Characterization of two sequential polytripeptides containing glycine and ethyl-glutamate residues

The polytripeptides poly(glu(OEt)-gly-gly) and poly(glu(OEt)-glu(OEt)-gly) have been synthesized and characterized using infrared linear dichroism, ultraviolet circular dichroism spectroscopy, and electron microscopy. Both polymers have been obtained in a cross β conformation with antiparallel chains, however, there appear to be significant differences in the stability of this conformation for the two polymers. Poly(glu(OEt)-gly-gly) has also been shown to exist in a 3₁ polyglycine II type helix in aqueous solution whereas films cast from this solution appear to be random. Finally this same polymer can be obtained in either a random or an extended form trifluoroacetic acid depending on the nature of the substrate used for film casting.     

Conformational Behavior of Chitosan in the Acetate Salt: An X-Ray Study

A well-defined X-ray fiber pattern of chitosan acetate was obtained by immersing a tendon chitosan, prepared from a crab tendon chitin by a solid-state N-deacetylation, in an aqueous acetic acid-isopropanol solution at 110°C. This pattern was very similar to that of chitosan salts with some inorganic acids, such as HF, HCl, and H₂SO₄, in which chitosan chains form an 8/5 helix, indicating that chitosan acetate also take up this conformation. This information may give an influential clue to the chitosan conformation in the aqueous acetic acid solution, the most popular solvent for chitosan. However, after one month of storage of the chitosan acetate, the fiber pattern, the density and its IR spectrum changed to those of the anhydrous polymorph of chitosan, suggesting that the acetic acid was removed accompanied with water molecules from the crystal during storage and that the polymorph can be obtained not only by annealing chitosan, but also through the chitosan acetate.     

Raman spectroscopic study of mechanically deformed poly-L-alanine

Raman spectroscopy has been used in investigating the conformational transitions of poly-L -alanine (PLA) induced by mechanical deformation. We see evidence of the alpha-helical, antiparallel beta-sheet, and a disordered conformation in PLA. The disordered conformation has not been discussed in previous infrared and X-ray diffraction investigations and may have local order similar to the left-handed 3₁ poly glycine helix. The amide III mode in the Raman spectrum of PLA is more sensitive than the amide I and II modes to changes in secondary structure of the polypeptide chain. Several lines below 1200 cm^?1 are conformationally sensitive and may generally be useful in the analysis of Raman spectra of proteins. A line at 909 cm^?1 decreases in intensity after deformation of PLA. In general only weak scattering is observed around 900 cm^?1 in the Raman spectra of antiparallel beta-sheet polypeptides. The Raman spectra of the amide N–H deuterated PLA and poly-L -leucine (PLL) in the alpha-helical conformation and poly-L -valine (PLV) in the beta-sheet conformation are presented. Splitting is observed in the amide III mode of PLV and the components of this mode are assigned. The Raman spectrum of an alpha-helical random copolymer of L -leucine and L -glutamic acid is shown to be consistent with the spectra of other alphahelical polypeptides.     

Conformational studies on Cu(II) polycomplexes of pyridoxal Schiff bases of polypeptides

Structures of Cu(II) complexes of pyridoxal Schiff bases with poly(L -lysine), poly(L -ornithine), and poly(L -α,γ-diaminobutyric acid) were investigated by absorption spectra, CD, and conformational analysis. Although the polypeptides retain their typical right-handed α-helical conformation, opposite Cotton effects were found for the poly(L -lysine) and poly(L -ornithine) polycomplexes in the whole range of wavelengths from 600 to 250 nm. As in the analogous derivatives of salicyladehyde, this effect seems to be due to a stereospecific binding of the square planar Cu(II)-bis-pyridoxylideneimine group to the α-helical matrix. Circular dichroism spectrum of poly(L -α,γ-diaminobutyric acid) polycomplex is similar to that found for poly(L -lysine) derivative, but indicates large tetrahedral distortion of the square-planar coordination of copper ion.     

