NMR studies of toxin III from the sea anemone Radianthus paumotensis and comparison of its secondary structure with related toxins

Nearly complete assignments of the proton nuclear magnetic resonance (NMR) spectrum of the polypeptide toxin III from the sea anemone Radianthus paumotensis (RP) are presented. The secondary structures of the related toxins RP II and RP III are described and are compared with each other and with another related toxin ATX Ia from Anemonia sulcata [Widmer, H., Wagner, G., Schweitz, H., Lazdunski, M., & Wüthrich, K. (1988) Eur. J. Biochem. 171, 177-192]. All of these proteins contain a highly twisted four-strand antiparallel beta-sheet core connected by loops of irregular structure. From the work done with AP-A from Anthopleura xanthogrammica [Gooley, P. R., & Norton, R. S. (1986) Biochemistry 25, 2349-2356], it is clear that this homologous toxin also has the same basic core. Some small differences are seen in the structures of these toxins, particularly in the position of the N-terminal residues that form one of the outside strands of the beta-sheet. In addition, the R. paumotensis toxins are two residues longer, extending the third strand of sheet containing the C-terminal residues. A comparison of chemical shifts for assigned residues is also presented, in general supporting the similarity of structure among these proteins.     

The status of tyrosyl residues in a Formosan cobra cardiotoxin.

Spectrophotometric titration of Formosan cobra cardiotoxin showed that two of the three tyrosyl residues were titrated freely with a normal apparent pKa of 9.6 whereas the remaining one ionized at pH above 11.0. Nitration of cardiotoxin in Tris . HCl buffer with tetranitromethane resulted in the selective nitration of tyrosine 11 and tyrosine 22. It also revealed that tyrosine 51 was the abnormal one in the spectrophotometric titration. Complete nitration occurred in the presence of 6.0 M guanidine hydrochloride. Compared with the conformation of native cardiotoxin, the peptide conformation of the partially nitrated cardiotoxin did not change significantly but the conformation of the completely nitrated cardiotoxin changed remarkably. The biological activity of cardiotoxin was indeed affected by nitration, but the immunological activity was nearly intact even when all the tyrosine residues were nitrated.     

Isolation and fluorescence studies on a lipophorin from the weevil diaprepes abbreviatus

Lipophorin was isolated from larvae of a root weevil, Diaprepes abbreviatus (Coleoptera: Curculionidae), using density gradient ultracentrifugation. D. abbreviatus lipophorin contained two apoproteins, apolipophorin-I (M_r = 226,000) and apolipophorin-II (M_r = 72,100) and had a density of 1.08. Relative to other larval lipophorins, D. abbreviatus lipophorin contained little cysteine (determined as cysteic acid) and methionine. Fluorescence spectroscopy of intrinsic tyrosine and tryptophan residues excited at 290 nm revealed a single broad emission peak at 330 nm. Upon denaturing and delipidating lipophorin in guanidine HCl, this peak resolved into two peaks with maxima at 305 and 350 nm. Excitation spectra suggested that the two peaks were due to tyrosine and tryptophan, respectively. Fluorescence quenching agents, iodide and acrylamide, were used to determine accessibility of tyrosine and tryptophan residues to the aqueous environment. Iodide, a polar quenching agent, did not quench fluorescent emission from native lipophorin; quenching by iodide increased to moderate levels when lipophorin was denatured in guanidine HCl. Acrylamide quenched the fluorescence of native lipophorin moderately and very efficiently quenched fluorescence of denatured lipophorin. No difference was observed between fluorescence quenching of denatured vs. denatured and delipidated lipophorin by either iodide or acrylamide.     

NMR studies on Ni(II) induced cyclization of a histidine-tagged peptide.

A linear decapeptide, HGASYQDLGH, was synthesized and used as a model to evaluate the effect of nickel addition upon non-covalent backbone cyclization. The NMR data, obtained for the peptide in the presence of the metal ion, support the existence of predominant folded structures in solution, where the two His residues are maintained close to each other. These results suggest that insertion of even a single His residue at each peptide terminus can be used efficiently to reduce peptide flexibility without any backbone modification.     

Azotobacter vinelandii rhodanese: selenium loading and ion interaction studies.

