Stability and folding kinetics of a ubiquitin mutant with a strong propensity for nonnative beta-hairpin conformation in the unfolded state

A F45W mutant of yeast ubiquitin has been used as a model system to examine the effects of nonnative local interactions on protein folding and stability. Mutating the native TLTGK G-bulged type I turn in the N-terminal beta-hairpin to NPDG stabilizes a nonnative beta-strand alignment in the isolated peptide fragment. However, NMR structural analysis of the native and mutant proteins shows that the NPDG mutant is forced to adopt the native beta-strand alignment and an unfavorable type I NPDG turn. The mutant is significantly less stable (approximately 9 kJ mol(-1)) and folds 30 times slower than the native sequence, demonstrating that local interactions can modulate protein stability and that attainment of a nativelike beta-hairpin conformation in the transition state ensemble is frustrated by the turn mutations. Surprising, alcoholic cosolvents [5-10% (v/v) TFE] are shown to accelerate the folding rate of the NPDG mutant. We conclude, backed-up by NMR data on the peptide fragments, that even though nonnative states in the denatured ensemble are highly populated and their stability further enhanced in the presence of cosolvents, the simultaneous increase in the proportion of nativelike secondary structure (hairpin or helix), in rapid equilibrium with nonnative states, is sufficient to accelerate the folding process. It is evident that modulating local interactions and increasing nonnative secondary structure propensities can change protein stability and folding kinetics. However, nonlocal contacts formed in the global cooperative folding event appear to determine structural specificity.     

Peptide models of folding initiation sites of bovine beta-lactoglobulin: identification of nativelike hydrophobic interactions involving G and H strands

In an attempt to characterize the early folding events in bovine beta-lactoglobulin (BLG), a set of peptides, covering the flexible N-terminal region and the stable C-terminus beta-core, was synthesized and analyzed by circular dichroism and by nuclear magnetic resonance in water, trifluoroethanol (TFE), and sodium dodecyl sulfate (SDS) below and above the critical micellar concentration. The role of local and long-range hydrophobic interactions in guiding the folding has been investigated. For the peptide fragment covering the more flexible N-terminal region of BLG (beta-strands A, B), where both theoretical predictions and kinetic refolding experiments suggested the formation of non-native alpha-helix, no native long-range contacts were identified, and a helical secondary structure was stabilized only in the presence of 25 mM SDS. At variance, in 50% (v/v) TFE, native, long-range hydrophobic interactions were observed in the peptide covering the core region comprising G and H beta-strands. The side chains involved in these interactions form a nativelike hydrophobic cluster, thus suggesting that the GH region may act as the folding initiation site for BLG. This result is reinforced by the identification, in the urea denaturated BLG, of residual structure located at the level of the GH interface, as evidenced by NMR analysis. These results, in excellent agreement with kinetic, thermodynamic, and cold denaturation folding data, once more underline the utmost importance of the GH region for the stability and folding of BLG. Severe aggregation effects prevented the structural analysis of the peptide covering the EFGH region, indicating that this larger segment does not represent an independent folding domain and that the terminal alpha-helix is necessary for stabilizing the BLG folding core.     

Nonnative helical motif in a chaperone-bound protein fragment

The effect of cotranslationally active chaperones on the conformation of incomplete protein chains is poorly understood. The secondary structure of a 77-residue chaperone-bound N-terminal protein fragment corresponding to the first five helices (A-E) of apomyoglobin (apoMb_1-77) is investigated here at the residue-specific level by multidimensional NMR. The substrate-binding domain of DnaK, DnaK-β, is employed as a chaperone model. By taking advantage of the improved spectral quality resulting from chaperone deuteration, we find that DnaK-β-bound apoMb_1-77 displays a region of nonnative helicity at residues away from the main chaperone binding site. The nonnative structural motif comprises portions of the native D and E helices and has similar characteristics to the reported nonnative DE helical region of acid-unfolded full-length apoMb. Upon incorporation of the missing C-terminal amino acids, a structural kink develops between residues 56 and 57, and two separate native D and E helices are generated. This work highlights, for the first time to our knowledge, the presence of a nonnative helical motif in a large chaperone-bound protein fragment under physiologically relevant solution conditions.     

