Characterization of a partially folded intermediate of stem bromelain at low pH.

Equilibrium studies on the acid included denaturation of stem bromelain (EC 3.4.22.32) were performed by CD spectroscopy, fluorescence emission spectroscopy and binding of the hydrophobic dye, 1-anilino 8-naphthalene sulfonic acid (ANS). At pH 2.0, stem bromelain lacks a well defined tertiary structure as seen by fluorescence and near-UV CD spectra. Far-UV CD spectra show retention of some native like secondary structure at pH 2.0. The mean residue ellipticities at 208 nm plotted against pH showed a transition around pH 4.5 with loss of secondary structure leading to the formation of an acid-unfolded state. With further decrease in pH, this unfolded state regains most of its secondary structure. At pH 2.0, stem bromelain exists as a partially folded intermediate containing about 42.2% of the native state secondary structure Enhanced binding of ANS was observed in this state compared to the native folded state at neutral pH or completely unfolded state in the presence of 6 m GdnHCl indicating the exposure of hydrophobic regions on the protein molecule. Acrylamide quenching of the intrinsic tryptophan residues in the protein molecule showed that at pH 2.0 the protein is in an unfolded conformation with more tryptophan residues exposed to the solvent as compared to the native conformation at neutral pH. Interestingly, stem bromelain at pH 0.8 exhibits some characteristics of a molten globule, such as an enhanced ability to bind the fluorescent probe as well as considerable retention of secondary structure. All the above data taken together suggest the existence of a partially folded intermediate state under low pH conditions.     

Temperature-induced formation of a non-native intermediate state of the All β-sheet protein CD2

Domain 1 of the cell adhesion protein CD2 (CD2-1) has an all β-structure typical of proteins belonging to the immunoglobin superfamily. It has a remarkable, ability to fold as a native monomer or a metastable intertwined dimer. To understand the origin of structural rearrangements of CD2-1, we have studied equilibrium unfolding of the protein using various biophysical spectroscopic techniques. At temperatures above approx 68°C, a partially folded state of CD2-1 (H state) with a distinct secondary structure, involving largely exposed aromatic and hydrophobic residues and a substantially perturbed tertiary structure, is observed. In contrast, an unfolded state (D state) of CD2-1 with random-coil-like secondary and tertiary structures is observed in 6 M GuHCl. This partially folded high-temperature state has increased negative molar ellipticity at 222 nm in far-ultraviolet CD spectra, implying formation of a non-native helical conformation. The existence of this non-native high-temperature intermediate is consistent with relatively high intrinsic helical propensities in the primary sequence of CD2-1. This conformation flexibility may be important in the observed domain swapping of CD2-1.     

Comparison of CD spectra in the aromatic region on a series of variant proteins substituted at a unique position of tryptophan synthase alpha-subunit

CD spectra in the aromatic region of a series of the mutant alpha-subunits of tryptophan synthase from Escherichia coli, substituted at position 49 buried in the interior of the molecule, were measured at pH 7.0 and 25 degrees C. These measurements were taken to gain information on conformational change produced by single amino acid substitutions. The CD spectra of the mutant proteins, substituted by Tyr or Trp residue in place of Glu residue at position 49, showed more intense positive bands due to one additional Tyr or Trp residue at position 49. The CD spectra of other mutant proteins also differed from that of the wild-type protein, despite the fact that the substituted residues at position 49 were not aromatic. Using the spectrum of the wild-type protein (Glu49) as a standard, the spectra of the other mutants were classified into three major groups. For 10 mutant proteins substituted by Ile, Ala, Leu, Met, Val, Cys, Pro, Ser, His, or Gly, their CD values of bands (due to Tyr residues) decreased in comparison with those of the wild-type protein. The mutant protein substituted by Phe also belonged to this group. These substituted amino acid residues are more hydrophobic than the original residue, Glu. In the second group, three mutant proteins were substituted by Lys, Gln, or Asn, and the CD values of tyrosyl bands increased compared to those of the wild-type proteins. These residues are polar. In the third group, the CD values of tyrosyl bands of two mutant proteins substituted by Asp or Thr were similar to those of the wild-type protein, except for one band at 276.5 nm. These results suggested that the changes in the CD spectra for the mutant proteins were affected by the hydrophobicity of the residues at position 49.     