Rhodanese is a sulfurtransferase which in vitro catalyzes the transfer of a sulfane sulfur from thiosulfate to cyanide. Ionic interactions of the prokaryotic rhodanese-like protein from Azotobacter vinelandii were studied by fluorescence and NMR spectroscopy. The catalytic Cys230 residue of the enzyme was selectively labelled using [15N]Cys, and changes in 1H and 15N NMR resonances on addition of different ions were monitored. The results clearly indicate that the sulfur transfer is due to a specific reaction of the persulfurated Cys residue with a sulfur acceptor such as cyanide and not to the presence of the anions. Moreover, the 1H-NMR spectrum of a defined spectral region is indicative of the status of the enzyme and can be used to directly monitor sulfur loading even at low concentrations. Selenium loading by the addition of selenodiglutathione was monitored by fluorescence and NMR spectroscopy. It was found to involve a specific interaction between the selenodiglutathione and the catalytic cysteine residue of the enzyme. These results indicate that rhodanese-like proteins may function in the delivery of reactive selenium in vivo.     

Denaturation and renaturation of myeloperoxidase. Consequences for the nature of the prosthetic group.

The effects of the chaotropic agent, guanidine HCl, on the chlorinating activity, optical absorption, EPR, and resonance Raman spectra of myeloperoxidase have been studied. In the presence of the agent the Soret optical absorption of the reduced enzyme (lambda max = 474 nm) is blue shifted to 448 nm, a position similar to heme alpha-containing enzymes. The chlorinating activity of the enzyme disappears, and EPR spectra show a loss of intensity of the rhombic high spin heme signals (gx = 6.9; gy = 5.4) and the appearance of a more axial high spin signal (gx = gy = 6.0). Surprisingly the effects of guanidine HCl are partly reversible. Upon decreasing the concentration of the chaotropic agents by dilution, both the chlorinating activity and the original optical spectrum of native reduced enzyme (lambda max = 474 nm) are partly restored. The resonance Raman spectra of denatured cyanomyeloperoxidase are less complicated than those of native myeloperoxidase, which have been interpreted previously to suggest an iron chlorin chromophore. The multiple lines in the oxidation state marker region are not seen in the spectra of the denatured species. The changes suggest that upon denaturation the macrocycle is converted into a more symmetric structure. Since the effects on the optical absorption spectrum are reversible we speculate that, in the native enzyme, an apparent porphyrin macrocycle undergoes a reversible interaction with amino acid residues in the protein which creates an asymmetry in the electronic distribution of the macrocycle. Comparison of the Raman spectra of denatured cyanomyeloperoxidase with those of analogous heme alpha model complexes suggests the presence of a formyl group in the denatured species; our data, however, demonstrate that the chromophore structure is not identical to heme alpha and may contain a different C beta substitution on the ring macrocycle.     

Conformational studies of a hyperthermostable enzyme

The structural features of the hyperthermophilic endo-beta-1,3-glucanase from Pyrococcus furiosus were studied using circular dichroism, steady-state and time-resolved fluorescence spectroscopy and anisotropy. Upon heat and chemical treatment the folded and denatured states of the protein were characterized by distinguishable spectral profiles that identified a number of conformational states. The fluorescence methods showed that the spectral differences arose from changes in the local environment around specific tryptophan residues in the native, partially folded, partially unfolded and completely unfolded state. A structural resemblance was observed between the native protein and the structurally perturbed state which resulted after heat treatment at 110 degrees C. The enzyme underwent disruption of the native secondary and tertiary structure only after incubation at biologically extremely high temperatures (i.e. 150 degrees C), whilst in the presence of 8 m of guanidine hydrochloride the protein was partially unfolded.     

Equilibrium unfolding of DLC8 monomer by urea and guanidine hydrochloride: Distinctive global and residue level features

We present circular dichroism (CD), steady state fluorescence and multidimensional NMR investigations on the equilibrium unfolding of monomeric dynein light chain protein (DLC8) by urea and guanidine hydrochloride (GdnHCl). Quantitative analysis of the CD and fluorescence denaturation curves reveals that urea unfolding is a two-state process, whereas guanidine unfolding is more complex. NMR investigations in the native state and in the near native states created by low denaturant concentrations enabled residue level characterization of the early structural and dynamic perturbations by the two denaturants. Firstly, (15)N transverse relaxation rates in the native state indicate that the regions around N10, Q27, the loop between beta2 and beta4 strands, and K87 at the C-terminal are potential unfolding initiation sites in the protein. Amide and (15)N chemical shift perturbations indicate different accessibilities of the residues along the chain and help identify locations of the early perturbations by the two denaturants. Guanidine and urea are seen to interact at several sites some of which are different in the two cases. Notable among the common interaction site is that around K87 which is in close proximity to W54 on the protein structure, but the interaction modes of the two denaturants are different. The secondary chemical shifts indicate that the structural perturbation by 1M urea is small, compared to that by guanidine which is more encompassing over the length of the chain. The probable (phi, psi) changes at the individual residues have been calculated using the TALOS algorithm. It appears that the helices in the protein are significantly perturbed by guanidine. Further, comparison of the spectral density functions of the native and the two near native states in the two denaturants implicate greater loosening of the structure by guanidine as compared to that by urea, even though the structures are still in the native state ensemble. These differences in the early perturbations of the native state structure and dynamics by the two denaturants might direct the protein along different pathways, as the unfolding progresses on further increasing the denaturant concentration.     

