Turn conformations in peptides containing the -Xaa-Ser- sequence.

The conformations of the protected dipeptides Boc-L-Pro-L-Ser-NHMe, Boc-L-Pro-D-Ser-NHMe, Boc-L-Val-L-Ser-NHMe and Boc-L-Val-D-Ser-NHMe have been explored through interpretation of their infrared spectra in CH2Cl2, DMSO and D2O solution. In CH2Cl2 solution the formation of a ten-membered ring (beta-turn) for each compound is signaled by characteristic shifts in both the urethane C = O and the terminal NH stretching frequencies. For each peptide, differences in the amide I absorption patterns for LL and LD isomers are consistent with the formation of type I and type II beta-turns respectively in CH2Cl2 solution. The amide I absorptions suggest substantial disruption of intramolecular hydrogen bonding in DMSO, and no intermolecular hydrogen bonding whatsoever in aqueous solution. In CH2Cl2 solution the OH stretching vibration is consistent with the formation of a hydrogen bond to the C = O of the serine group; however, two additional absorptions at frequencies characteristic of "free' OH groups also appear in all spectra. Implications regarding the serine in stabilizing the beta-turn are discussed.

A search for the ideal type I beta-turn.

In 1968 C. Venkatachalam (Biopolymers, Vol. 6, pp. 1425-1436) predicted the ideal forms of beta-turns (type I, type II, etc.) based entirely on theoretical calculations. Subsequently, over a thousand x-ray structures of different globular proteins have been analyzed, with results suggesting that the most important form among the hairpin conformers is the type I beta-turn. For the latter type of hairpin conformation, the original computations had predicted phi i+I = -60 degrees, psi i+1 = -30 degrees, phi i+2 = -90 degrees, and psi i +2 = 0 degrees as backbone torsion angle values, and these have been used from that time as reference values for the identification of the type I beta-turn. However, it has never been clarified whether these "ideal" backbone torsion angle values exist in real structures, or whether these torsion angles are only "theoretical values." Using the most recent release of the Protein Data Bank (1994), a survey has been made to assign amino acid pairs that approach the ideal form of the type I beta-turn. The analysis resulted in four sequences where the deviation from ideal values for any main-chain torsion angles was less than 2 degrees. In order to determine whether such a backbone fold is possible only in proteins owing to fortuitous cooperation of different folding effects, or whether it occurs even in short peptides, various attempts have been made to design the optimal amino acid sequence. Such a peptide model compound adopting precisely the predicted torsion angle values [phi i+1 = -60 degrees, psi i +1 = -30 degrees, phi i +2 = -90 degrees, and psi i+2 = 0 degrees] could provide valuable information. The solid state conformation of cyclo[(delta)Ava-Gly-Pro-Thr(OtBu)-Gly] reported herein, incorporating the -Pro-Thr- subunit, yields values suggesting that the "ideal" type I beta-turn is even possible for a peptide where there are no major environmental effects present.

Chaperone SecB: conformational changes demonstrated by circular dichroism.

The chaperone SecB, which is involved in protein export in Escherichia coli, is shown by circular dichroism measurements to contain a high content of beta-pleated sheets. Prediction of the secondary structure of SecB is in good agreement with the observed content of beta-sheet. In accordance with the previous studies in which changes in conformation were assessed indirectly [Randall (1992), Science 257, 241-245], here we show that the conformation of SecB changes with the concentration of salt in the milieu and also when SecB interacts with a peptide ligand.

Complexes of aluminium with peptide ligands: a Fourier transform IR spectroscopic study.

