Different molten globule-like folding intermediates of hen egg white lysozyme induced by high pH and tertiary butanol

We have provided evidence that hen egg white lysozyme (HEWL) existed in alpha helical and beta structure dominated molten globule (MG) states at high pH and in the presence of tertiary butanol, respectively. Circular dichroism (CD), intrinsic fluorescence, ANS binding and acrylamide-induced fluorescence quenching techniques have been used to investigate alkali-induced unfolding of HEWL and the effect of tertiary butanol on the alkaline-induced state. At pH 12.75, HEWL existed as molten globule like intermediate. The observed MG-like intermediate was characterized by (i) retention of 77% of the native secondary structure, (ii) enhanced binding of ANS (approximately 5 times) compared to native and completely unfolded state, (iii) loss of the tertiary structure as indicated by the tertiary structural probes (near-UV, CD and Intrinsic fluorescence) and (iv) acrylamide quenching studies showed that MG state has compactness intermediate between native and completely unfolded states. Moreover, structural properties of the protein at isoelectric point (pI) and denatured states have also been described. We have also shown that in the presence of 45% tertiary butanol (t-butanol), HEWL at pH 7.0 and 11.0 (pI 11.0) existed in helical structure without much affecting tertiary structure. Interestingly, MG state of HEWL at pH 12.7 transformed into another MG state (MG2) at 20% t-butanol (v/v), in which secondary structure is mainly beta sheets. On further increasing the t-butanol concentration alpha helix was found to reform. We have proposed that formation of both alpha helical and beta sheet dominated intermediate may be possible in the folding pathway of alpha + beta protein.     

Effect of structural changes in thyroxine-binding globulin on its biological activity and immunochemical properties

Using spectroscopic, electrophoretic and microcalorimetric techniques, the changes in the spatial structure of human thyroxine-binding globulin (TBG) induced by exposure of protein solutions to high temperatures (45-90 degrees C) and low pH (2.5-6.0) were studied. Simultaneously the biological activity and immunoreactivity of TBG samples were measured. The structural changes were manifested at 52 degrees C or at pH 4.0 and were then aggravated with a rise in temperature or a decrease of pH. The circular dichroism spectra showed that the molecular ellipticity had a maximum decrease (by 10%) at 218-222 nm. In fluorescence spectra excitable at 280 nm the band half-width increased by 4-6 nm; their intensity decreased by 30-40%, whereas the position of the maxima did not change significantly. After addition of an equimolar amount of thyroxine to inactivated TBG the protein fluorescence was quenched by 25-40%. The electrophoregrams of treated preparations contained additional protein bands possessing no biological activity, whose mobility was less than that of native TBG. Microcalorimetric assays of native TBG revealed a thermoabsorption peak with a maximum at 62.5 degrees C and a half-width of 7.1 degrees C. The thermodynamic parameters of melting of TBG spatial structure were consistent with a model of a two-domain structure of the molecule. The biological activity and immunoreactivity of TBG showed a coordinated decrease with a rise in the degree of protein denaturation, However, the formation of TBG complex with antibodies did not screen the thyroxine-binding center of TBG and did not alter its affinity. Possible mechanisms of structural transition of TBG and its effect on the biological properties of TBG are discussed.     

Structural characterisation of the human alpha-lactalbumin molten globule at high temperature

Molten globules are partially folded forms of proteins thought to be general intermediates in protein folding. The 15N-1H HSQC NMR spectrum of the human alpha-lactalbumin (alpha-LA) molten globule at pH 2 and 20 degrees C is characterised by broad lines which make direct study by NMR methods difficult; this broadening arises from conformational fluctuations throughout the protein on a millisecond to microsecond timescale. Here, we find that an increase in temperature to 50 degrees C leads to a dramatic sharpening of peaks in the 15N-1H HSQC spectrum of human alpha-LA at pH 2. Far-UV CD and ANS fluorescence experiments demonstrate that under these conditions human alpha-LA maintains a high degree of helical secondary structure and the exposed hydrophobic surfaces that are characteristic of a molten globule. Analysis of the H(alpha), H(N) and 15N chemical shifts of the human alpha-LA molten globule at 50 degrees C leads to the identification of regions of native-like helix in the alpha-domain and of non-native helical propensity in the beta-domain. The latter may be responsible for the observed overshoot in ellipticity at 222 nm in kinetic refolding experiments.     