Denaturation behavior of phaseolin in urea, guanidine hydrochloride, and sodium dodecyl sulfate solutions

The denaturation behavior of phaseolin in urea, guanidine hydrochloride, and sodium dodecyl sulfate solutions was examined by monitoring changes in the intrinsic fluorescence of tryptophan and tyrosyl residues. Changes in various fluorescence parameters, such as quantum yield, emission maximum, spectral half-width, fluorescence depolarization, and fluorescence quenching by acrylamide, have indicated that while phaseolin is relatively stable up to 8 M urea, it is completely destabilized in 6 M guanidine hydrochloride and 6 mM sodium dodecyl sulfate. Furthermore, while the denaturation of phaseolin in urea solutions followed a two-step process, that in guanidine hydrochloride and sodium dodecyl sulfate followed a single-step process. While the accessibility of tryptophan residues to the nonionic acrylamide quencher is almost 100% in 6 M guanidine hydrochloride and 6 mM sodium dodecyl sulfate, only about 72% was accessible in 8 M urea compared to 52% in native phaseolin. The results presented here suggest that the protomeric structure of phaseolin is quite stable to changes in the environment. This structural stability may be partly responsible for its resistance to proteolysis by various proteinases.     

Conformational changes in pennisetin using intrinsic fluorescence spectroscopy

Fluorescence spectra of native pennisetin resulted in a single emission peak at 335 nm at excitation wavelength of 274 and 295 nm with quantum yield values for tyrosine and tryptophan as 0.086 and 0.097, respectively. These results indicate the presence of tryptophan residues in a polar environment and quenching of tyrosine residues in the native state of pennisetin. In the presence of an increasing concentration of guanidine hydrochloride (Gdn · HCl), changes such as red shift in emission peak from 335 to 344 nm, decrease in relative fluorescence intensity and increase in quantum yield value were observed, suggesting unfolding of the pennisetin molecule during denaturation. The quenching of tryptophanyl fluorescence by acrylamide and iodide further showed the presence of a single kind of tryptophanyl residue and its polar environment in pennisetin molecule.     

Effect of pH and denaturants on the folding and stability of murine interleukin-6.

The conformation and stability of a recombinant mouse interleukin-6 (mIL-6) has been investigated by analytical ultracentrifugation, fluorescence spectroscopy, urea-gradient gel electrophoresis, and near- and far-ultraviolet circular dichroism. On decreasing the pH from 8.0 to 4.0, the tryptophan fluorescence of mIL-6 was quenched 40%, the midpoint of the transition occurring at pH 6.9. The change in fluorescence quantum yield was not due to unfolding of the molecule because the conformation of mIL-6, as judged by both urea-gradient gel electrophoresis and CD spectroscopy, was stable over the pH range 2.0-10.0. Sedimentation equilibrium experiments indicated that mIL-6 was monomeric, with a molecular mass of 22,500 Da over the pH range used in these physicochemical studies. Quenching of tryptophan fluorescence (20%) also occurred in the presence of 6 M guanidine hydrochloride upon going from pH 7.4 to 4.0 suggesting that an amino acid residue vicinal in the primary structure to one or both of the two tryptophan residues, Trp-36 and Trp-160, may be partially involved in the quenching of endogenous fluorescence. In this regard, similar results were obtained for a 17-residue synthetic peptide, peptide H1, which corresponds to an N-terminal region of mIL-6 (residues Val-27-Lys-43). The pH-dependent acid quenching of endogenous tryptophan fluorescence of peptide H1 was 30% in the random coil conformation and 60% in the presence of alpha-helix-promoting solvents. Replacement of His-33 with Ala-33 in peptide H1 alleviated a significant portion of the pH-dependent quenching of fluorescence suggesting that the interaction of the imidazole ring of His-33 with the indole ring of Trp-36 is a major determinant responsible for the quenching of the endogenous protein fluorescence of mIL-6.     