Aluminium has been recognized to be a neurotoxic agent and a risk factor in Alzheimer's disease and other neuronal dysfunctions. CD spectroscopic studies on two synthetic fragments of the human neurofilament protein midsized subunit (NF-M), and their alanine-for-serine-substituted and/or serine-phosphorylated derivatives showed the formation of stable, citric acid resistant complexes of Al3+ with peptide ligands [M. Hollósi, Z. M. Shen, A. Perczel, and G. D. Fasman (1994) Proc. Natl. Acad. Sci. USA, vol. 9, pp. 4902-4906]. In the case of Ser-phosphorylated fragments, a beta-sheet inducing effect of Ca2+ and Al3+ ions was observed. However, the serine-containing parent peptides, NF-M13 (KSPVPKSPVEEKG) and NF-M17 (EEKGKSPVPKSPVEEKG), failed to show CD spectral changes reflecting beta-sheet formation upon addition of Al3+ ions. On the basis of the amide I region of the Fourier transform ir spectra, in trifluoroethanol, the peptide backbone of NF-M17 and NF-M17 (A6A11) shows marked changes in the presence of Al3+. The most significant spectral differences are seen in the carboxyl region (> 1700 cm-1). The high-frequency component bands above 1760 cm-1 in both spectra belong to the C = O of undissociated CF3COOH. Another strong band at 1710 cm-1 which appears only in the spectrum of NF-M17 (A6A11)(NF-M17 with Ser6 and Ser11 replaced by Ala) can be assigned to the side chain or C-terminal COOH groups. The differential protonation state of the carboxyl groups in the two peptides suggests the formation of Al3+ complexes of different structure and stability.(ABSTRACT TRUNCATED AT 250 WORDS)

A poly(ethylene glycol) water-soluble conjugate of porin: refolding to the native state.

Porin, from Rhodabacter capsulatus, was chemically modified with methoxypoly(ethylene glycol) (m-PEG; molecular mass = 5000 Da) succinimidyl carbonate to yield methoxypoly(ethylene glycol)-porin (m-PEG-SC-Porin), as previously reported for bacteriorhodopsin [Sirokman, G., & Fasman, G. D. (1993) Protein Sci. 3, 1101-1170]. The m-poly(ethylene glycol)-porin (m-PEG-SC-Porin 50) conjugate, containing one poly(ethylene glycol) chain, was water soluble. The secondary structure of the conjugate in water was mainly random coil. Circular dichroism spectroscopy showed it was predominantly in the beta-pleated sheet structure in 0.6% octyltetraoxyethylene and 0.3 M LiCl, as was porin. A proteoliposome, containing the isolated porin conjugate, was prepared to measure permeability of the sugar stachyose. The m-PEG-SC-Porin 50 proteoliposome of porin maintained the permeability for the sugar, as did the proteoliposome of porin. The swelling rate of the conjugate versus the sugar was lower than it was for porin. This indicated that a pore in the conjugate exists but perhaps with a slightly different pore size. The refolding of the conjugate was studied by stepwise addition of trifluoroethanol (TFE) to lower the dielectric constant, simulating the insertion of porin into the membrane. An alpha-helical structure that did not exist in the native porin was formed with the m-PEG-SC-Porin 50, upon the addition of TFE, and the helicity increased with increasing concentrations of TFE. The m-PEG-SC-Porin 50 could be stepwise refolded to the native conformation, predominantly in the beta-sheet conformation, by the addition of hexafluoro-2-propanol in the 5-10% concentration range.(ABSTRACT TRUNCATED AT 250 WORDS)

Solubilization of beta-amyloid-(1-42)-peptide: reversing the beta-sheet conformation induced by aluminum with silicates.

Plaques are one of the two lesions found in the brain of patients with Alzheimer disease. Using a synthetic peptide corresponding to rat beta-amyloid-(1-42) (beta A4), circular dichroism (CD) analyses were performed to examine the effect of Na4SiO4 on the conformational state produced by Al3+. A previous study on fragments of neuronal proteins involved in tangle formation had shown a conformational transition from a beta-pleated sheet to a soluble random coil upon addition of Na4SiO4. In the present study, CD measurements showed that the beta-pleated sheet conformation of beta A4 induced by Al3+ was reversed to the random coil soluble form by the addition of Na4SiO4. The tight binding of SiO4(4-) with Al3+ provides the mechanism for this transition. These results provide insight into the role of aluminum in the Alzheimer diseased brain and suggests that investigation of the use of silicates as a therapeutic agent.

The solubilization of model Alzheimer tangles: reversing the beta-sheet conformation induced by aluminum with silicates.