Raman spectroscopic study of poly (β-benzyl-L-aspartate) and sequential polypeptides

Poly-β-benzyl-L -aspartate (poly[Asp(OBzl)]) forms either a lefthanded α-helix, β-sheet, ω-helix, or random coil under appropriate conditions. In this paper the Raman spectra of the above poly[Asp(OBzl)] conformations are compared. The Raman active amide I line shifts from 1663 cm^?1 to 1679 cm^?1 upon thermal conversion of poly[Asp(OBzl)] from the α-helical to β-sheet conformation while an intense line appearing at 890 cm^?1 in the spectrum of the α-helix decreases in intensity. The 890 cm^?1 line also displays weak intensity when the polymer is dissolved in chloroform–dichloroacetic acid solution and therefore is converted to the random coil. This line probably arises from a skeletal vibration and is expected to be conformationally sensitive. Similar behavior in the intensity of skeletal vibrations is discussed for other polypeptides undergoing conformational transitions. The Raman spectra of two cross-β-sheet copolypeptides, poly(Ala-Gly) and poly(Ser-Gly), are examined. These sequential polypeptides are model compounds for the crystalline regions of Bombyx mori silk fibroin which forms an extensive β-sheet structure. The amide I, III, and skeletal vibrations appeared in the Raman spectra of these polypeptides at the frequencies and intensities associated with β-sheet homopolypeptides. Since the sequential copolypeptides are intermediate in complexity between the homopolypeptides and the proteins, these results indicate that Raman structure–frequency correlations obtained from homopolypeptide studies can now be applied to protein spectra with greater confidence. The perturbation scheme developed by Krimm and Abe for explaining the frequency splitting of the amide I vibrations in β-sheet polyglycine is applied to poly(L -valine), poly-(Ala-Gly), poly(Ser-Gly), and poly[Asp(OBzl)]. The value of the “unperturbed” frequency, V₀, for poly[Asp(OBzl)] was significantly greater than the corresponding values for the other polypeptides. A structural origin for this difference may be displacement of adjacent hydrogen-bonded chains relative to the standard β-sheet conformation.     

Conformational analysis of an ionizable polydipeptide

A semi-empirical conformational energy calculation has been performed on the ionizable polydipeptide poly(Glu-Ala). The results indicate that; (1)the ionized polymer assumes the lefthanded extended helix conformation is aqueous solution; (2) the poly(Glu-Ala) extended helix is less stable than that of polyglutamic acid; (3) the unionized polydipeptide will preferentially assume the “β-helical” conformation (isolated 2₁ degenerate helix) in aqueous solution. These conclusions are supported by experiment.     

Conformational study on poly [gamma-(alpha-phenethyl)-L-glutamate] using vibrational circular dichroism spectroscopy

Vibrational circular dichroism (VCD) and IR absorption spectra are obtained in a chloroform solution for poly[gamma-((R)-alpha-phenethyl)-L-glutamate] (PRPLG) and poly[gamma-((S)-alpha-phenethyl)-L-glutamate] (PSPLG), whose only structural difference is an opposite chiral center in the side chain. Their characteristic amide A, I, and II bands show VCD patterns quite similar to those of poly[gamma-benzyl-L-glutamate] (PBLG), indicating that the secondary structure of these polypeptides is a right-handed alpha-helix. The VCD spectra in the CH stretching region exhibit different patterns for PRPLG and PSPLG, reflecting the chirality difference in the side chains. This difference is interpreted on the basis of the additivity of optical activity contributions from the main chain conformation and the chirality difference in the side chains. The results indicate that a VCD difference spectrum of the CH stretching region is a useful diagnostic tool for elucidating local chirality differences.     

Carrier design: Conformational studies of amino acid (X) and oligopeptide (X-DL-Alam) substituted poly(L-lysine)