Spectral properties of human placental aldose reductase and aldehyde reductase II

Circular dichroism and fluorescence spectra of aldose reductase (E.C.1.1.1.21) and aldehyde reductase II (E.C.1.1.1.19) purified to homogeneity from human placenta have been studied. The alpha helical content of aldose reductase and aldehyde reductase II was 51% and 56%, respectively, whereas no beta helical structure was found in either case. In the case of aldose reductase, the secondary structure was unaffected at alkaline pH (9.5), whereas a drastic alteration in the structure was observed at 58 degrees C. The secondary structure of aldehyde reductase II, on the other hand, remained unaffected at higher pH and temperature.     

Spectroscopic study on the structure and stability of beef liver arginase

The secondary and tertiary structure of the oligomeric arginase (EC 3.5.3.1) from beef liver was investigated by circular dichroism (CD) and fluorescence measurements. The far-ultraviolet CD spectrum of the enzyme at neutral pH is indicative of high helical content. The intrinsic fluorescence emission of the protein is due to tryptophan, the contribution of tyrosine being small. Upon excitation at 295 nm, the maximum of emission occurs at 330 nm, implying that the tryptophan residues are rather buried in a hydrophobic interior of the protein. Ethylenediaminetetraacetic acid (EDTA), which inactivates the enzyme by removing the functional Mn2+-ion from the enzyme, does not dissociate the enzyme into subunits, nor affect noticeably its secondary and tertiary structure. Inactivation occurs in the acid pH range, being complete at pH below 4. However, acidification up to pH 1.5 produced only limited changes in the far-ultra-violet CD spectrum and intrinsic fluorescence emission properties. The enzyme shows noteworthy thermal stability, as shown by measuring the residual activity after heating and by evaluating the temperature dependence of the CD signal at 220 nm and the intensity of emission fluorescence. A temperature of half inactivation (Tm) of 77 degrees was determined upon heating the enzyme at pH 7.5 in the presence of Mn2+-ions for 10 min; in the presence of EDTA, Tm is shifted to 55 degrees. Taken together, these observations indicate that the structural stability of beef liver arginase arises from a clustering of hydrophobic amino acids and from Mn2+-ion binding.     

Thermal denaturation of a recombinant mouse amelogenin: circular dichroism and differential scanning calorimetric studies

Conformational analyses of a recombinant mouse tooth enamel amelogenin (rM179) were performed using circular dichroism (CD), fluorescence, differential scanning calorimetry, and sedimentation equilibrium studies. The results show that the far-UV CD spectra of rM179 at acidic pH and 10 degrees C are different from the spectra of random coil in 6 M GdnHCl. A near-UV CD spectrum of rM179 at 10 degrees C is similar to that of rM179 in 6 M GdnHCl, which indicates that aromatic residues of native structure are exposed to solvent and rotate freely. Far-UV CD values of rM179 at 80 degrees C are different from that of random-coil structure in 6 M GdnHCl, which suggests that rM179 at 80 degrees C has specific secondary structures. A gradual thermal transition was observed by far-UV CD, which is interpreted as a weak cooperative transition from specific secondary structures to other specific secondary structures. The fluorescence emission maximum for the spectrum due to Trp residues in rM179 at 10 degrees C shows the same fluorescence emission maximum as rM179 in 6 M GdnHCl and amino acid Trp, which indicates that the three Trp in rM179 are exposed to solvent. Deconvolution of differential scanning calorimetry curve gives the population of three states (A, I, and C states). These results indicate that three states (A, I, and C) have specific secondary structures, in which hydrophobic and Trp residues are exposed to the solvent. The thermodynamic characteristics of rM179 are unique and different from a typical globular protein, proline-rich peptides, and a molten globule state.     

The effect of evolution on homologous proteins: a comparison between the chromophore microenvironments of Italian water buffalo (Bos bubalus, L.) and sperm whale apomyoglobin.