Circular dichroic investigation of the native and non-native conformational states of the growth factor receptor-binding protein 2 N-terminal src homology domain 3: effect of binding to a proline-rich peptide from guanine nucleotide exchange factor

SH3 (src homology domain 3) domains are small protein modules that interact with proline-rich peptides. The structure of the N-terminal SH3 domain from growth factor receptor-binding protein 2 (Grb2), an adapter protein in the intracellular signaling pathway to Ras, was investigated by circular dichroic (CD) spectroscopy. The compact native beta-barrel conformation, previously elucidated by NMR spectroscopy, was largely predominant at pH = 4.8, in the absence of salt. From the structural changes induced by varying pH, ionic strength, temperature, or hydrophobicity of the environment, evidence for the existence of distinct nonnative conformations was obtained in the far- and near-UV domains. Along the free energy scale, these appear to distribute into two conformational ensembles, depending on the extent of structural and thermodynamic differences compared to the native conformation. The first ensemble consists of non-native conformations with a nativelike secondary structure, and the second is composed of partially unfolded conformations having short alpha-helical fragments or turnlike motifs in their nonnative secondary structure. Most of the observed nonnative conformations exist in mild or nondenaturing conditions. They probably have distinct compactness of their inner structure, depending on the strength of nonlocal interactions, but only the native all-beta conformation possesses a condensed protein exterior, appropriate for the binding to the VPPPVPPRRR decapeptide from Sos. Upon binding, the native conformation undergoes a local tertiary structure change in a hydrophobic pocket at the binding site. This is accompanied by the PP-II helix folding of the proline-rich peptide. Interestingly, in the near-UV domain, a significant change in the spectral contribution of an aromatic exciton was observed, thus allowing quantitative tracking of the binding process.     

Folding and domain-domain interactions of the chaperone PapD measured by 19F NMR

The folding of the two-domain bacterial chaperone PapD has been studied to develop an understanding of the relationship between individual domain folding and the formation of domain-domain interactions. PapD contains six phenylalanine residues, four in the N-terminal domain and two in the C-terminal domain. To examine the folding properties of PapD, the protein was both uniformly and site-specifically labeled with p-fluoro-phenylalanine ((19)F-Phe) for (19)F NMR studies, in conjunction with those of circular dichroism and fluorescence. In equilibrium denaturation experiments monitored by (19)F NMR, the loss of (19)F-Phe native intensity for both the N- and C-terminal domains shows the same dependence on urea concentration. For the N-terminal domain the loss of native intensity is mirrored by the appearance of separate denatured resonances. For the C-terminal domain, which contains residues Phe 168 and Phe 205, intermediate as well as denatured resonances appear. These intermediate resonances persist at denaturant concentrations well beyond the loss of native resonance intensity and appear in kinetic refolding (19)F NMR experiments. In double-jump (19)F NMR experiments in which proline isomerization does not affect the refolding kinetics, the formation of domain-domain interactions is fast if the protein is denatured for only a short time. However, with increasing time of denaturation the native intensities of the N- and C-terminal domains decrease, and the denatured resonances of the N-terminal domain and the intermediate resonances of the C-terminal domain accumulate. The rate of loss of the N-terminal domain resonances is consistent with a cis to trans isomerization process, indicating that from an equilibrium denatured state the slow refolding of PapD is due to the trans to cis isomerization of one or both of the N-terminal cis proline residues. The data indicate that both the N- and C-terminal domains must fold into a native conformation prior to the formation of domain-domain interactions.     

Nicked apomyoglobin: a noncovalent complex of two polypeptide fragments comprising the entire protein chain

Limited proteolysis of the 153-residue chain of horse apomyoglobin (apoMb) by thermolysin results in the selective cleavage of the peptide bond Pro88-Leu89. The N-terminal (residues 1-88) and C-terminal (residues 89-153) fragments of apoMb were isolated to homogeneity and their conformational and association properties investigated in detail. Far-UV circular dichroism (CD) measurements revealed that both fragments in isolation acquire a high content of helical secondary structure, while near-UV CD indicated the absence of tertiary structure. A 1:1 mixture of the fragments leads to a tight noncovalent protein complex (1-88/89-153, nicked apoMb), characterized by secondary and tertiary structures similar to those of intact apoMb. The apoMb complex binds heme in a nativelike manner, as given by CD measurements in the Soret region. Second-derivative absorption spectra in the 250-300 nm region provided evidence that the degree of exposure of Tyr residues in the nicked species is similar to that of the intact protein at neutral pH. Also, the microenvironment of Trp residues, located in positions 7 and 14 of the 153-residue chain of the protein, is similar in both protein species, as given by fluorescence emission data. Moreover, in analogy to intact apoMb, the nicked protein binds the hydrophobic dye 1-anilinonaphthalene-8-sulfonate (ANS). Taken together, our results indicate that the two proteolytic fragments 1-88 and 89-153 of apoMb adopt partly folded states characterized by sufficiently nativelike conformational features that promote their specific association and mutual stabilization into a nicked protein species much resembling in its structural features intact apoMb. It is suggested that the formation of a noncovalent complex upon fragment complementation can mimic the protein folding process of the entire protein chain, with the difference that the folding of the complementary fragments is an intermolecular process. In particular, this study emphasizes the importance of interactions between marginally stable elements of secondary structure in promoting the tertiary contacts of a native protein. Considering that apoMb has been extensively used as a paradigm in protein folding studies for the past few decades, the novel fragment complementing system of apoMb here described appears to be very useful for investigating the initial as well as late events in protein folding.     