Pyridoxal 5'-phosphate, a fluorescent probe in the active site of aspartate transcarbamylase.

Pyridoxal-P reacts specifically with a single lysine residue at the active site of Escherichia coli aspartate transcarbamylase (Greenwell, P., Jewett, S. L., and Stark, G. R. (1973) J. Biol. Chem. 248, 5994-6001). Reduction of the Schiff base with sodium borohydride, succinylation of the remaining lysine residues, and digestion with trypsin result in formation of a single pyridoxyl peptide, which was purified to homogeneity after chromatography on DEAE-cellulose, treatment with alkaline phosphatase, and rechromatography. Amino acid composition and the results of limited sequential degradation showed that this peptide corresponds to residues 62 to 98 in the sequence of Konigsberg and co-workers, and contains 2 residues of lysine (Henderson, L., Roy, D., Martin, D., and Konigsberg, W., personal communication). By similar isolation, a second peptide was obtained from unsuccinylated catalytic subunit, containing only the pyridoxylated lysine, which corresponds to Lys-80. Derivatives of catalytic subunit containing an average of either one, two, or three pyridoxamine-P moieties per trimer have been prepared by reduction. These species, which retain catalytic activity in proportion to their unmodified active sites, were recombined with regulatory subunit to prepare partially modified derivatives of native aspartate transcarbamylase. At pH 8, fluorescence emission bands were observed at 340 nm, due to aromatic amino acids in the protein, and at 395 nm, due to the pyridoxamine-P moiety. Upon excitation at 280 nm energy transfer from protein to pyridoxamine-P was approximately 15%. The properties of the probe were used to study changes accompanying the binding of substrates and inhibitors. The effects of CTP and ATP were small. With the transition state analog N-(phosphonacetyl)-L-aspartate (PALA) or the substrate carbamyl-P, two types of response were observed. Derivatives of catalytic subunit and native enzyme which contain some unmodified sites and hence retain partial catalytic activity gave large increases in fluorescence at 395 nm. However, fully modified inactive derivatives gave much smaller increases. A derivative of native enzyme containing one triply modified and one unmodified catalytic subunit behaved like the other partially modified species. These results indicate that there is communication among the active sites of different catalytic trimers in modified native enzyme, as well as among active sites within the same modified catalytic trimer. The increases in fluorescence result from a red shift of the absorption maximum of the pyridoxamine-P moiety from 315 to 325 nm, which increases the absorbance at the excitation wavelength for fluorescence. At pH 7, the absorption spectrum is already shifted and, consequently, the binding of PALA and carbamyl-P has little effect on the fluorescence. Therefore, the binding of these compounds at pH 8.0 must cause a structural change in the protein, which in turn causes protonation of a group in the modified active sites, altering the spectral properties.     

Selective determination of arginine-containing and tyrosine-containing peptides using capillary electrophoresis and laser-induced fluorescence detection.

The use of two different amino acid-selective fluorogenic reagents for the derivatization of peptides is investigated. One such scheme utilizes a selective reaction of benzoin with the guanidine moiety to derivatize arginine residues occurring in a peptide. The second scheme involves the formylation of tyrosine, followed by reaction with 4-methoxy-1,2-phenylenediamine. The use of capillary electrophoresis and laser-induced fluorescence detection allows enhanced efficiencies and sensitivities to be obtained for the separations of either arginine- or tyrosine-containing peptides. A helium-cadmium laser (325 nm) is ideally suited for the laser-based detection system due to a close match of the excitation maxima of derivatized peptides from both reactions. A detection limit of 270 amol is achieved for model arginine-containing peptides, while the detection limit for model tyrosine-containing peptides is measured at 390 amol. Both derivatization reactions are found to be useful for high-sensitivity peptide mapping applications in which only the peptides containing the derivatized amino acids are detected.     

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.     