The perturbing effect of guanidium hydrochloride and pH on the molecular structure of water buffalo apomyoglobin has been investigated by circular dichroism in the far and near ultraviolet and by fluorescence. In the wavelength region between 320 and 260 nm the circular dichroic spectrum of the globin is highly structured and the contributions of the aromatic chromophores have been resolved. Buffalo apomyoglobin undergoes a structural transition at neutral pH which involves elements of the secondary and tertiary structure, as indicated by changes of dichroic activity of the peptide and aromatic chromophores and the fluorescence of the two tryptophanyl residues. The possibility of charge-transfer complex between indole and imidazole is discussed. A major structural transition with abrupt unfolding takes place in the pH region between 5.6 and 4.3. Below pH 4.3 the peptide helical residues, which survive the acid transition, appear to be resistent to further acidification to pH 2.0 while tryptophanyl emission is quenched and shifted to longer wavelengths. A structural transition occurs also in alkali above pH 10, which has been detected by the same techniques. The relationships between buffalo and sperm whale apomyoglobin are discussed.     

Environmentally induced reversible conformational switching in the yeast cell adhesion protein alpha-agglutinin

The yeast cell adhesion protein alpha-agglutinin is expressed on the surface of a free-living organism and is subjected to a variety of environmental conditions. Circular dichroism (CD) spectroscopy shows that the binding region of alpha-agglutinin has a beta-sheet-rich structure, with only approximately 2% alpha-helix under native conditions (15-40 degrees C at pH 5.5). This region is predicted to fold into three immunoglobulin-like domains, and models are consistent with the CD spectra as well as with peptide mapping and site-specific mutagenesis. However, secondary structure prediction algorithms show that segments comprising approximately 17% of the residues have high alpha-helical and low beta-sheet potential. Two model peptides of such segments had helical tendencies, and one of these peptides showed pH-dependent conformational switching. Similarly, CD spectroscopy of the binding region of alpha-agglutinin showed reversible conversion from beta-rich to mixed alpha/beta structure at elevated temperatures or when the pH was changed. The reversibility of these changes implied that there is a small energy difference between the all-beta and the alpha/beta states. Similar changes followed cleavage of peptide or disulfide bonds. Together, these observations imply that short sequences of high helical propensity are constrained to a beta-rich state by covalent and local charge interactions under native conditions, but form helices under non-native conditions.     

Conformational studies on the beta subunits of human hemoglobin and their arginyl-COOH peptides.

The beta subunits of hemoglobin upon alkylation of the cysteinyl residues with iodoacetamide showed a sedimentation velocity with an S20w, near 1.8 as for monomeric subunits. They reacted with alpha chains to give a tetrameric hemoglobin with a sedimentation constant near 4.4. Their CD spectrum was indistinguishable from that of untreated beta chains below 270 nm, otherwise they showed some deviation that became pronounced in the Soret region, where the optical activity of the alkylated subunits was definitely lower than that of the native subunits. Upon removal of the heme the apo-beta subunits showed a decreased optical activity in the far-uv region of the spectrum indicating a substantial loss of helical content. Their sedimentation behavior was consistent with the presence of large aggregates, which dissociates into monomers upon reconstitution with cyanoheme. The apo-beta subunits could be renatured from 6 M guanidine hydrochloride. They showed a stoichiometric reaction with heme in the molar ratio 1:1. Upon reconstitution with the heme their optical activity became similar to that of the native beta chains in the far-uv region of the spectrum, but remained lower in the near-uv and Soret regions. After acylation of the lysyl residues with citraconic anhydride the apo-beta subunits were digested with trypsin and the arginyl-COOH peptides beta(1-30), beta(31-40), beta(41-104), and beta(105-146) were separated by gel chromatography. With the exception of the peptide beta/105-146), which was insoluble at neutral pH, the sedimentation behavior of the other peptides showed the presence of small polymers. The sedimentation behavior of the peptide beta(31-40) was not tested. The percentage of alpha helix, beta conformation, and of random coil (or unordered structure) of the various proteins and peptides was measured fitting their CD spectra in the far-uv region with the parameter published by Y.H. Chen et al. ((1974), Biochemistry 13, 3350) and by N. Greenfield and G.D. Fasman ((1969), Biochemistry 8, 4108). In this way the helical content of the native and reconstituted alkylated beta subunits appeared to be near 76%, a value very near to that present in the same subunits in the hemoglobin crystal. The helical content of the apo-beta subunits in 0.04 M borate buffer at pH 9.6 decreased to a value near 45%. The helical content of the isolated peptides in electrolyte solutions was in any case near 10% indicating an almost complete loss of the structure that they have in the hemoglobin crystal. Cyanoheme reacted with the peptide beta(41-104), however, the reaction was not stoichiometric indicating a low affinity of the heme for the peptide. With the exception of the peptide beta(31-104), all of the other peptides recovered some of their helical structure when dissolved in 50% methanol. Notably also the apo-beta subunits did so suggesting that the loss of structure upon the removal of the heme could be in part due to the exposure of the heme pocket to water.     