Neurofibrillary tangles are one of two lesions found in the brain of Alzheimer disease victims. With synthetic peptide fragments of human neurofilament NF-M17 (Glu-Glu-Lys-Gly-Lys-Ser-Pro- Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly, phosphorylated and unphosphorylated), CD studies were done to examine the effect of sodium orthosilicate on the conformational state produced by Al3+ on fragments of neuronal proteins. Previous studies had shown a conformational transition from alpha-helix and random to beta-pleated sheet upon addition of Al3+ to both phosphorylated and unphosphorylated peptides. If sufficient quantities of Al3+ are added, the peptide precipitates from solution. The ability to reverse or slow the progression of aggregation was examined. Al3+ binding was reversed with 1-2 molar equivalents of sodium orthosilicate (with respect to Al3+), altering the conformation from beta-sheet to random coil and resulting in a CD spectrum similar to that of the initial peptide. The tight binding of the SiO4(4-) with the Al3+ provides the mechanism for this transition. These results provide additional information toward understanding the role of aluminum in the Alzheimer diseased brain and suggest the investigation of the possible use of silicates as a therapeutic agent.

Study of Al3+ binding and conformational properties of the alanine-substituted C-terminal domain of the NF-M protein and its relevance to Alzheimer's disease.

NF-M13 [H-(Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly)-OH], NF-M17 [H-(Glu-Glu-Lys-Gly-Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly) -OH], and their phosphorylated derivatives, representing the C-terminal phosphorylation domain of the neurofilament protein midsize subunit, have four possible binding sites for metal ions: the COO- group of glutamate, the OH group of the serine residue, the PO3H- group of phosphoserine (when present), and the COO- at the terminus of the peptide chain. The CD titration of the phosphorylated neurofilament fragments with Al3+ and Ca2+ yielded a significant conformational change that resulted in conformations containing high beta-pleated-sheet contents, which precipitate on standing (intermolecular complex). Al3+ binding to the unphosphorylated NF-M13 and NF-M17 did not exhibit this behavior. Several alanine analogues of the parent NF-M17 peptide were synthesized in order to determine the relationship between metal ions and possible binding sites. CD titration of analogues with Ca2+ indicated that the critical residues of NF-M17 for Ca(2+)-induced conformational changes, from random to beta-pleated sheet, are the N-terminal serine or both phosphorylated serines. Al(3+)-induced conformational changes suggest that the critical sites of NF-M17 yielding the beta-pleated-sheet structure are the four glutamates or phosphorylated serines, especially the C-terminal SerP. On the basis of the titration data, it is very likely that analogues with a serine in position 11 form a stable intramolecular complex with Al3+ that, however, does not result in the adoption of the beta-conformation. Back-titration with citric acid fails to reverse the Al(3+)-induced conformational changes of the phosphorylated peptides. The above results, especially the possible formation of intramolecular and intermolecular Al3+ complexes, may have relevance to the molecular mechanism, through which the neurotoxin Al3+ gives rise to the formation of neurofilament tangles.

Stable intrachain and interchain complexes of neurofilament peptides: a putative link between Al3+ and Alzheimer disease.

The etiologic role of Al3+ in Alzheimer disease has been controversial. Circular dichroism (CD) spectroscopic studies on two synthetic fragments of human neurofilament protein mid-sized subunit (NF-M), NF-M13 (KSPVPKSPVEEKG) and NF-M17 (EEKGKSPVPKSPVEEKG), and their alanine-substituted and/or serine-phosphorylated derivatives were carried out in an attempt to find a molecular mechanism for the effect of Al3+ to induce aggregation of neuronal proteins or their catabolic fragments. Al3+ and Ca2+ ions were found to induce beta-pleated sheet formation in the phosphorylated fragments. The cation sensitivity depended on the length and charge distribution of the sequence and site of phosphorylation. Al3+-induced conformational changes were irreversible to citric acid chelation, whereas Ca(2+)-induced conformational changes were reversible with citric acid. Studies of the alanine derivatives demonstrated which residues affected Al3+ or Ca2+ binding. Peptides containing at least one free (nonphosphorylated) serine residue were shown to form an intramolecular Al3+ complex, rather than an intermolecular one. In the intramolecular (intrachain) complex, the ligand function of the deprotonated serine hydroxyl was delineated [(Al.pepH-1)-type complex]. Ca2+ ions did not show a tendency for intramolecular complexing. The potential role of Al3+ in Alzheimer disease tangle and plaque formation is strongly suggested.

CD and Fourier transform ir spectroscopic studies of peptides. II. Detection of beta-turns in linear peptides.