The present study was undertaken to examine the influence of the reversal of the sidechain sequential order on the conformation of branched polypeptides. At the same time, the influence of the optically active amino acid joined directly to the poly (L -Lys) backbone and the DL -Ala oligomer grafted as chain-terminating fragment were separately analyzed. Therefore two sets of polypeptides were synthesized corresponding to the general formula poly [Lys-(X_i,)] (XK) and poly[Lys-(DL -Ala_m-X_i)] (AXK) when X = Ala, D -Ala, Leu, D -Leu, Phe, D -Phe, Ile, Pro, Glu.,D -Glu, or His. For coupling amino acid X to polylysine, three types of active ester methods were compared: the use of pentafluorophenyl or pentachlorophenyl ester, and the effect of the addition of an equimolar amount of 1-hydroxybenzotriazole. After cleavage of protecting groups, AXK polypeptides were synthesized by grafting short oligo (DL -Ala) chains to XK by using N-carboxy-DL -Ala anhydride. The CD measurements performed in water solutions of various pH values and ionic strengths were used for classification of the polypeptide conformations as either ordered (helical) or unordered. Different from what was observed with the unsubstituted poly (L -Lys), poly[Lys-(X_i)] type polypeptides can adopt ordered structure even under nearly physiological conditions (pH 7.3, 0.2M NaCl). These data suggest that the introduction of amino acid residue with either (ar) alkyl side chain (Ala, Leu, Phe) or negatively charged side chain (Glu) promotes markedly the formation of ordered structure. Comparison of chiroptical properties of poly [Lys- (DL -Ala_m-X_i)] and of poly [Lys- (X_i)] reveals that side-chain interactions play an important role in the stabilization of ordered solution conformation of AXK type branched polypeptides. The results give rather conclusive evidence that not only hydrophobic interactions, but also ionic attraction, can be involved in the formation and stabilization of helical conformation of branched polypeptides. © 1993 John Wiley & Sons, Inc.     

Circular dichroism of collagen, gelatin, and poly(proline) II in the vacuum ultraviolet.

Circular dichroism spectra for acid-soluble calfskin collagen, gelatin, and poly(proline) II in solution have been extended into the vacuum ultraviolet region. The extended spectrum of gelatin reveals that the circular dichroism of this unordered polymer is more closely related to the spectrum of charged polypeptides than might be evident from near ultraviolet work. A short-wavelength band is found at about 172 nm, which corresponds in position, magnitude, and sign to a band recorded earlier for poly(L -glutamic acid) at pH 8.0. This band is observed in a helical structure for the first time in the vacuum ultraviolet circular dichroism and absorption spectra of poly(proline) II. Both circular dichroism and absorption spectra point to the assignement of this band as the nσ*. Neither the nσ* nor the expected positive lobe of the ππ* helix band is observed in the extended circular dichroism spectrum of collagen. We postulate that these two bands cancel here in analogy to the case of α-helical poly(L -glutamic acid).     

Tat cell-penetrating peptide has the characteristics of a poly(proline) II helix in aqueous solution and in SDS micelles.

Tat cell-penetrating peptide (GRKKRRQRRRPPQG) is able to translocate and carry molecules across cell membranes. Using CD spectroscopy the conformation of this synthetic peptide was studied in aqueous and membrane-mimicking, micellar SDS solutions at different temperatures. The CD spectrum of the Tat cell-penetrating peptide in SDS micellar solution was virtually unchanged from that in aqueous solution, and at low temperature it was close to that of a poly(proline) II helix.     

Sequential polypeptides containing arginine as histone models: synthesis and conformational studies

The sequential polypeptides (L -Arg-X-Gly)_n, where X represents amino acid residues Ala, Val, and Leu, were prepared as models of arginine-rich histones to be used in studying their structure and their interactions with DNA. The polymerization was carried out on the pentachlorophenyl active esters of the appropriate tripeptides, while the toluene-4-sulfonyl group was used for protecting the arginine guanido group. CD was employed to investigate the conformation of (L -Arg-X-Gly)_n polymers in aqueous solutions, at different pH, as well as in trifluoroenthanol and hexafluoroisopropyl alcohol solutions. In aqueous solutions (at pH 7 and 12) the prepared sequential polymers behaved as a random coil. The CD spectra in various trifluoroethanol–water or hexafluoroisopropyl alcohol–water mixtures indicated that the degree of helical conformation of the studied polytripeptides increased in the order of Ala → Val → Leu. The opposite was true for the β-structure. Characteristics of β-turn are excluded from the poly(L -Arg-L -Leu-Gly), which assumed the most pronounced helical conformation. The poly(L -Arg-L -Val-Gly) exerts a significant preference to the β-turn structure compared to that of poly(L -Arg-L -Ala-Gly). Thus the probability for helical, β-structure or β-turn conformations of the polymers was analyzed in relation to the bulkiness and length, and to the special features of the X-residue side chain (β-branching). We concluded that the prepared sequential arginine-containing polypeptides are plausible models for histone fractions, f₃ and f₂α₁.     