Equilibrium and kinetic measurements of the conformational transition of reduced thioredoxin

The single disulfide bond in Escherichia coli thioredoxin was reduced by reaction with a 20-fold excess of reduced dithiothreitol at neutral pH and 25 degrees C. For some measurements, reduced thioredoxin was further reacted with iodoacetamide to alkylate the cysteinyl residues. The denaturation transitions of oxidized, reduced, and reduced alkylated thioredoxin were observed by using far-ultraviolet circular dichroic and exclusion chromatographic measurements. Cleavage of the disulfide bond lowers the stability of the native thioredoxin to denaturation by about 2.4 kcal/mol, and subsequent alkylation lowers the stability by a further 1.6 kcal/mol. The kinetics of the conformational change of reduced thioredoxin in guanidine hydrochloride were observed by using exclusion chromatography at moderate pressure and 2 degrees C. Analyses of single and multimixing protocols are consistent with a predominant nonnative configuration in the denatured state and the transient accumulation of a compact nativelike intermediate during refolding. The intermediate can incorporate the nonnative configuration and can accommodate its isomerization. No compelling chromatographic evidence was found for a conformation having an elution time different from that characteristic for either the native or the denatured protein.     

Acid-induced equilibrium folding intermediate of human platelet profilin

The acid-induced unfolding of human platelet profilin (HPP) can be minimally modeled as a three-state process. Equilibrium unfolding studies have been performed on human platelet profilin1 (HPP) and monitored by far-UV circular dichroism, tryptophan fluorescence, ANS binding, and NMR spectroscopy. Far-UV CD measurements obtained by acid titration demonstrate that HPP unfolds via a three-state mechanism (N --> I --> U), with a highly populated intermediate between pH 4 and 5. Approximately 80% of native helical secondary structural content remains at pH 4, as indicated by monitoring the CD signal at 222 nm. The stability (DeltaGH2O) of the native conformation at pH 7.0 (obtained by monitoring the change in tryptophan signal as a function of urea concentration) is 5.56 +/- 0.51 kcal mol-1; however, the DeltaGH2O for the intermediate species at pH 4 is 2.01 +/- 0.47 kcal mol-1. The calculated m-values for the pH 7.0 and pH 4.0 species were 1.64 +/- 0.15 and 1.34 +/- 0.17 kcal mol-1 M-1, respectively, which is an indication that the native and intermediate species are similarly compact. Additionally, translational diffusion measurements obtained by NMR spectroscopy and ANS binding studies are consistent with a globular and compact conformation at both pH 7.0 and 4.0. The pKa values for the two histidine (His) residues located on helix 4 of HPP were determined to be 5.6 and 5.7 pH units. These pKa values coincide with the midpoint of the far-UV CD acid titration curve and suggest that the protonation of one or both His residues may play a role in the formation of the unfolding intermediate. Stable intermediate species populate the 2D 1H-15N HSQC NMR spectra between pH 4 and 5. A number of backbone and side-chain resonances show significant perturbations relative to the native spectrum; however, considerable nativelike tertiary contacts remain. Interestingly, the residues on HPP that are significantly altered at low pH coincide with segments of the G-actin binding surface and poly-l-proline binding interface. The earlier reports that a decrease in pH below 6.0 induces structural alterations in profilin, favoring dissociation of the profilin-actin complex, corresponds with the structural alterations observed in the partially unfolded species. Our findings suggest that a novel mechanism for pH induced disruption of the profilin-G-actin complex involve a nativelike unfolding intermediate of profilin.     