Circular dichroism studies of the HIV-1 Rev protein and its specific RNA binding site

The circular dichroism (CD) spectrum of the Rev protein from HIV-1 indicates that Rev contains about 50% alpha helix and 25% beta sheet at 5 degrees C in potassium phosphate buffer, pH 3, and 300 mM KF. The spectrum is independent of protein concentration over a 20-fold range. At neutral pH, Rev is relatively insoluble but can be brought into solution by binding to its specific RNA binding site, the Rev-responsive element (RRE), at a Rev:RNA ratio of about 3:1. Nonspecific binding to tRNA does not solubilize Rev. As judged by difference CD spectra, the conformation of Rev when bound to the RRE at neutral pH is similar to the conformation of unbound Rev at pH 3, although changes in the RNA may also contribute to the difference spectrum. Indeed, some difference is observed near 260 nm, consistent with a conformational change of the RRE upon Rev binding. Rev alone at pH 3 shows irreversible aggregation as the temperature is raised, while Rev bound to the RRE at neutral pH shows a reversible transition with a Tm of 68 degrees C.     

Conformational changes in subfractions of calf thymus histone H1.

This paper presents the first study of conformational changes in the subfractions of calf thymus H1. H1 was fractionated by the method of Kincade and Cole (Kincade, J. M., and Cole, R.D. (1966), J. Biol. Chem. 241. 5790) using a very shallow Gdn-HC1 gradient. A possible new H1 subfraction, about 5--8% of the H1, has been found and characterized by amino acid analysis and electrophoresis. The effects of salt concentration and pH on the conformation of each of the four major subfractions have been studied by measuring the fluorescence anisotropy of the tyrosine emission and the circular dichroism (CD) of the peptide bond. Upon the addition of salt to aqueous solutions at neutral pH, all four subfractions show an instantaneous change in fluorescence anisotropy, fluorescence intensity, tyrosine absorbance, and CD. The folding associated with this instantaneous change is highly cooperative, and involves the region of the molecule containing the lone tyrosine, which becomes buried in the folded form. The folding of subfraction 3a is more sensitive to salt than the other major subfractions. Upon folding, approximately 13% of the residues of subfractions 1b and 2 form alpha and beta structure; 3a and 3b have approximately 16% of the residues in alpha and beta structures. There is no evidence for interactions between the subfractions. In salt-free solutions, each of the four major subfractions show very little change in conformation in going from low to neutral pH, but each shows a very sharp transition near pH 9. This transition gives rise to a marked increase in fluorescence anisotropy and fluorescence intensity, and involves the formation of both alpha and beta strucute in a manner similar to that of the salt-induced state.     