Nuclear magnetic resonance titration curves of histidine ring protons. Ribonuclease S-peptide and S-proteins.

The histidine C-2 proton NMR titration curves of ribonuclease S-peptide (residues 1 to 20) and S-protein (residues 21 to 124) are reported. Although S-protein contains 3 histidine residues, four discrete resonances are observed to titrate. One of these arises from the equivalent histidine residues of unfolded S-protein. The variation in area of the four resonances indicate that there is a reversible pH-dependent equilibrium between the folded and unfolded forms of S-protein, with some unfolded material being present at most pH values. Two of the resonances of the folded S-protein can be assigned to 2 of the histidine residues, 48 and 105, from the close similarity of their titration curves to those in ribonuclease. These similarities indicate a homology of portions of the folded conformation of S-protein to that of ribonuclease in solution. These results indicate that the complete amino acid sequence is not required to produce a folded conformation similar to the native globular protein, and they appear to eliminate the possibility that proteins fold from their NH2 terminus during protein synthesis. The low pH inflection present in the titration curve assigned to histidine residue 48 in ribonuclease is absent from this curve in S-protein. This is consistent with our previous conclusion that this inflection arises from the interaction of histidine 48 with aspartic acid residue 14, which is also absent in S-protein. The third titrating resonance of native S-protein is assigned to the remaining histidine residue at position 119. The properties of this resonance are not identical with either of the titration curves of the active site histidine residues 12 and 119 of ribonuclease. The resonance assigned to histidine 119 is the only one significantly affected on the addition of sodium phosphate to S-protein, indicating that some degree of phosphate binding occurs. In both the absence and presence of phosphate this curve also lacks the low pH inflection observed in the histidine 119 NMR titration curve in ribonuclease. This difference presumably arise from a conformational between ribonuclease and the folded S-protein involving a carboxyl group.     

Structure-based substitutions for increased solubility of a designed protein

Manipulation of protein solubility is important for many aspects of protein design and engineering. Previously, we designed a series of consensus ankyrin repeat proteins containing one, two, three and four identical repeats (1ANK, 2ANK, 3ANK and 4ANK). These proteins, particularly 4ANK, are intended for use as a universal scaffold on which specific binding sites can be constructed. Despite being well folded and extremely stable, 4ANK is soluble only under acidic conditions. Designing interactions with naturally occurring proteins requires the designed protein to be soluble at physiological pH. Substitution of six leucines with arginine on exposed hydrophobic patches on the surface of 4ANK resulted in increased solubility over a large pH range. Study of the pH dependence of stability demonstrated that 4ANK is one of the most stable ankyrin repeat proteins known. In addition, analogous leucine to arginine substitutions on the surface of 2ANK allowed the partially folded protein to assume a fully folded conformation. Our studies indicate that replacement of surface-exposed hydrophobic residues with positively charged residues can significantly improve protein solubility at physiological pH.     

De novo backbone and sequence design of an idealized alpha/beta-barrel protein: evidence of stable tertiary structure

We have designed, synthesized, and characterized a 216 amino acid residue sequence encoding a putative idealized alpha/beta-barrel protein. The design was elaborated in two steps. First, the idealized backbone was defined with geometric parameters representing our target fold: a central eight parallel-stranded beta-sheet surrounded by eight parallel alpha-helices, connected together with short structural turns on both sides of the barrel. An automated sequence selection algorithm, based on the dead-end elimination theorem, was used to find the optimal amino acid sequence fitting the target structure. A synthetic gene coding for the designed sequence was constructed and the recombinant artificial protein was expressed in bacteria, purified and characterized. Far-UV CD spectra with prominent bands at 222nm and 208nm revealed the presence of alpha-helix secondary structures (50%) in fairly good agreement with the model. A pronounced absorption band in the near-UV CD region, arising from immobilized aromatic side-chains, showed that the artificial protein is folded in solution. Chemical unfolding monitored by tryptophan fluorescence revealed a conformational stability (DeltaG(H2O)) of 35kJ/mol. Thermal unfolding monitored by near-UV CD revealed a cooperative transition with an apparent T(m) of 65 degrees C. Moreover, the artificial protein did not exhibit any affinity for the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (ANS), providing additional evidence that the artificial barrel is not in the molten globule state, contrary to previously designed artificial alpha/beta-barrels. Finally, 1H NMR spectra of the folded and unfolded proteins provided evidence for specific interactions in the folded protein. Taken together, the results indicate that the de novo designed alpha/beta-barrel protein adopts a stable three-dimensional structure in solution. These encouraging results show that de novo design of an idealized protein structure of more than 200 amino acid residues is now possible, from construction of a particular backbone conformation to determination of an amino acid sequence with an automated sequence selection algorithm.     