Comparative CD and Fourier transform ir (FTIR) spectroscopic data on N-Boc protected linear peptides with or without the (Pro-Gly) beta-turn motif (e.g., Boc-Tyr-Pro-Gly-Phe-Leu-OH and Boc-Tyr-Gly-Pro-Phe-Leu-OH) are reported herein. The CD spectra, reflecting both backbone and aromatic contributions, were not found to be characteristic of the presence of beta-turns. In the amide I region of the FTIR spectra, analyzed by self-deconvolution and curve-fitting methods, the beta-turn band showed up between 1639 and 1633 cm-1 in trifluoroethanol (TFE) but only for models containing the (Pro-Gly) core. This band was also present in the spectra in chloroform but absent in dimethylsulfoxide. These findings, in agreement with recent ir data on cyclic models and 3(10)-helical polypeptides and proteins in D2O [see S. J. Prestrelski, D. M. Byler, and M. P. Thompson (1991), International Journal of Peptide and Protein Research, Vol. 37, pp. 508-512; H. H. Mantsch, A. Perczel, M. Hollósi, and G. D. Fasman (1992), FASEB Journal, Vol. 6, p. A341; H. H. Mantsch, A. Perczel, M. Hollósi, and G. Fasman (1992), Biopolymers, Vol. 33, pp. 201-207; S. M. Miick, G. V. Martinez, W. R. Fiori, A. P. Todd, and G. L. Millhauser (1992), Nature, Vol. 359, pp. 653-655], suggest that the amide I band, with a major contribution from the acceptor C = O of the 1<--4 intramolecular H bond of beta-turns, appears near or below 1640 cm-1, rather than above 1660 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)

FT-IR spectroscopy indicates that Ca(2+)-binding to phosphorylated C-terminal fragments of the midsized neurofilament protein subunit results in beta-sheet formation and beta-aggregation.

The Fourier-transform infrared (FT-IR) spectra in trifluoroethanol (TFE) of phosphorylated peptides, NF-M17(Ser6P) and NF-M17(Ser11P) representing the C-terminal repeating domain of the midsized neurofilament protein subunit (NF-M) have been measured. In the absence of Ca2+ ions both phosphopeptides adopt predominantly aperiodic conformation with minor amounts of beta-turns, 3(10) helix or beta-pleated sheet. Addition of Ca(ClO4)2 results in opaque solutions, and in the case of the Ser6P peptide, precipitation. The infrared spectra of the supernatants reflect the presence of unordered and beta-sheet structure. The infrared spectrum of the solid Ca(2+)-complex of NF-M17 (Ser6P) in a KBr pellet shows amide component bands at 1654 (alpha-helix or loops) and 1626.5 cm-1 (beta-sheet). The high intensity of the beta-sheet component suggests extensive beta-aggregation. The data reported herein give an infrared spectroscopic support to our previous findings that upon Ca2+ binding, phosphorylated NF-M fragments undergo a marked conformational change which gives rise to partial beta-sheet formation and beta-aggregation. This observation may have relevance to the molecular events which lead to the accumulation of abnormal proteineous structures in Alzheimer's disease.

Distinguishing transmembrane helices from peripheral helices by circular dichrosim.

The interpretation of the circular dichroism (c.d.) spectra of proteins to date requires additional secondary structural information of the proteins to be analysed (e.g., X-ray or n.m.r. data). Therefore these methods are inappropriate for a c.d. database whose secondary structures are unknown, as in the case of the membrane proteins. The Convex Constraint Analysis algorithm [Perczel, Hollósi, Tusnady, and Fasman (1991) Convex constraint analysis: a natural deconvolution of circular dichroism curves of proteins. Protein Eng. 4, 669-679] operates on a collection of c.d. spectral data to extract the common spectral components with their spectral weights. The linear combinations of these derived 'pure' c.d. curves can reconstruct the original data set with great accuracy. For a membrane protein data set, the five-component spectra so obtained from the deconvolution consisted of two different types of alpha-helices (the alpha-helix in the soluble domain and the alpha1-helix, for the transmembrane alpha-helix), a beta-pleated sheet, a class-C-like spectrum related to beta-turns and a spectrum correlated with the unordered conformation. The deconvoluted c.d. spectrum for the alpha1-helix was characterized by a positive red-shifted band in the range 195-200 nm (+95,000 degrees.cm2-dmol-1), with the intensity of the negative band at 208 nm being slightly less negative than that of the 222 nm band (-50,000 and -60,000 degrees.cm2.dmol-1 respectively) in comparison with the regular alpha-helix, with a positive band at 190 nm and two negative bands at 208 and 222 nm with magnitudes of +70,000, -30,000 and -30,000 degrees.cm2.dmol-1 respectively.

