions. When the correct dipole transition moment direction for peptides is used in calculations of the circular dichroism of the alpha-helix, the results are much improved over those from earlier calculations that used the direction for primary amides. Studies that consider the mixing of the NV(1) transition with CT transitions are reviewed. These indicate that such mixing is likely to have a significant effect on the absorption and CD spectra of the alpha-helix and other types of peptide conformation. Nevertheless, the independent systems model gives a reasonable first approximation to the absorption and CD spectra of the alpha-helix.
Asunto(s)
Amidas/química , Péptidos/química , Dicroismo Circular , Microanálisis por Sonda ElectrónicaRESUMEN
Intermolecular structures are important factors for understanding the conformational properties of amyloid fibrils. In this study, vacuum-ultraviolet circular dichroism (VUVCD) spectroscopy and circular dichroism (CD) theory were used for characterizing the intermolecular structures of ß2-microglobulin (ß2m) core fragments in the amyloid fibrils. The VUVCD spectra of ß2m20-41, ß2m21-31, and ß2m21-29 fragments in the amyloid fibrils exhibited characteristic features, but they were affected not only by the backbone conformations but also by the aromatic side-chain conformations. To estimate the contributions of aromatic side-chains to the spectra, the theoretical spectra were calculated from the simulated structures of ß2m21-29 amyloid fibrils with various types of ß-sheet stacking (parallel or antiparallel) using CD theory. We found that the experimental spectrum of ß2m21-29 fibrils is largely affected by aromatic-backbone couplings, which are induced by the interaction between transitions within the aromatic and backbone chromophores, and these couplings are sensitive to the type of stacking among the ß-sheets of the fibrils. Further theoretical analyses of simulated structures incorporating mutated aromatic residues suggested that the ß2m21-29 fibrils are composed of amyloid accumulations in which the parallel ß-sheets stack in an antiparallel manner and that the characteristic Phe-Tyr interactions among the ß-sheet stacks affect the aromatic-backbone coupling. These findings indicate that the coupling components, which depend on the characteristic intermolecular structures, induce the spectral differences among three fragments in the amyloid fibrils. These advanced spectral analyses using CD theory provide a useful method for characterizing the intermolecular structures of protein and peptide fragment complexes.
Asunto(s)
Amiloide/química , Dicroismo Circular , Modelos Moleculares , Espectrometría de Fluorescencia , Microglobulina beta-2/química , Simulación por Computador , Estructura Secundaria de ProteínaRESUMEN
Channelrhodopsins (ChRs) are of great interest currently because of their important applications in optogenetics, the photostimulation of neurons. The absorption and circular dichroism (CD) spectra of C1C2, a chimera of ChR1 and ChR2 of Chlamydomonas reinhardtii, have been studied experimentally and theoretically. The visible absorption spectrum of C1C2 shows vibronic fine structure in the 470 nm band, consistent with the relatively nonpolar binding site. The CD spectrum has a negative band at 492 nm (Δε(max) = -6.17 M(-1) cm(-1)) and a positive band at 434 nm (Δε(max) = +6.65 M(-1) cm(-1)), indicating exciton coupling within the C1C2 dimer. Time-dependent density functional theory (TDDFT) calculations are reported for three models of the C1C2 chromophore: (1) the isolated protonated retinal Schiff base (retPSB); (2) an ion pair, including the retPSB chromophore, two carboxylate side chains (Asp 292, Glu 162), modeled by acetate, and a water molecule; and (3) a hybrid quantum mechanical/molecular mechanical (QM/MM) model depicting the binding pocket, in which the QM part consists of the same ion pair as that in (2) and the MM part consists of the protein residues surrounding the ion pair within 10 Å. For each of these models, the CD of both the monomer and the dimer was calculated with TDDFT. For the dimer, DeVoe polarizability theory and exciton calculations were also performed. The exciton calculations were supplemented by calculations of the coupling of the retinal transition with aromatic and peptide group transitions. For the dimer, all three methods and three models give a long-wavelength C2-axis-polarized band, negative in CD, and a short-wavelength band polarized perpendicular to the C2 axis with positive CD, differing in wavelength by 1-5 nm. Only the retPSB model gives an exciton couplet that agrees qualitatively with experiment. The other two models give a predominantly or solely positive band. We further analyze an N-terminal truncated mutant because it was assumed that the N-terminal domain has a crucial role in the dimerization of ChRs. However, the CD spectrum of this mutant has an exciton couplet comparable to that of the wild-type, demonstrating that it is dimeric. Patch-clamp experiments suggest that the N-terminal domain is involved in protein stabilization and channel kinetics rather than dimerization or channel activity.