Environment-specific amino acid substitution tables: tertiary templates and prediction of protein folds.

The local environment of an amino acid in a folded protein determines the acceptability of mutations at that position. In order to characterize and quantify these structural constraints, we have made a comparative analysis of families of homologous proteins. Residues in each structure are classified according to amino acid type, secondary structure, accessibility of the side chain, and existence of hydrogen bonds from the side chains. Analysis of the pattern of observed substitutions as a function of local environment shows that there are distinct patterns, especially for buried polar residues. The substitution data tables are available on diskette with Protein Science. Given the fold of a protein, one is able to predict sequences compatible with the fold (profiles or templates) and potentially to discriminate between a correctly folded and misfolded protein. Conversely, analysis of residue variation across a family of aligned sequences in terms of substitution profiles can allow prediction of secondary structure or tertiary environment.     

Stepwise proteolytic removal of the beta subdomain in alpha-lactalbumin. The protein remains folded and can form the molten globule in acid solution.

Bovine alpha-lactalbumin (alpha-LA) is an alpha/beta protein which adopts partly folded states when dissolved at low pH (A-state), by removal of the protein-bound calcium at neutral pH and low salt concentration (apo-state), as well as in aqueous trifluoroethanol. Previous spectroscopic studies have indicated that the A-state of alpha-LA at pH 2.0, considered a prototype molten globule, has a native-like fold in which the helical core is mostly retained, while the beta subdomain is less structured. Here, we investigate the conformational features of three derivatives of alpha-LA characterized by a single peptide bond fission or a deletion of 12 or 19/22 amino-acid residues of the beta subdomain of the native protein (approximately from residue 34 to 57). These alpha-LA derivatives were obtained by limited proteolysis of the protein in its partly folded state(s). A nicked alpha-LA species consisting of fragments 1-,3-40 and 41-123 (nicked-LA) was prepared by thermolytic digestion of the 123-residue chain of alpha-LA in 50% (v/v) aqueous trifluoroethanol. Two truncated or gapped protein species given by fragments 1-40 and 53-123 (desbeta1-LA) or fragments 1-34 and 54-,57-123 (desbeta2-LA) were obtained by digestion of alpha-LA with pepsin in acid or with proteinase K at neutral pH in its apo-state, respectively. The two protein fragments of nicked or gapped alpha-LA are covalently linked by the four disulfide bridges of the native protein. CD measurements revealed that, in aqueous solution at neutral pH and in the presence of calcium, the three protein species maintain the helical secondary structure of intact alpha-LA, while the tertiary structure is strongly affected by the proteolytic cleavages of the chain. Temperature effects of CD signals in the far- and near-UV region reveal a much more labile tertiary structure in the alpha-LA derivatives, while the secondary structure is mostly retained even upon heating. In acid solution at pH 2.0, the three alpha-LA variants adopt a conformational state essentially identical to the molten globule displayed by intact alpha-LA, as demonstrated by CD measurements. Moreover, they bind strongly the fluorescent dye 8-anilinonaphthalene-1-sulfonate, which is considered a diagnostic feature of the molten globule of proteins. Therefore, the beta subdomain can be removed from the alpha-LA molecule without impairing the capability of the rest of the chain to adopt a molten globule state. The results of this protein dissection study provide direct experimental evidence that in the alpha-LA molten globule only the alpha domain is structured.     