Cytochrome c reconstituted from two peptide fragments displays native-like redox properties.

Recombination of two fragments of horse cytochrome c (the heme-containing N-fragment, residues 1-56, and the C-fragment, residues 57-104), which are substantially unstructured at neutral pH, gives rise to a 1:1 fragment complex with a compact conformation, in which the alpha helical structure and the native Met80-Fe(III) axial bond are recovered. With respect to the native protein, the ferric complex shows a less rigid atomic packing and a decreased stability [Delta(DeltaG(o))D = 14.7 kJ.mol(-1)], ascribed to perturbations involving the Trp59 microenvironment and, to a lower extent, the heme pocket region. The redox potential, E1/2 = 234 +/- 5 mV vs. normal hydrogen electrode at 25 degrees C, is close to that of the intact protein, consistent with recovery of the native Met80-heme Fe(III) axial bond. Furthermore, the fragment complex shows reactivity similar to intact cytochrome c, in the reaction with cytochrome c oxidase. We conclude that the absence in the complex of some native cross-links and interlocked packing important for protein rigidity and stability is not as relevant for maintaining the native redox properties of the protein, provided that some structural requirements (i.e. recovering of the native-like alpha helical structure) are fulfilled and coordination of Met80 to the heme-iron is restored.     

Conformational analysis of a set of peptides corresponding to the entire primary sequence of the N-terminal domain of the ribosomal protein L9: evidence for stable native-like secondary structure in the unfolded state

There is considerable interest in the structure of the denatured state and in the role local interactions play in protein stability and protein folding. Studies of peptide fragments provide one method to assess local conformational preferences which may be present in the denatured state under native-like conditions. A set of peptides corresponding to the individual elements of secondary structure derived from the N-terminal domain of the ribosomal protein L9 have been synthesized. This small 56 residue protein adopts a mixed alpha-beta topology and has been shown to fold rapidly in an apparent two-state fashion. The conformational preferences of each peptide have been analyzed by proton nuclear magnetic resonance spectroscopy and circular dichroism spectroscopy. Peptides corresponding to each of the three beta-stands and to the first alpha-helix are unstructured as judged by CD and NMR. In contrast, a peptide corresponding to the C-terminal helix is remarkably structured. This 17 residue peptide is 53 % helical at pH 5.4, 4 degrees C. Two-dimensional NMR studies demonstrate that the helical structure is distributed approximately uniformly throughout the peptide, although there is some evidence for fraying at the C terminus. Detailed analysis of the NMR spectra indicate that the helix is stabilized, in part, by a native N-capping interaction involving Thr40. A mutant peptide which lacks Thr40 is only 32 % helical. pH and ionic strength-dependent studies suggested that charge charge interactions make only a modest net contribution to the stability of the peptide. The protein contains a trans proline peptide bond located at the first position of the C-terminal helix. NMR analysis of the helical peptide and of a smaller peptide containing the proline residue indicates that only a small amount of cis proline isomer (8 %) is likely to be populated in the unfolded state.     

Structural and dynamic characterization of an unfolded state of poplar apo-plastocyanin formed under nondenaturing conditions

Plastocyanin is a predominantly beta-sheet protein containing a type I copper center. The conformational ensemble of a denatured state of apo-plastocyanin formed in solution under conditions of low salt and neutral pH has been investigated by multidimensional heteronuclear NMR spectroscopy. Chemical shift assignments were obtained by using three-dimensional triple-resonance NMR experiments to trace through-bond heteronuclear connectivities along the backbone and side chains. The (3)J(HN,Halpha) coupling constants, (15)N-edited proton-proton nuclear Overhauser effects (NOEs), and (15)N relaxation parameters were also measured for the purpose of structural and dynamic characterization. Most of the residues corresponding to beta-strands in the folded protein exhibit small upfield shifts of the (13)C(alpha) and (13)CO resonances relative to random coil values, suggesting a slight preference for backbone dihedral angles in the beta region of (phi,psi) space. This is further supported by the presence of strong sequential d(alphaN)(i, i + 1) NOEs throughout the sequence. The few d(NN)(i, i + 1) proton NOEs that are observed are mostly in regions that form loops in the native plastocyanin structure. No medium or long-range NOEs were observed. A short sequence, between residues 59 and 63, was found to populate a nonnative helical conformation in the unfolded state, as indicated by the shift of the (13)C(alpha), (13)CO, and (1)H(alpha) resonances relative to random coil values and by the decreased values of the (3)J(HN,Halpha) coupling constants. The (15)N relaxation parameters indicate restriction of motions on a nanosecond timescale in this region. Intriguingly, this helical conformation is present in a sequence that is close to but not in the same location as the single short helix in the native folded protein. The results are consistent with earlier NMR studies of peptide fragments of plastocyanin and confirm that the regions of the sequence that form beta-strands in the native protein spontaneously populate the beta-region of (phi,psi) space under folding conditions, even in the absence of stabilizing tertiary interactions. We conclude that the state of apo-plastocyanin present under nondenaturing conditions is a noncompact unfolded state with some evidence of nativelike and nonnative local structuring that may be initiation sites for folding of the protein.     