Asunto(s)
Fotorreceptores de Plantas/química , Acetatos/química , Animales , Chlamydomonas reinhardtii , Dicroismo Circular , Dimerización , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Modelos Químicos , Modelos Moleculares , Proteínas Mutantes Quiméricas/química , Técnicas de Placa-Clamp , Estabilidad Proteica , Teoría Cuántica , Células Sf9 , Análisis Espectral , Spodoptera , Transfección , Agua/químicaRESUMEN
The visible CD spectrum of bacteriorhodpsin (bR) in purple membrane has a negative CD band at ~600 nm and a positive band at ~530 nm and has been variously interpreted as resulting from exciton coupling within the bR trimer, heterogeneity in protein conformation, or the presence of two distinct low-energy electronic transitions in bR. We have performed time-dependent density functional theory (TDDFT) calculations on the protonated Schiff base of retinal (retPSB) in bR to predict the intrinsic CD. The resulting spectroscopic parameters have been used to predict the long-wavelength CD spectrum of retPSB trimers. TDDFT, exciton theory, and classical polarizability (DeVoe) predict a strong negative couplet centered near 570 nm, with a magnitude in good agreement with experiment. Coupling of the retPSB chromophore with aromatic and peptide chromophores has been considered by means of perturbation theory and is responsible for the net positive CD of the 570 nm band. The visible CD spectrum of bR is dominated by exciton interactions.
Asunto(s)
Bacteriorodopsinas/química , Dicroismo Circular , Modelos Moleculares , Conformación Proteica , Teoría Cuántica , Retinaldehído/química , Bases de Schiff/química , Factores de TiempoAsunto(s)
Dicroismo Circular/métodos , Pliegue de Proteína , Proteínas/química , Animales , Apoproteínas/química , Apoproteínas/metabolismo , Bacillus cereus/enzimología , Bovinos , Pollos/metabolismo , Citocromos c/química , Citocromos c/metabolismo , Interpretación Estadística de Datos , Cinética , Lactalbúmina/química , Lactalbúmina/metabolismo , Muramidasa/química , Muramidasa/metabolismo , Mioglobina/química , Mioglobina/metabolismo , Proteínas/metabolismo , beta-Lactamasas/química , beta-Lactamasas/metabolismoRESUMEN
We have recently investigated by far-UV circular dichroism (CD) the effects of Ca(2+) binding and the phosphorylation of Ser 81 for the synthetic peptide CaM [54-106] encompassing the Ca(2+)-binding loops II and III and the central alpha helix of calmodulin (CaM) (Arrigoni et al., Biochemistry 2004, 43, 12788-12798). Using computational methods, we studied the changes in the secondary structure implied by these spectra with the aim to investigate the effect of Ca(2+) binding and the functional role of the phosphorylation of Ser 81 in the action of the full-length CaM. Ca(2+) binding induces the nucleation of helical structure by inducing side chain stacking of hydrophobic residues. We further investigated the effect of Ca(2+) binding by using near-UV CD spectroscopy. Molecular dynamics simulations of different fragments containing the central alpha-helix of CaM using various experimentally determined structures of CaM with bound Ca(2+) disclose the structural effects provided by the phosphorylation of Ser 81. This post-translational modification is predicted to alter the secondary structure in its surrounding and also to hinder the physiological bending of the central helix of CaM through an alteration of the hydrogen bond network established by the side chain of residue 81. Using quantum mechanical methods to predict the CD spectra for the frames obtained during the MD simulations, we are able to reproduce the relative experimental intensities in the far-UV CD spectra for our peptides. Similar conformational changes that take place in CaM [54-106] upon Ca(2+) binding and phosphorylation may occur in the full-length CaM.