13C NMR structural investigation of scleroglucan

This paper concerns the ¹³C NMR signal assignment in the DMSO of a neutral polysaccharide, scleroglucan. The previously proposed chemical structure is confirmed. The ¹³C NMR spectrum shows that scleroglucan is a regular poly (A, B, C, D) type glucan. The relaxation times of the different series of carbon atoms demonstrate that a single, pendant glucose group is attached to each third monomer along the main chain of what is a β(1 → 3)-glucan. Partial acid hydrolysis gives a spectrum analogous to that of the β(1 → 3)-d-glucan, curdlan, and confirms the structure of the polymer backbone.In aqueous solution, no signal has been obtained due to the existence of a rigid, ordered conformation as demonstrated by optical rotation; in the presence of sodium hydroxide, a conformational transition is produced just as with curdlan. The conclusion is that the behaviour of scleroglucan in solution is similar to that of other β(1 → 3)-d-glucans even though it is more soluble.     

Heparin-polypeptide interactions in aqueous solution

Circular dichroism spectroscopy has been used to study the interactions between heparin and cationic polypeptides in dilute aqueous solution at neutral pH. The results indicate that poly(l-lysine), poly(l-arginine), and poly(l-ornithine) adopt the α-helical conformation in the presence of heparin, rather than the “charged coil” form observed for the polypeptide alone under the same conditions. Maximum interaction for the poly(l-lysine) and poly(l-ornithine) systems occur at an amino acid: disaccharide residue ratio of 2.3 ± 0.1:1, which correlates with the analytical data of 2.3 sulfates per heparin disaccharide. For poly(l-arginine), maximum interaction occurs at a residue ratio of 3.3 ± 0.1:1, and indicates that all the anionic groups (sulfate and carboxyl) of the heparin are involved in this case.The interactions of heparin are analogous to those observed previously for six connective tissue mucopolysaccharides, except that none of the latter had any effect on the conformation of poly(l-ornithine). The poly(l-ornithine)-heparin system shows a thermal “melting” transition at T_m = 56.0 ± 1.0 °C, at which point the polypeptide reverts to the “charged coil” form; the interactions with poly(l-lysine) and poly(l-arginine) are stable up to temperatures > 90 °C. The high thermal stability of these conformation-directing effects indicate a stronger interaction for heparin than the other mucopolysaccharides, which is probably due to the high sulfate content.     

Étude thermodynamique de la transition hélice–pelote statistique en solvants mixtes de différents esters de l'acide poly(L-glutamique)

Helix–coil transition of poly(γ-methyl-L -glutamate), poly(γ-ethyl-L -glutamate), and poly(γ-benzyl-L -glutamate) has been studied in mixed solvents by calorimetry, polarimetry, and viscometry. The experimental data have allowed the evaluation of solvation enthalpy Δh_b, equilibrium constant K for hydrogen bond formation between the active solvent component and CO and NH groups, and the cooperativity parameter σ. The conformational transition of polypeptides in solution in a mixed solvent containing enough active solvent to maintain the coiled conformation has been produced by dilution with the helix-supporting solvent for the measurements of enthalpy of transition Δh_s. The average value for Δh_s is 3550 ± 300 J/mol and is practically independent of the nature of the side chain for the dichloroacetic acid-ethylene dichloride solvent pair at 25°C. A noticeable concentration effect exists in the case of poly(γ-benzyl-L -glutamate). The helical conformation is less stable for poly(γ-ethyl-L -glutamate), and this is explained by a steric effect hindering the access of dichloroacetic acid to side chains. Constant K has been calculated using polarimetric data and also from values of Δh_s obtained at different temperatures using the Bixon and Lifson theory on the one hand and that of Sayama and coworkers on the other hand. Values of σ for poly(γ-ethyl-L -glutamate) have been calculated according to both theories mentioned, and the results show that the two sets of values are quite similar. The constant σ depends on the nature of the active solvent, on temperature, and on the binary-solvent composition. These conclusions are confirmed by viscometric results. Values of Δh_b calculated from constant K are 5230 J/mol when Bixon and Lifson theory is used and 5569 J/mol when the theory at Sayama and coworkers is used. In both cases the value for Δh_b is much lower than that of an intramolecular hydrogen bond. Experimental results suggest that the solvation mechanism would proceed in a manner so that mechanisms described in both theories are involved.     