Structural characterization of the ribosome maturation protein, RimM

The RimM protein has been implicated in the maturation of the 30S ribosomal subunit. It binds to ribosomal protein S19, located in the head domain of the 30S subunit. Multiple sequence alignments predicted that RimM possesses two domains in its N- and C-terminal regions. In the present study, we have produced Thermus thermophilus RimM in both the full-length form (162 residues) and its N-terminal fragment, spanning residues 1 to 85, as soluble proteins in Escherichia coli and have performed structural analyses by nuclear magnetic resonance spectroscopy. Residues 1 to 80 of the RimM protein fold into a single structural domain adopting a six-stranded beta-barrel fold. On the other hand, the C-terminal region of RimM (residues 81 to 162) is partly folded in solution. Analyses of 1H-15N heteronuclear single quantum correlation spectra revealed that a wide range of residues in the C-terminal region, as well as the residues in the vicinity of a hydrophobic patch in the N-terminal domain, were dramatically affected upon complex formation with ribosomal protein S19.     

1H-n.m.r. studies of the isolated activation segment from pig procarboxypeptidase A.

The isolated activation segment (asA) from pig pancreatic procarboxypeptidase A was studied by 1H-n.m.r. spectroscopy over a wide range of solution conditions. Isolated asA shows many characteristics of compactly folded globular proteins, such as the observation of perturbed positions for resonances from methyl groups, alpha-carbon atoms, histidine residues and the tyrosine residue. The single tyrosine residue (Tyr-70) exhibits a very high pKa, and both histidine and tyrosine residues show slow chemical modification (deuteration and iodination). In contrast, asA shows rapid NH exchange. Analysis of the spectra by pH titration and nuclear Overhauser effects revealed several residue interactions. Quantitative analysis of deuterium and tritium exchange allowed the assignment of the histidine C-2-H resonances to their respective residues in the sequence. His-66, the closest to the sites of proteolytic attack in the proenzyme, is shown to be the most accessible to solvent in procarboxypeptidase A. It was also shown that asA is thermally very stable ['melting' temperature (Tm) 88 degrees C] and requires a high urea concentration for denaturation (6.25 M, at pH 7.5). Evidence is presented for some degree of conformational flexibility in the premelting range, a feature that could be ascribed to the preponderance of helical secondary structure and to the lack of disulphide bridges. The free solution structure of asA is probably unchanged when it binds to carboxypeptidase A.     

Electrochemical, kinetic, and circular dichroic consequences of mutations at position 82 of yeast iso-1-cytochrome c

Replacement of Phe-82 in yeast iso-1-cytochrome c with Tyr, Leu, Ile, Ser, Ala, and Gly produces a gradation of effects on (1) the reduction potential of the protein, (2) the rate of reaction with Fe(EDTA)2-, and (3) the CD spectra of the ferricytochromes in the Soret region under conditions where contributions from the alkaline forms of these proteins are absent. The reduction potential of cytochrome c is lowered by as little as 10 mV (Tyr-82) or by as much as 43 mV (Gly-82; pH 6.0) as the result of these substitutions. The second-order rate constants for reduction of these cytochromes range from a low of 6.20 (2) x 10(4) for the Tyr-82 variant to a high of 14.8 x 10(4) M-1 s-1 for the Ser-82 variant [pH 6.0, 25 degrees C, mu = 0.1 M (sodium phosphate)]. Analysis of these rates by use of relative Marcus theory produces values of k11corr that range from 10.9 M-1 s-1 for the wild-type protein to 190 M-1 s-1 for the Gly-82 mutant [25 degrees C, mu = 0.1 M, pH 6.0 (sodium phosphate)]. Reinvestigation of the effect of substituting Phe-82 by a Tyr residue on the CD spectrum of the protein now reveals little alteration of the intense, negative Cotton effect in the Soret CD spectrum of ferricytochrome c. On the other hand, substitution of nonaromatic residues of various sizes at this position results in loss of this spectroscopic feature, consistent with previous findings.(ABSTRACT TRUNCATED AT 250 WORDS)     