Dynamic structural and functional relationships in recombinant plasminogen activator inhibitor-1 (rPAI-1).

The conformational characteristics of active, latent, and denatured recombinant plasminogen activator inhibitor-1 (rPAI-1) were compared using UV spectroscopy, spectrofluorimetry and circular dichroism (CD) techniques. The UV absorbance wavelength maxima in all preparations approximated 280 nm, while the extinction coefficients of active and latent rPAI-1 differed by up to 60%. When incubated at 37 degrees C, the A280 of latent rPAI-1 was quite stable while the A280 of active rPAI-1 spontaneously increased, eventually approximating that of latent rPAI-1. Alkali difference spectroscopy yielded markedly divergent titration patterns for active and latent rPAI-1, suggesting that the tyrosine residues present in active and latent rPAI-1 differ in terms of solvent exposure. At an excitation wavelength of 280 nm, active rPAI-1 exhibited the greatest relative fluorescence quantum yield. The relative fluorescence of latent and denatured rPAI-1 were less than that of active PAI-1, and the emission maxima of both species were slightly red-shifted in comparison to that of active rPAI-1, suggesting that at least one of the four tryptophan residues present in rPAI-1 is less exposed to the aqueous environment in the active form of the molecule. In contrast, the derived secondary structures based on CD of active and latent rPAI-1 were nearly identical, with both moieties exhibiting approx. 40% alpha-helix and 15% beta-sheet. Taken together, these spectroscopic data provide evidence supporting the hypothesis that active and latent PAI-1 differ in terms of their tertiary conformation and aromatic residue exposure, while their secondary structures appear generally comparable. Furthermore, denaturant-induced reactivation of latent rPAI-1 produces a partially active rPAI-1 with spectroscopic properties similar to that of latent rPAI-1, suggesting that denatured rPAI-1 more closely resembles the latent rPAI-1 conformation after refolding. The spontaneous spectroscopic changes observed in rPAI-1 may reflect conformational transitions that are critical to the regulation of endogenous PAI-1 activity.

Refolding and proton pumping activity of a polyethylene glycol-bacteriorhodopsin water-soluble conjugate.

Bacteriorhodopsin (BR), from the purple membrane (PM) of Halobacterium halobium, was chemically modified with methoxypolyethylene glycol (m-PEG; molecular weight = 5,000 Da) succinimidyl carbonate. The polyethylene glycol-bacteriorhodopsin (m-PEG-SC-BR33) conjugate, containing one polyethylene glycol chain, was water soluble. The secondary structure of the conjugate in water appeared partially denatured, but was shown to contain alpha-helical segments by circular dichroism spectroscopy. The isolated bacteriorhodopsin conjugate, with added retinal, was refolded in a mixed detergent-lipid micelle and had an absorption maximum at 555 nm. The refolded conjugate was transferred into vesicles that pumped protons, upon illumination, as efficiently as did native BR. Modification of the PM with m-PEG did not alter the native structure or inhibit proton pumping, and therefore it is suggested that the glycol polymer is present as a moiety covalently linked to residues unnecessary for proton pumping and proper folding. The site of attachment of m-PEG was determined to be at either Lys 129 or Lys 159, with position Lys 129 the most probable site of attachment. The m-PEG-SC-BR33 could be stepwise refolded to the native conformation by the addition of trifluoroethanol to lower the dielectric constant, simulating the insertion of the BR into the phospholipid bilayer.

Synthesis and conformational analysis of N-glycopeptides. II. CD, molecular dynamics, and NMR spectroscopic studies on linear N-glycopeptides.