Sequential 1H-NMR assignments and secondary structure of the sea anemone polypeptide anthopleurin-A

The sequence-specific assignment of resonances in the 500-MHz 1H-NMR spectrum of a cardioactive sea anemone polypeptide, anthopleurin-A, is described. The assignment procedure involved analysis of two-dimensional phase-sensitive multiple-quantum-filtered, double-quantum, homonuclear Hartmann-Hahn and nuclear Overhauser effect spectra. Using sequential information, specific assignments have been made for resonances arising from all 49 amino acid residues. Resonances arising from a number of residues in a minor conformer present in solution are also assigned. These results greatly extend previous resonance assignments made from spectra acquired at 300 MHz [Gooley, P. R. and Norton, R. S. (1985) Eur. J. Biochem. 153, 529-539] and provide the basis for a more accurate definition of the conformation of anthopleurin-A in aqueous solution. The secondary structure includes a four-stranded antiparallel beta-sheet encompassing residues 2-4, 21-23, 34-36 and 45-49, and possibly a beta-bulge located at Ser-19 and Gly-20. A type II beta-turn is formed by residues 30-33. These structural elements also occur within other related sea anemone polypeptides, but the conformation of the small loop region containing Pro-41 appears to be unique to anthopleurin-A.     

A Solid Phase Vibrational Circular Dichroism Study of Polypeptide–Surfactant Interaction

We studied the interaction of poly‐l ‐lysine (PLL) and poly‐l ‐arginine (PLAG) with sodium dodecyl sulfate (SDS) surfactant and the interaction of poly‐l‐ glutamic acid (PLGA) and poly‐l ‐aspartic acid (PLAA) with tetradecyltrimethylammonium bromide (TTAB) surfactant using vibrational circular dichroism (VCD) spectroscopy in the region of C‐H stretching vibration and in the Amide I region both in solution and in mulls. A chirality transfer from polypeptides to achiral surfactants was observed in the C‐H stretching region, where measurements in solution were impossible. This observation was enabled by a special sample treatment technique using lyophilization and the preparation of mulls. This technique demonstrated itself as an interesting and beneficial tool for VCD measurements. In addition, we observed that SDS changed the secondary structure of PLL to the β‐sheet and of PLAG to the α‐helix. TTAB disrupted the PLGA and PLAA structure. These results were obtained in the mull but were confirmed by the VCD spectra measured in solution and by electronic circular dichroism. The chirality transfer from the polypeptides to SDS was caused by polypeptides ordered into a specific conformation during the interaction, while in the TTBA system it was induced primarily by the chirality of the amino acid residues. Chirality 27:965–972, 2015. © 2015 Wiley Periodicals, Inc.     

Synthesis and physicochemical properties in aqueous solution of the sequential polypeptide poly(Tyr-Ala-Glu)

A polypeptide having the repealing sequence (Tyr-Ala-Glu)_n was synthesized by the polymerization of the N-hydroxysuccinimide ester of O-benzyl-L -tyrosyl-L -alanyl-γ-benzyl-L -glutamate, followed by the removal of the benzyl groups by means of hydrogen bromide. The main fraction obtained on gel filtration had an average molecular weight of over 60, 000, corresponding to over 500 amino acid residues per polypcptide chain. The polymer is soluble in water above pH 5.5, and precipitates on lowering the pH. The x-ray powder photographs show features of an α-helical structure. The dependence of the ultraviolet absorption spectrum, the optical rotatory dispersion, and the fluorescence of poly(Tyr-Ala-Glu) on pH, in salt-free as well as in salt-containing aqueous solutions, was compared with the corresponding properties of a copolymer containing equimolar proportions of tyrosine, alanine, and glutamic acid in a random sequence. From these measurements it was concluded that poly(Tyr-Ala-Glu ) has a helical con formation at low pH and a random coil conformation at high pH, the transition taking place at pH 6 in the absence of salt and pH II in the presence of salt. Thus, in the range pH 7 to l0. random coil-to-helix transition can be achieved by merely increasing the ionic strength. A model is proposed for the structure of the helical poly peptide which accounts for the Stability of the helical conformation by assuming hydrogen bonding between the carboxylate group of the ith glutamic acid residue and the hydroxyl group of the (i + 4 )th tyrosine residue. The complex ORD of helical poly(Tyr-Ala-Glu) is explained as being due to a superposition of the ORD of an α-helix and that of a regular array of phenolic ehroniopholes originating from the immobilization of the aromatic rings in the specific structure of the polymer.     