A polar, solvent-exposed residue can be essential for native protein structure

BACKGROUND: A large energy gap between the native state and the non-native folded states is required for folding into a unique three-dimensional structure. The features that define this energy gap are not well understood, but can be addressed using de novo protein design. Previously, alpha(2)D, a dimeric four-helix bundle, was designed and shown to adopt a native-like conformation. The high-resolution solution structure revealed that this protein adopted a bisecting U motif. Glu7, a solvent-exposed residue that adopts many conformations in solution, might be involved in defining the unique three-dimensional structure of alpha(2)D. RESULTS: A variety of hydrophobic and polar residues were substituted for Glu7 and the dynamic and thermodynamic properties of the resulting proteins were characterized by analytical ultracentrifugation, circular dichroism spectroscopy, and nuclear magnetic resonance spectroscopy. The majority of substitutions at this solvent-exposed position had little affect on the ability to fold into a dimeric four-helix bundle. The ability to adopt a unique conformation, however, was profoundly modulated by the residue at this position despite the similar free energies of folding of each variant. CONCLUSIONS: Although Glu7 is not involved directly in stabilizing the native state of alpha(2)D, it is involved indirectly in specifying the observed fold by modulating the energy gap between the native state and the non-native folded states. These results provide experimental support for hypothetical models arising from lattice simulations of protein folding, and underscore the importance of polar interfacial residues in defining the native conformations of proteins.     

Molten globule-like folding intermediate of asialofetuin at acidic pH

In our earlier communication on acid-induced unfolding of bovine serum fetuin (BSF), we showed the existence of a molten globule (MG)-like state of BSF at pH 1.8. The MG state was characterized by higher content of secondary structure than native and almost complete loss of tertiary structure and more solvent exposed hydrophobic surface [Biochim. Biophys. Acta 1649 (2003) 164]. In this work we have shown the presence of an MG-like partially folded intermediate of asialofetuin at around pH 1.8, which is much different from the MG state observed in BSF in secondary structure contents. The results show that asialofetuin at pH 1.8 retains approximately 45% secondary structure, as evident from far-UV CD spectra. The near-UV CD spectra showed almost complete loss of tertiary structure. The intrinsic fluorescence and acrylamide quenching of the lone tryptophan residue showed that in acid-induced state, it is buried in the interior in a nonpolar environment. The temperature dependence of far-UV CD signal of asialofetuin at pH 1.8 exhibits a weak cooperative thermal transition. A significant increase in ANS fluorescence showed extensive solvent exposure of nonpolar cluster. Size exclusion chromatography (SEC) indicates a slight increase in the hydrodynamic size of acid-induced protein. These results suggest that asialofetuin at pH 1.8 represents the MG-like folding intermediate. Moreover, our results showed that glycosylation might play a role in stabilization of secondary structure during acid and/or thermal denaturation.     

pH-induced conformational changes in the soluble manganese-stabilizing protein of photosystem II

In this paper, we analyzed the pH-induced changes in the conformational states of the manganese-stabilizing protein (MSP) of photosystem II. Distinct conformational states of MSP were identified using fluorescence spectra, far-UV circular dichroism, and pressure-induced unfolding at varying suspension pH values, and four different conformational states of MSP were clearly distinguished using the center of fluorescence spectra mass when suspension pH was altered from 2 to 12. MSP was completely unfolded at a suspension pH above 11 and partly unfolded below a pH of 3. Analysis of the center of fluorescence spectral mass showed that the MSP structure appears stably folded around pH 6 and 4. The conformational state of MSP at pH 4 seems more stable than that at pH 6. Studies of peak positions of tryptophan fluorescence and MSP-bound 1-anilinonaphthalene-8-sulfonic acid fluorescence spectra supported this observation. A decrease in the suspension pH to 2 resulted in significant alterations in the MSP structure possibly because of protonation of unprotonated residues at lower pH, suggesting the existence of a large number of unprotonated amino acid residues at neutral pH possibly useful for proton transport in oxygen evolution. The acidic pH-induced conformational changes of MSP were reversible upon increase of pH to neutral pH; however, N-bromosuccinimide modification of tryptophan (Trp241) blocks the recovery of pH-induced conformational changes in MSP, implying that Trp241 is a key residue for the unfolded protein to form a functional structure. Thus, pH-induced structural changes of stable MSP (pH 6-4) may be utilized to analyze its functionality as a cofactor for oxygen evolution.     