Accessibility of tryptophan residues in Na,K-ATPase

The accessibility of the tryptophans in dog kidney Na,K-ATPase was studied with the technique of quenching by acrylamide. By use of a modified Stern-Volmer equation, fa, the effective fraction of tryptophans most exposed to quencher, and Ka, the effective quenching constant, were calculated. The direct Stern-Volmer plots are nonlinear under nondenaturing conditions, indicating that the tryptophan residues are unequally accessible to quencher. Modified Stern-Volmer plots revealed marked differences in the exposure of tryptophans in the E1 and E2 states. In the presence of Na or ADP, ligands that stabilize E1, these plots curve downward, indicating that the in addition to buried (unquenched) tryptophans, there is a heterogeneous class of tryptophans. In the presence of K or ouabain, conditions that favor E2, the modified Stern-Volmer plots are linear, consistent with a homogeneous population of tryptophans. Treatment with chymotrypsin to block the E1 to E2 transition results in a new set of quenching parameters which are unchanged with Na or K. Even after detergent denaturation (1% sodium dodecyl sulfate for 30 min), Stern-Volmer plots are nonlinear, and a significant fraction of tryptophan residues remain inaccessible to quencher. Denaturation with urea or guanidine HCl plus dithiothreitol increases the fraction of quenchable fluorescence even more, but still a small fraction, about 7-13%, is buried. The observed changes in exposure of the tryptophan residues would seem to account for the differences in intrinsic fluorescence seen on adding K and Na to Na,K-ATPase. The present results provide new evidence that a significant rearrangement of amino acid residues results from the E1 to E2 transition. Furthermore, a region of the molecule is inaccessible even after denaturation; this may correspond to highly hydrophobic stretches that are normally buried in the membrane.     

Fluorescence and 13C NMR determination of side-chain and backbone dynamics of synthetic melittin and melittin analogues in isotropic solvents

The dynamics in isotopic solvents of selectively 13C labeled synthetic melittin and three analogues have been investigated by using NMR and fluorescence techniques both separately and in combination. In conjunction with the "model-free" approach to interpretation of NMR relaxation data [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4570], the availability of steady-state fluorescence anisotropy and lifetime data augment T1, T2, and NOE data to provide quantitative information about fluorophore dynamics in these peptides. A method is presented for using combined fluorescence and NMR data to obtain technique- and model-independent values for parameters describing local motion of 13C-labeled fluorophores in peptides and proteins. The dynamics of melittin and melittin analogues are found to be consistent with structural characteristics inferred from CD, fluorescence, and NMR spectral information presented in the preceding paper (Weaver et al., 1989). In particular, the mobility of the random coil peptide monomers is shown to be quite similar, while side-chain as well as peptide backbone motion in the aggregated or oligomeric species differs markedly among the analogues. For melittin itself, experimentally determined overall rotational correlation times for the monomer and tetramer agree very well with values predicted on the basis of solvent-accessible protein surface area. The local dynamics of selectively 13C-labeled Trp-19 and Gly-12 residues of melittin are also found to be consistent with peptide structure. In random coil melittin monomer, a specific model for the motion indicates that the Trp side chain moves through an approximate angle of +/- 71 degrees about the beta-gamma bond with a correlation time of 159 +/- 24 ps. In melittin tetramer, the indole moiety is spatially more confined with a flip angle of +/- 37 degrees, yet demonstrates an increased rate of motion with a correlation time of 56 +/- 8 ps. The constrained mobility of the Trp-19 side chain is consistent with motional constraints inferred from the X-ray structure of melittin tetramer. These results show that protein side-chain motion, even of moieties as large as indole, can occur on the picosecond time scale and that these motions are reasonably similar to those inferred from molecular dynamics simulations.     

Cooperative assembly of a nativelike ubiquitin structure through peptide fragment complexation: energetics of peptide association and folding

Peptide fragments corresponding to the N- and C-terminal portions of bovine ubiquitin, U(1-35) and U(36-76), are shown by NMR to associate in solution to form a complex of modest stability (Kassn approximately 1.4 x 10(5) M(-1) at pH 7.0), with NMR features characteristic of a nativelike structure. The complex undergoes cold denaturation, with temperature-dependent estimates of stability from NMR indicating a DeltaC(p) degrees for fragment complexation in good agreement with that determined for native ubiquitin, suggesting that fragment association results in the burial of a similar hydrophobic surface area. The stability of the complex shows appreciable pH dependence, suggesting that ionic interactions on the surface of the protein contribute significantly. However, denaturation studies of native ubiquitin in the presence of guanidine hydrochloride (Gdn.HCl) show little pH dependence, suggesting that ionic interactions may be "screened" by the denaturant, as recently suggested. Examination of the conformation of the isolated peptide fragments has shown evidence for a low population of nativelike structure in the N-terminal beta-hairpin (residues 1-17) and weak nascent helical propensity in the helical fragment (residues 21-35). In contrast, the C-terminal peptide (36-76) shows evidence in aqueous solution, from some Halpha chemical shifts, for nonnative phi and psi angles; nonnative alpha-helical structure is readily induced in the presence of organic cosolvents, indicating that tertiary interactions in both native ubiquitin and the folded fragment complex strongly dictate its structural preference. The data suggest that the N-terminal fragment (1-35), where interaction between the helix and hairpin requires the minimum loss of conformational entropy, may provide the nucleation site for fragment complexation.     