Asunto(s)
Calcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Dicroismo Circular , Cristalografía por Rayos X , Humanos , Técnicas In Vitro , Modelos Moleculares , Fragmentos de Péptidos/síntesis química , Fosforilación , Conformación Proteica , Estructura Secundaria de Proteína , TermodinámicaRESUMEN
The structural basis of lipid acyl-chain selection by membrane-intrinsic enzymes is poorly understood because most integral membrane enzymes of lipid metabolism have proven refractory to structure determination; however, robust enzymes from the outer membranes of gram-negative bacteria are now providing a first glimpse at the underlying mechanisms. The methylene unit resolution of the phospholipid:lipid A palmitoyltransferase PagP is determined by the hydrocarbon ruler, a 16-carbon saturated acyl-chain-binding pocket buried within the transmembrane beta-barrel structure. Substitution of Gly88 lining the floor of the hydrocarbon ruler with Ala or Met makes the enzyme select specifically 15- or 12-carbon saturated acyl chains, respectively, indicating that hydrocarbon ruler depth determines acyl-chain selection. However, the Gly88Cys PagP resolution does not diminish linearly because it selects both 14- and 15-carbon saturated acyl chains. We discovered that an exciton, emanating from a buried Tyr26-Trp66 phenol-indole interaction, is extinguished by a local structural perturbation arising from the proximal Gly88Cys PagP sulfhydryl group. Site-specific S-methylation of the single Cys afforded Gly88Cys-S-methyl PagP, which reasserted both the exciton and methylene unit resolution by specifically selecting 13-carbon saturated acyl chains for transfer to lipid A. Unlike the other Gly88 substitutions, the Cys sulfhydryl group recedes from the hydrocarbon ruler floor and locally perturbs the subjacent Tyr26 and Trp66 aromatic rings. The resulting hydrocarbon ruler expansion thus occurs at the exciton's expense and accommodates an extra methylene unit in the selected acyl chain. The hydrocarbon ruler-exciton juxtaposition endows PagP with a molecular gauge for probing the structural basis of lipid acyl-chain selection in a membrane-intrinsic environment.
Asunto(s)
Aciltransferasas/química , Sustitución de Aminoácidos , Aciltransferasas/genética , Aciltransferasas/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Sitios de Unión/genética , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Cisteína/química , Electroforesis en Gel de Poliacrilamida , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glicina/química , Lípido A/metabolismo , Metilación , Modelos Moleculares , Fosfolípidos/metabolismo , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Espectrometría de Masa por Ionización de Electrospray , Especificidad por Sustrato/genética , TermodinámicaRESUMEN
Core and linker histones are the most abundant protein components of chromatin. Even though they lack intrinsic structure, the N-terminal "tail" domains (NTDs) of the core histones and the C-terminal tail domain (CTD) of linker histones bind to many different macromolecular partners while functioning in chromatin. Here we discuss the underlying physicochemical basis for how the histone terminal domains can be disordered and yet specifically recognize and interact with different macromolecules. The relationship between intrinsic disorder and amino acid composition is emphasized. We also discuss the potential structural consequences of acetylation and methylation of lysine residues embedded in intrinsically disordered histone tail domains.
Asunto(s)
Aminoácidos/química , Histonas/química , Proteínas/química , Animales , Bioquímica/métodos , Supervivencia Celular , Cromatina/química , Humanos , Modelos Biológicos , Conformación Proteica , Isoformas de Proteínas , Estructura Terciaria de ProteínaRESUMEN
Detectable exciton couplets arising from heme-heme interactions in the hemoglobin (Hb) tetramers of HbO(2) and deoxyHb were predicted by DeVoe theory. This prediction was supported by the observation of an exciton couplet in the CD difference spectrum between the Hb tetramer and the alphabeta dimer of HbCO. In this paper, DeVoe theory is used to calculate the heme-heme interactions in the CO complex of the Hb tetramer (alpha(2)beta(2)) and dimer (alphabeta), the systems studied by Goldbeck et al. The couplet strength of the resulting theoretical CD difference spectrum agrees well with experiment, thus confirming that heme-heme interactions contribute significantly to the CD of HbCO. Given that the heme-heme distances in HbCO are 25 A and more, it is highly likely that heme-heme interactions also contribute significantly to the CD of other multi-heme proteins, e.g., cytochrome c(3), cytochrome oxidase, cytochrome bc(1), etc., where the hemes are in closer proximity.
Asunto(s)
Hemo/química , Hemoglobinas/química , Subunidades de Proteína/química , Dicroismo Circular , Dimerización , MatemáticaRESUMEN
The far-UV circular dichroism spectra of a series of amino acid derivatives containing single peptide bonds have been measured. The N-acetyl-alanine displays a polyproline (PP) II-like spectrum, but alaninamide shows a very weak positive signal. Similarly Gly-Ala shows a PPII spectrum, but Ala-Gly does not. On heating, the spectrum shows a two-state transition also shown by long PPII polypeptides. Thus the characteristic PPII negative maximum at <200 nm results from the coupling of a peptide bond N-terminal to the chiral alpha-carbon, and therefore the simplest peptide bonds have a preferred conformation that defines the spectrum of disordered proteins of any size.