Trifluoroethanol-induced "molten globule" state in stem bromelain

2,2,2-Trifluoroethanol (TFE) denatures proteins but also stabilizes/induces alpha helical conformation in partially/completely unfolded proteins. As reported earlier from this laboratory, stem bromelain is known to exist as a partially folded intermediate (PFI) at pH 2.0. The effect of increasing concentration of TFE on the PFI of bromelain has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino 8-naphthalene sulfonic acid (ANS), and near-UV CD temperature transition. Far-UV CD spectra show considerable accumulation of secondary structure at 70% (v/v) concentration of TFE with spectral features resembling the pH 7.0 preparation. Interestingly the partially folded intermediate regained significant tertiary structure/interactions, with increasing concentration of TFE, and at 60% (v/v) TFE approached almost that of the pseudo native (pH 7.0) state. Further increase to 70% (v/v) TFE, however, resulted in complete loss of tertiary structure/interactions. Studies on tryptophan fluorescence also suggested the induction of some compact structure at 60% (v/v) concentration of TFE. The partially folded intermediate showed enhanced binding of the fluorescent probe (ANS) in the presence of 60% (v/v) TFE. Taken together these observations suggest a "molten globule" state between 60 and 70% (v/v) TFE. Thermal transition studies in the near-UV CD region indicated cooperative transition for PFI in the presence of 60% (v/v) TFE changing to noncooperative transition at 70% (v/v) TFE. This was accompanied by a shift in the midpoint of thermal denaturation (T(m)) from 58 to 51 degrees C. Gradual transition and loss of cooperative thermal unfolding in the 60-70% (v/v) range of TFE also support the existence of the molten globule state.     

On the nature of the unfolded intermediate in the in vitro transition of the colicin E1 channel domain from the aqueous to the membrane phase.

The transition of the colicin E1 channel polypeptide from a water-soluble to membrane-bound state occurs in vitro at acid pH values that are associated with an unfolded channel structure whose properties qualitatively resemble those of a "molten globule," or "compact unfolded," intermediate state. The role of such a state for activity was tested by comparing the pH dependence of channel-induced solute efflux and the amplitude of the near-UV CD spectrum. The requirement of a partly unfolded state for activity was shown by the coincidence of the onset of channel activity measured for 4 different lipid compositions with the decrease in near-UV CD amplitude as a function of pH. Tertiary constraints on the 3 tryptophans of the colicin channel, assayed by the amplitude of the near-UV CD spectrum, are retained over the pH range 3-4 where channel activity could be measured and, as well, at pH 2. In addition, the tryptophan fluorescence emission spectrum is virtually unchanged over the pH range 2-6. The temperature independence of the near-UV spectrum at pH 3-6 up to 70 degrees C implies that the colicin E1 channel polypeptide is more stable than that of colicin A. A transition between 53 and 58 degrees C in the amplitude of the near-UV CD is consistent with preservation of part of the hydrophobic core in a destabilized state at pH 2. Thus, the unfolded state associated with colicin activity at acidic pH has the properties of a "compact unfolded" state, having some, but not all of the properties of a "molten globule."(ABSTRACT TRUNCATED AT 250 WORDS)     

Hints of nonhierarchical folding of acidic fibroblast growth factor

We have analyzed by circular dichroism (CD) and proton nuclear magnetic resonance (NMR) the helical propensity of the all-beta protein acidic fibroblast growth factor (aFGF) and two peptides corresponding to beta-strand 8 (beta8 peptide, amino acids 95-107) and the beta-strand 8/turn/beta-strand 9 hairpin (beta8/9 peptide, amino acids 95-114), which has been involved in receptor binding. A secondary structure prediction of aFGF carried out by several procedures labels the 95-104 sequence as predominantly alpha-helical. A titration of aFGF with 2,2,2-trifluoroethanol (TFE) induces a change in the far-UV CD spectrum of the protein giving rise to a prominent alpha-helical shape (22% alpha-helix). The cooperativity of the transition and the moderate TFE concentrations used (midpoint at 24%) suggest that the effect of TFE is specific. Moreover, a titration performed at pH 2 yields a higher amount of alpha-helix (55%) at a smaller TFE concentration. Synthetic peptides containing the beta8 and beta8/9 sequences display a random coil conformation at pH 7 but acquire alpha-helical structure in the presence of TFE, methanol, and SDS micelles. At pH below 3.0 a significant amount (20-30%) of alpha-helical conformation is present in both the beta8 and beta8/9 peptides even in the absence of other solvent additives. The secondary structure of the peptides was determined by proton nuclear magnetic resonance (1H NMR). These results suggest that the 95-114 sequence of aFGF has helical propensity and that the protein may fold nonhierarchically in the early steps of folding, acquiring its final beta-structure by a later interaction with the rest of the polypeptide.     