NMR and circular dichroic studies on the solution conformation of a synthetic peptide derived from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase

The solution conformation of a synthetic peptide of 20 amino acids (P235-254) derived from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase was studied by proton two-dimensional NMR spectroscopy and circular dichroism. Based on the standard techniques we have assigned all the resonances in the NMR spectrum to the corresponding protons of the peptide. Analysis of the secondary chemical shift distribution and of the nuclear Overhauser effect connectivities showed no evidence for a highly populated regular conformation but suggested the tendency to form an alpha-helix around the unique Trp residue. The propensity for a helical structure is in agreement with the results of circular dichroic spectroscopy showing a slight negative band at 222 nm which was cancelled by 6 M guanidine hydrochloride. Increasing amounts of 2,2,2-trifluoroethanol (up to 40%) increase considerably the helical population of the peptide as reflected in the circular dichroic spectra. Analysis of the present results shows that the free peptide P235-254 has the tendency to form a basic amphiphilic helix. The presence of two acid residues, Glu236 and Asp239, on the hydrophilic side of the alpha-helix, which is mainly composed by basic residues, may explain the lower affinity of this peptide for calmodulin as compared with other peptides derived from calmodulin-activated enzymes.     

Structural stability of glycophorin. Effects of heat and guanidine . HCl

The effects of guanidine hydrochloride and high temperature on human glycophorin and sialic acid-free glycophorin were monitored by circular dichroism, viscosity, and fluorescence of 1-anilino-8-naphthalane sulfonate (ANS). The following observations were made: 1. Glycophorin and its sialic acid-free counterpart are unusually stable to both guanidine . HCl and heat. 2. CD and viscosity measurements indicate that guanidine . HCl neither causes a cooperative unfolding nor generates a random coil. 3. The ANS binding site is much more sensitive to guanidine . HCl than the ellipticity at 220 nm (theta 220). 4. The effect of temperature on CD is reversible whereas the effect of guanidine . HCl is not. 5. The carbohydrate moiety influences the viscosity, and also contributes to the changes in theta 220 when solutions of glycophorin are heated. These unusual properties indicate a complex mechanism of unfolding for this structurally stable macromolecule.     

The structure of human acidic fibroblast growth factor and its interaction with heparin.

The secondary and tertiary structure of recombinant human acidic fibroblast growth factor (aFGF) has been characterized by a variety of spectroscopic methods. Native aFGF consists of ca. 55% beta-sheet, 20% turn, 10% alpha-helix, and 15% disordered polypeptide as determined by laser Raman, circular dichroism, and Fourier transform infrared spectroscopy; the experimentally determined secondary structure content is in agreement with that calculated by the semi-empirical methods of Chou and Fasman (Chou, P. Y., and Fasman, G. C., 1974, Biochemistry 13, 222-244) and Garnier et al. (Garnier, J. O., et al., 1978, J. Mol. Biol. 120, 97-120). Using the Garnier et al. algorithm, the major secondary structure components of aFGF have been assigned to specific regions of the polypeptide chain. The fluorescence spectrum of native aFGF is unusual in that it is dominated by tyrosine fluorescence despite the presence of a tryptophan residue in the protein. However, tryptophan fluorescence is resolved upon excitation above 295 nm. The degree of tyrosine and tryptophan solvent exposure has been assessed by a combination of ultraviolet absorption, laser Raman, and fluorescence spectroscopy; the results suggest that seven of the eight tyrosine residues are solvent exposed while the single tryptophan is partially inaccessible to solvent in native aFGF, consistent with recent crystallographic data. Denaturation of aFGF by extremes of temperature or pH leads to spectroscopically distinct conformational states in which contributions of tyrosine and tryptophan to the fluorescence spectrum of the protein vary. The protein is unstable at physiological temperatures. Addition of heparin or other sulfated polysaccharides does not affect the spectroscopic characteristics of native aFGF. These polymers do, however, dramatically stabilize the native protein against thermal and acid denaturation as determined by differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The interaction of aFGF with such polyanions may play a role in controlling the activity of this growth factor in vivo.