Carrier design: Synthesis and conformational studies of poly (L-lysine) based branched polypeptides with hydroxyl groups in the side chains

In the present study the development of a new series of branched polypeptides that contain hydroxyl groups in side chains is reported. Serine or threonine were attached by 1-hydroxy-benzotriazole catalyzed active ester method to the N-terminals of oligo (DL -alanine) chains grafted to a polylysine backbone resulted in poly[Lys-(Ser₁-DL -Ala_m)] (SAK) and poly-[Lys-(Thr_i-DL -Ala_m)] (TAK). Ser was coupled also directly to the η-amino groups of polylysine followed by polymerization of N-carboxy-DL -alanine anhydride resulting oligo (DL -Ala) chain terminals. In this way a reverse sequence was built up in the side chain corresponding to the poly[Lys-(DL -Ala_m-Ser_i)] (ASK). The number of hydroxyl groups in the polymer was increased by the synthesis of a branched polypeptide with oligo (DL -serine) branches instead of oligo (DL -alanine) ones—poly[Lys-(DL -Ser_m)] (SK). Classification of solution conformations of branched polypeptides was carried out by CD spectroscopy performed in water solution of various pH values and ionic strengths. Incorporation of single Ser residues in poly[Lys-(X_i)]-type polypeptides markedly promotes the formation of ordered structure without resulting precipitation even in high salt concentration. The presence of branches with multiple DL -Ser residues resulted in a slightly decreased ability of the polypeptide backbone to adopt an ordered conformation. Comparison of the CD properties of the SAK-ASK pair demonstrates that these compounds are similar, showing an increased tendency to form an ordered spatial arrangement in solution at elevated pH or ionic strength; however, differences in their CD spectra suggest that SAK has higher capability to form regular conformation under comparable conditions. The replacement of Ser by the Thr residue in poly[Lys-(X_i-DL -Ala_m)] induced a conformational transition and TAK exhibited a more helical structure. These results might indicate that not only hydrophobic or ionic attraction, but also H-bond interaction, can play a role in the formation and/or stabilization of ordered conformation of branched polypeptides. Findings with the hydroxyl group containing polymers reported in this paper can also explain their prolonged shelf stability and high water solubility. © 1997 John Wiley & Sons, Inc. Biopoly 42: 719–730, 1997     

Conformational Analysis of the β-amyloid Peptide Fragment, β(12–28)

Abstract

NMR and CD spectroscopy have been used to examine the conformation of the peptide, β(12–28), (VHHQKLVFFAEDVGSNK) in aqueous and 60% TFE/40% H₂0 solution at pH 2.4. In 60% TFE solution, the peptide is helical as confirmed by the CD spectrum and by the pattern of the NOE cross peaks detected in the NOESY spectrum of the peptide. In aqueous solution, the peptide adopts a more extended and flexible conformation. Broadening of resonances at low temperature, temperature-dependent changes in the chemical shifts of several of the CH_α resonances and the observation of a number of NOE contacts between the hydrophobic side-chain protons of the peptide are indicative of aggregation in aqueous solution. The behavior of β(12–28) in 60% TFE and in aqueous solution are consistent with the overall conformation and aggregation behavior reported for the larger peptide fragment, β(1–28) and the parent β-amyloid peptide.     

Raman scattering of chymotrypsinogen A, ribonuclease, and ovalbumin in the aqueous solution and solid state

The Raman spectra of three globular proteins, beef pancreas chymotrypsinogen A, beef pancreas ribonuclease, and hen egg white ovalbumin have been obtained in the solid state and aqueous solution. X-ray diffraction and circular dichroism evidence have indicated that these proteins have a low α-helical content and a large fraction of the residues in the unordered and β-sheet conformation. The frequencies and intensities of the amide I and amide III lines are consistent with assignments based on the Raman spectra of polypeptides. The intense amide III lines observed in all the spectra would be expected for proteins with a low fraction of the residues in the α-helical conformation. Several spectra changes occur upon dissolution of the proteins in water and may be associated with further hydration of the proteins. The spectrum of thermally denatured chymotrypsinogen is presented. A 3 cm^–1 decrease in the frequency of the amide I line of the protein dissolved in D₂O upon heating was observed. This observation is consistent with a denaturation mechanism allowing only slight changes in the secondary structure but an increase in solvent penetration upon going from the native to the reversibly denatured state.