Study of the role of tryptophan residues in streptokinase molecules using a chemical modification method

Incubation of streptokinase in an H2O2-dioxane-bicarbonate buffer (pH 8.5) system leads to the oxidation of tryptophan residues as can be evidenced from the changes in absorption and tryptophan fluorescence spectra. A complete oxidation of tryptophan residues of the protein takes place within 3 hours, the number of the residues is 4. The first tryptophanyl of the protein is oxidized the most easily; the activity of streptokinase decreases thereby by 50%. Modification of the second residue leads to complete inactivation of streptokinase. The rate constants for the oxidation of the first, of the two first and of the third plus fourth tryptophanyls are equal to 1.5.10(-2) min-1, 1,1.10(-2) min-1 and 0.5.10(-2) min -1, respectively. The complete oxidation of tryptophan residues is concomitant with the inability of streptokinase to form stable equimolar complexes with human plasminogen, but in does not result (as can be judged from the CD spectroscopy data) in the breakdown of the protein secondary structure. The specificity of oxidation of the protein tryptophan residues is discussed. The importance of readily oxidized tryptophan residues for the streptokinase function is postulated.     

Subdomain interactions as a determinant in the folding and stability of T4 lysozyme.

The folding of large, multidomain proteins involves the hierarchical assembly of individual domains. It remains unclear whether the stability and folding of small, single-domain proteins occurs through a comparable assembly of small, autonomous folding units. We have investigated the relationship between two subdomains of the protein T4 lysozyme. Thermodynamically, T4 lysozyme behaves as a cooperative unit and the unfolding transition fits a two-state model. The structure of the protein, however, resembles a dumbbell with two potential subdomains: an N-terminal subdomain (residues 13-75), and a C-terminal subdomain (residues 76-164 and 1-12). To investigate the effect of uncoupling these two subdomains within the context of the native protein, we created two circular permutations, both at the subdomain interface (residues 13 and 75). Both variants adopt an active wild-type T4 lysozyme fold. The protein starting with residue 13 is 3 kcal/mol less stable than wild type, whereas the protein beginning at residue 75 is 9 kcal/mol less stable, suggesting that the placement of the termini has a major effect on protein stability while minimally affecting the fold. When isolated as protein fragments, the C-terminal subdomain folds into a marginally stable helical structure, whereas the N-terminal subdomain is predominantly unfolded. ANS fluorescence studies indicate that, at low pH, the C-terminal subdomain adopts a loosely packed acid state. An acid state intermediate is also seen for all of the full-length variants. We propose that this acid state is comprised of an unfolded N-terminal subdomain and a loosely folded C-terminal subdomain.     

pH jump studies of the folding of the multidomain ribosomal protein L9: the structural organization of the N-terminal domain does not affect the anomalously slow folding of the C-terminal domain

The folding kinetics of the multidomain ribosomal protein L9 were studied using pH jump stopped-flow fluorescence and circular dichroism (CD) in conjunction with guanidine hydrochloride (GdnHCl) jump stopped-flow CD experiments. Equilibrium CD and 1D (1)H NMR measurements demonstrated that the C-terminal domain unfolds below pH 4 while the N-terminal domain remains fully folded. Thus, the N-terminal domain remains folded during the pH jump experiments. The folding rate constant of the C-terminal domain was determined to be 3.5 s(-1) by pH jump experiments conducted in the absence of denaturant using stopped-flow CD and fluorescence. CD-detected GdnHCl jump measurements showed that the N- and C-terminal domains fold independently each by an apparent two-state mechanism. The folding rate constant for the N-terminal domain and the C-terminal domain in the absence of denaturant were calculated to be 760 and 4. 7 s(-1), respectively. The good agreement between the pH jump and the denaturant concentration jump experiments shows that the folding rate of the C-terminal domain is the same whether or not the N-terminal domain is folded. This result suggests that the slow folding of the C-terminal domain is not a consequence of unfavorable interactions with the rest of the protein chain during refolding. This is an interesting result since contact order analysis predicts that the folding rate of the C-terminal domain should be noticeably faster. The folding rate of the isolated N-terminal domain was also measured by stopped-flow CD and was found to be the same as the rate for the domain in the intact protein.     

A proton-nuclear-magnetic-resonance study of human somatotropin (growth hormone). Assignment and properties of the histidine residues.