Phage T4 lysozyme. Physical properties and reversible unfolding.

Phage T4 lysozyme has been used extensively in studies of the genetic code. However, little work has been done on the characterization of the purified enzyme. Therefore, we determined the spectral properties of native T4 lysozyme and used these properties to follow the unfolding transition. The ultraviolet absorption spectrum and solvent perturbation difference spectrum indicate that the aromatic amino acids are extensively exposed to solvent. The CD and ORD spectra are characteristic of a high fraction of helix. Guanidine hydrochloride denaturation results show that over a T4 lysozyme concentration range of 0.07-1 g/l the c-m equals 2.7 M guanidine hydrochloride at pH 5 and that the transition is 100% reversible as judged by enzymatic assay and four different spectrophotometric criteria: CD at 295 nm, CD at 223 nm, fluorescence intensity at 350 nm and wavelength of maximum fluorescence. Guanidine hydrochloride denaturation at pH 2.5 was followed using fluorescence emission and has a c-m equals 1.7 M guanidine hydrochloride, indicating a strong pH dependence of chemical unfolding. Reversible thermal denaturation conditions were located at acid pH, 0.2 M NaCl, 10-4 M dithiothreitol and 10-6 M T4 lysozyme. The CD signal at 223 nm was used to measure the unfolding. Thermodynamic analysis of the thermal data showed an increase in T-m, increment H-unf and increment S-unf with increasing pH.     

Helical and expanded conformation of equine beta-lactoglobulin in the cold-denatured state

The thermal unfolding transition of equine beta-lactoglobulin (ELG) was investigated by circular dichroism (CD) over a temperature range of -15 degrees C to 85 degrees C. In the presence of 2 M urea, a cooperative unfolding transition was observed both with increasing and decreasing temperature. The CD spectrum indicated that the heat and cold-denatured states of ELG have substantial secondary structures but lack persistent tertiary packing of the side-chains. In order to clarify the relation between the heat or cold-denatured state and the acid-denatured (A) state characterized previously, we have attempted to observe the temperature dependence of the CD spectrum at pH 1.5. The CD spectrum in the heat-denatured state is similar to that in the A state. The CD spectrum in the A state does not change cooperatively with increasing temperature. These results indicate that the heat-denatured state and the A state are the same structural state. On the other hand, the CD intensity at acid pH cooperatively increased with decreasing temperature. The CD spectrum at low temperature and acid pH is consistent with that in the cold-denatured state. Therefore, the cold-denatured state is distinguished from the heat-denatured state or the A state, and ELG assumes a larger amount of non-native alpha-helices in the cold-denatured state. Small angle X-ray scattering and analytical ultracentrifugation have indicated that ELG assumes an expanded chain-like conformation in the cold-denatured state in contrast to the compact globular conformation in the A state. The relation between the molecular size and the helical content in the partially folded states is discussed.     

Correlation between quaternary structure and ligand dissociation kinetics for fully liganded hemoglobin.

The quaternary structures of fully liganded adult hemoglobin and hemoglobin Kansas (alpha2beta2 102 Asn-thr) bound by carbon monoxide or nitric oxide were spectroscopically characterized using high-resolution nuclear magnetic resonance (NMR) and ultraviolet circular dichroism (CD). The spectral markers used for the quarternary transition were the line in the NMR spectrum in H2O-14 ppm downfield from 2,2-dimethyl-2-silapentane-5-sulfonate and the negative peak at 285 nm in the ultraviolet CD spectrum. In the nitrosyl derivatives, these two structural markers were compared with the electron paramagnetic resonance (EPR) spectrum at room temperature for the purpose of correlating structural changes in the protein with changes at the heme...     