The comprehensive structural analysis reported herein of eight N-glycopeptides, in three different solvents, is based on quantitative CD experiments, homonuclear nuclear Overhauser effect measurements, and molecular dynamics (MD) calculations. Although several orientations of the two amide planes attached to the carbohydrate pyranose ring are possible, according to NOE, CD data, and MD simulations, of all of the glycopeptide models, regardless of the type of the carrier peptide, only one dominant conformer population was found. This conformer is characterized by a nearly trans orientation of the CH and NH hydrogens of both acetamido groups. This finding is in perfect agreement with x-ray crystallographic data on the solid state conformation of the 1-N-acetyl- and 1-N-(beta-aspartyl)-2-acetamido-2-deoxy-beta-D-glucopyranosyla min e. The precise identification of this dominant conformer of N-glycopeptides in solution was the major question addressed herein by the structural analyses. A "CD additivity" experiment was carried out using an equimolar solution of Boc-Pro-Asp-NHCH3 and 1-N-acetyl-3,4,6- tri-O-acetyl-2-acetamido-2-deoxy-beta-D-glucopyranosylamine at ambient temperature in acetonitrile. The CD spectrum obtained from the equimolar solution of the above two molecules (the "spectroscopic sum") was identical with the CD curve obtained from the algebraic summation of the individually recorded CD spectra of the peptide and the carbohydrate moiety ("mathematical sum"). The global picture of the CD spectral analyses of the eight parent peptides with the eight N-glycopeptides revealed that in trifluoroethanol and acetonitrile, the side-chain modification of the Asn models (natural N-glycopeptide analogues) by N-glycosylation has a significant effect on the conformation of the carrier peptide, resulting in a decrease in the original type I beta-turn content. Simultaneously, the type II beta-turn conformational percentage increased to approximately 20%. Such a conformational ratio change seems to be larger than the expected errors arising from the CD analyses, and agrees with the results of MD calculations. N-glycosylation of Asn residues causes perturbations, not only through the covalent bond, but also through specific hydrogen bonds between the backbone and side chain atoms. CD spectroscopy, augmented by efficient CD curve deconvolution techniques, has proved to be a useful tool for studying multicomponent conformer mixtures of small linear peptides in solution and changes of conformational equilibria caused by N-glycosylation.

The evaluation of type I and type II beta-turn mixtures. Circular dichroism, NMR and molecular dynamics studies.

Circular dichroism (CD) and 1H-(1H)NOE spectra were obtained for Piv-Pro-Ser-NHCH3 (1), [Piv-(CH3)3-C-CO], Boc-Pro-Ser-NHCH3 (2) and Boc-Val-Ser-NHCH3 (3), to determine the solution conformation of these beta-turn models. In the crystal, 1 and 3 adopt an ideal type I beta-turn, while 2 is characterized by a semifolded backbone geometry incorporating a cis Boc-Pro tert-amide bond. The predominance of a beta-turn conformation in solution was suggested for models 1-3 on the basis of 1H-(1H)NOE data. In a nonpolar solvent the prevailing trans rotamer form (> 80%) of 2 has a beta-turn conformation according to heteronuclear NOE measurement. Positive 1H-(1H)NOEs were detected between the H alpha(Pro)/NH(Ser), H alpha(Ser)/NH(Ser) and NH(NHCH3)/HN(Ser) protons in the trans Boc-Pro rotamer form of 2 at -20 degrees in CDCl3. Similar positive homonuclear NOE enhancements were also observed on the appropriate proton signals in other models, such as Boc-Val-Ser-NHCH3 (3), Boc-Val-D-Ser-NHCH3 (4) and Boc-Pro-D-Ser-NHCH3 (5), in various solvents. The 1H-(1H)NOE experiments carried out in CD3CN clearly showed that besides the type I (or III) beta-turn structure, one of the main conformations of models 1-5 is close to the type II beta-turn backbone geometry in a nonpolar solvent. Unexpectedly, the conformational mixture of models 1-3 were characterized by class C (helix-like) CD spectra, although class C spectra are generally only correlated with the type I beta-turn conformation. These acyclic models are the first carefully investigated examples of -L-L- triamide systems, containing a significant amount of a type II beta-turn, as well as the type I beta-turn and, however, yielding a class C circular dichroism spectra. The CD spectra recorded for 3 and 4 in acetonitrile were 'calibrated' using the 1H-(1H)NOE data. Such a "calibration", as well as the semi-quantitative CD and NMR comprehensive analyses, demonstrated that class C, class B, as well as class C' CD spectra may be obtained from the linear combination of the same two-component spectra, with different conformational weights. Therefore, it is suggested that the extraction of the conformational components of such models, simply on the basis of their CD spectra, must be made with caution.