The 1H-n.m.r. spectra of human somatotropin (growth hormone) show perturbed peaks from individual aromatic and aliphatic apolar residues, characteristic of a specifically folded globular structure. The imidazole C-2-H resonances of the histidine residues (at positions 18, 21 and 151 in the somatotropin sequence) were individually resolved, and their titration behaviour in the pH range 1.2-11.5 was investigated. The imidazole C-2-H resonance of histidine-151 is assigned, by comparison of its titration behaviour in human somatotropin and desamido-somatotropin (Asn-152 leads to Asp-152). The C-2-H resonances of all three histidine residues are assigned, by comparison of their relative deuterium-exchange rates (determined by n.m.r.) and the relative tritium-exchange rates of the histidine residues (determined by tryptic digestion of tritiated human somatotropin and reversed-phase high-pressure liquid-chromatographic separation of the histidine-containing tryptic peptides). There is evidence that histidine-18 forms an ion-pair bond with a glutamic acid or aspartic acid residue. The globular structure does not appear to change from pH3 to 11.5, though there is evidence for an unfolding of a region of the structure (involving histidine-21 and a tyrosine residue) below pH3.     

Spatial separation of beta-sheet domains of beta-amyloid: disruption of each beta-sheet by N-methyl amino acids

In a recent model of beta-amyloid (Abeta) fibrils, based mainly on solid-state NMR data, a molecular layer consists of two beta-sheets (residues 12-23 and 31-40 of Abeta1-40), folded onto one another by a connecting "bend" structure (residues 25-29) in the side-chain dimension. In this paper, we use two N-methyl amino acids to disrupt each of the two beta-sheets individually (2NMe(NTerm), residues 17 and 19; and 2NMe(CTerm), residues 37 and 39), or both of them at the same time (4NMe, with the above four N-methylated residues). Our data indicate that incorporation of two N-methyl amino acids into one beta-sheet is sufficient to disrupt that sheet while leaving the other, unmodified beta-sheet intact and able to form fibrils. We show, however, that disruption of each of the two beta-sheets has strikingly different effects on fibrillogenesis kinetics and fibril morphology. Both 2NMe(NTerm) and 2NMe(CTerm) form fibrils at similar rates, but more slowly than that of unmodified Abeta1-40. Electron microscopy shows that 2NMe(NTerm) forms straight fibrils with fuzzy amorphous material coating the edges, while 2NMe(CTerm) forms very regular, highly twisted fibrils-in both cases, distinct from the morphology of Abeta1-40 fibrils. Both 2NMe peptides show a "CMC" approximately four times greater than that of Abeta1-40. CD spectra of these peptides also evolve differently in time: whereas the CD spectra of 2NMe(NTerm) evolve little over 10 days, those of 2NMe(CTerm) show a transition to high beta-sheet content at about day 4-5. We also show that disruption of both beta-sheet domains, as in 4NMe, prevents fibril formation altogether, and renders Abeta1-40 highly water soluble and monomeric, and with solvent-exposed side chains. In summary, our data show (1) that the two beta-sheet domains fold in a semiautonomous manner, since disrupting each one still allows the other to fold; (2) that disruption of the N-terminal beta-sheet has a more profound effect on fibrillogenesis than disruption of the C-terminal beta-sheet, suggesting that the former is the more critical for the overall structure of the fibril; and (3) that disruption of both beta-sheet domains renders the peptide monomeric and unable to form fibrils.     

Partially folded states of the cytolytic protein sticholysin II

Sticholysin II (Stn II) is a cytolytic protein produced by the sea anemone Stichodactyla helianthus, its effect being related to pore formation. The conformation of the protein and its temperature-induced transitions, in the 1.5-12.0 pH range and in the 0-0.5 M NaCl concentration interval, have been studied by circular dichroism and fluorescence spectroscopy. At temperature < 35 degrees C, the protein maintains the same, high beta-structure content, folded conformation in the 1.5-11.0 pH range and ionic strength up to 0.5 M. In the 1.5-3.5 pH range and ionic strength > or = 0.1 M, Stn II shows a thermal transition, resulting in a partially folded state characterized by: (i) a native-like content of regular secondary structure, as detected by far-UV CD; (ii) a largely disordered tertiary structure, as detected by near-UV CD, with partially exposed tryptophan residues according to their fluorescence emission; and (iii) ability to bind the hydrophobic probe 2-anilinonaphthalene-6-sulfonic acid. In the pH range 4.0-10.5, thermally-induced protein aggregation occurs. The obtained results demonstrate the existence of partially folded state of Stn II, which may contribute to the pore formation ability of this cytolysin.