Structural differences between Saccharomyces cerevisiae ribosomal stalk proteins P1 and P2 support their functional diversity

The eukaryotic acidic P1 and P2 proteins modulate the activity of the ribosomal stalk but playing distinct roles. The aim of this work was to analyze the structural features that are behind their different function. A structural characterization of Saccharomyces cerevisaie P1 alpha and P2 beta proteins was performed by circular dichroism, nuclear magnetic resonance, fluorescence spectroscopy, thermal denaturation, and protease sensitivity. The results confirm the low structure present in both proteins but reveal clear differences between them. P1 alpha shows a virtually unordered secondary structure with a residual helical content that disappears below 30 degrees C and a clear tendency to acquire secondary structure at low pH and in the presence of trifluoroethanol. In agreement with this higher disorder P1 alpha has a fully solvent-accessible tryptophan residue and, in contrast to P2 beta, is highly sensitive to protease degradation. An interaction between both proteins was observed, which induces an increase in the global secondary structure content of both proteins. Moreover, mixing of both proteins causes a shift of the P1 alpha tryptophan 40 signal, pointing to an involvement of this region in the interaction. This evidence directly proves an interaction between P1 alpha and P2 beta before ribosome binding and suggests a functional complementation between them. On a whole, the results provide structural support for the different functional roles played by the proteins of the two groups showing, at the same time, that relatively small structural differences between the two stalk acidic protein types can result in significant functional changes.     

Conformational stability of ribonuclease T1. I. Thermal denaturation and effects of salts.

The thermal transition of RNase T1 was studied by two different methods; tryptophan residue fluorescence and circular dichroism. The fluorescence measurements provide information about the environment of the indole group and CD measurements on the gross conformation of the polypeptide chain. Both measurements at pH 5 gave the same transition temperature of 56 degrees C and the same thermodynamic quantities, delta Htr (= 120 kcal/mol) and delta Str (= 360 eu/mol), for the transition from the native state to the thermally denatured state, indicating simultaneous melting of the whole molecule including the hydrophobic region where the tryptophan residue is buried. Stabilization by salts was observed in the pH range from 2 to 10, since the presence of 0.5 m NaCL caused an increase of about 5 degrees C to 10 degrees C in the transition temperature, depending on the pH. The fluorescence measurements on the RNase T1 complexed with 2'-GMP showed a transition with delta Htr =167 kcal/mol and delta Str =497 eu/mol at a transition temperature about 6 degrees C higher than that for the free enzyme. The large value of delta Htr for RNase T1 indicates the highly cooperative nature of the thermal transition; this value is much higher than those of other globular proteins. Analysis of the CD spectrum of thermally denatured RNase T1 suggests that the denatured state is not completely random but retains some ordered structures.     

Conformational intermediates in the folding of a coiled-coil model peptide of the N-terminus of tropomyosin and alpha alpha-tropomyosin.

Circular dichroism was used to study the folding of alpha alpha-tropomyosin and AcTM43, a 43-residue peptide designed to serve as a model for the N-terminal domain of tropomyosin. The sequence of the peptide is AcMDAIKKKMQMLKLDVENLLDRLEQLEADLKALEDRYKQLEGGC. The peptide appeared to form a coiled coil at low temperatures (< 25 degrees C) in buffers with physiological ionic strength and pH. The folding and unfolding of the peptide, however, were noncooperative. When CD spectra were examined as a function of temperature, the apparent degree of folding differed when the ellipticity was followed at 222, 208, and 280 nm. Deconvolution of the spectra suggested that at least three component curves contributed to the CD in the far UV. One component curve was similar to the CD spectrum of the coiled-coil alpha-helix of native alpha alpha-tropomyosin. The second curve resembled the spectrum of single-stranded short alpha-helical segments found in globular proteins. The third was similar to that of polypeptides in the random coil conformation. These results suggested that as the peptide folded, the alpha-helical content increased before most of the coiled coil was formed. When the CD spectrum of striated muscle alpha alpha-tropomyosin was examined as a function of temperature, the unfolding was also not totally cooperative. As the temperature was raised from 0 to 25 degrees C, there was a decrease in the coiled coil and an increase in the conventional alpha-helix type spectrum without formation of random coil. The major transition, occurring at 40 degrees C, was a cooperative transition characterized by the loss of all of the remaining coiled coil and a concomitant increase in random coil.