Ca(2+)-induced conformational transitions of phosphorylated peptides.

CD spectroscopic studies on protected peptides containing lysine and serine, or phosphoserine, and on serine-containing fragments of the neurofilament protein midsized subunit, both in the unphosphorylated and phosphorylated form, are reported. The introduction of the phosphoryl group was not found to have a significant spectral effect in aqueous solution. In trifluoroethanol (TFE), spectral shifts toward unordered (type U) spectra or the appearance of distorted spectra likely reflect the adoption of aperiodic polypeptide conformations due to salt bridge(s) between negatively charged phosphoserine and positive lysine side-chain groups. A turn-stabilizing effect of phosphorylation was also observed. CD-monitored titration experiments in TFE revealed a high conformational sensitivity of phosphopeptides toward Ca2+ ions. The appearance of the unordered spectra or spectral shifts were the sign of a bulk disordering effect of Ca2+ ions. Spectra with specific spectroscopic features reflect the formation of Ca2+ complexes and the adoption of ordered unique backbone conformations. When ordered structures were obtained on addition of Ca2+ ions, the observed CD curves showed a resemblance to the spectrum of beta-pleated sheets. This may originate from chain extension and the formation of beta-pleated sheet segments fixed by Ca2+ bridges between PO3H-1 groups of adjacent peptide chains. The data clearly show that the effect of the Ca2+ ions is highly specific: the sequence, chain length, presence and distribution of charged side-chain groups, degree and site of phosphorylation, and environmental factors appear to be determining in the process of chain extension or beta-sheet formation.

Conformational change of chaperone Hsc70 upon binding to a decapeptide: a circular dichroism study.

The conformation of bovine Hsc70, a 70-kDa heat shock cognate protein, and its conformational change upon binding to decapeptides, was studied by CD spectroscopy and secondary structure prediction (Chou, P.Y. & Fasman, G.D., 1974, Biochemistry 13, 222-245). The CD spectra were analyzed by the LINCOMB method, as well as by the convex constraint analysis (CCA) method (Perczel, A., Park, K., & Fasman, G.D., 1992, Anal. Biochem. 203, 83-93). The result of the CD analysis of Hsc70 (15% alpha-helix, 24% beta-sheet, 24% beta-turn, and 38% remainder) was very similar to the predicted secondary structure for the beta-sheet (24%) and the beta-turn (29%). However, there is disagreement between the alpha-helical content by CD analysis (15%) and the predicted structure (30%). In spite of the fact that the decapeptides contained a considerable amount of beta-sheet (22%), the interaction of the heat shock protein with the peptide resulted in an overall decrease in the content of beta-sheet conformation (-15%) of the complex. This may be due to induction of a molten globule state. The result of the CCA analysis indicated that the Hsc70 undergoes a conformational change upon binding the decapeptides.

Characterization of beta-turns in cyclic hexapeptides in solution by Fourier transform IR spectroscopy.

The beta-turn represents a structural element frequently encountered in globular proteins. However, in spite of various theoretical and experimental studies the ir signature bands of pure beta-turns are still not established beyond doubt. Although considerable information exists now on the ir spectra of alpha-helical and beta-sheet structures, the lack of knowledge concerning turn structures in general, and that of beta-turns in particular, presents a major uncertainty in the estimation of global protein secondary structures from ir spectroscopic data. To obtain more specific information about the characteristic amide bands in beta-turns, we report herein an ir spectroscopic analysis of a series of five cyclic pseudo-hexapeptides known to form beta-turns from previous CD and nmr studies [A. Perczel, M. Hollósi, B. M. Foxman, and G. D. Fasman (1991) Journal of the American Chemical Society, Volume 113, pp. 9772-9784]. We show here that in these cyclic peptides the amide groups involved in beta-turns that comprise a ten-membered hydrogen-bonded ring (and represent the first H-bond pair in a beta-sheet), give rise to characteristic amide I bands in the range 1638-1646 cm-1, with the exact position depending on the solvent and the nature of the side-chain substituents.