NMR evidence for forming highly populated helical conformations in the partially folded hNck2 SH3 domain

Recent studies of several proteins implied that the folding of β-proteins may follow a nonhierarchical mechanism in which two major transitions are essential, i.e., the collapse of a random coil to form a nonnative helical intermediate, followed by a transformation into the native β-structure. We report that the first hNck2 SH3 domain, assuming an all-β barrel in the native form, can be reversibly transformed into a stable and nonnative helical state by acid-unfolding. We also conducted extensive NMR and mutagenesis studies that led to two striking findings: 1), NMR analysis reveals that in the helical state formed at pH 2.0, the first and last β-strands in the native form become unstructured, whereas the rest is surprisingly converted into two highly populated helices with a significantly limited backbone motion; and 2), a conserved four-residue sequence is identified on the second β-strand, a mutation of which suddenly renders the SH3 domain into a helical state even at pH 6.5, with NMR conformational and dynamic properties highly similar to those of the wild-type at pH 2.0. This observation implies that the region might contribute key interactions to disrupt the helical state, and to facilitate a further transformation into the native SH3 fold in the second transition.     

Conformational analyses of a partially-folded bioactive prodomain of human furin

The 81-residue multifunctional prodomain of human furin adopts only a partially-folded conformational state under near physiological conditions. By use of NMR spectroscopy, we demonstrate that the N-terminal residues 1-46 of the prodomain in 50% trifluoroethanol (TFE) populates backbone conformations containing a short helix, a beta-strand and a helix-loop-helix super-secondary structure with elements of tertiary interactions. (15)N NMR relaxation measurements indicate that the helix-loop-helix region has similar motional characteristics in the fast picosecond to nanosecond timescales. On the other hand, the intervening segment (residues 47-65) is predominantly unstructured with a long and highly flexible region surrounding the protease 'activation loop' followed by a partially helical segment in the C-terminal end. Interestingly, the helix-loop-helix "fold" was found to be populated even when excised out of the full-length prodomain, since a peptide fragment derived from residues Pro16-Arg49 can also form the helix-loop-helix structure in aqueous solution in the absence of TFE. Structure analyses reveal that two helices orient in an antiparallel fashion directed by the sharing of hydrophobic residues involved in helix-capping interactions. Very importantly, a positively-charged Lys residue replacing His43 in the 16-49 fragment imparts stability to the super-secondary structure at both acidic and neutral pH, while a hydrophobic residue Leu at position 43 appears to destabilize the helical conformation in the 31-44 region. As such, this study provides valuable insights into the structural properties of the furin prodomain in relation to its role in the folding of the furin zymogen and its inhibitory action toward furin.     

A monomeric, biologically active, full-length human apolipoprotein E

Apolipoprotein E (apoE) is an exchangeable apolipoprotein that plays an important role in lipid/lipoprotein metabolism and cardiovascular diseases. Recent evidence indicates that apoE is also critical in several other important biological processes, including Alzheimer's disease, cognitive function, immunoregulation, cell signaling, and infectious diseases. Although the X-ray crystal structure of the apoE N-terminal domain was solved in 1991, the structural study of full-length apoE is hindered by apoE's oligomerization property. Using protein-engineering techniques, we generated a monomeric, biologically active, full-length apoE. Cross-linking experiments indicate that this mutant is nearly 95-100% monomeric even at 20 mg/mL. CD spectroscopy and guanidine hydrochloride denaturation demonstrate that the structure and stability of the monomeric mutant are identical to wild-type apoE. Monomeric and wild-type apoE display similar lipid-binding activities in dimyristoylphosphatidylcholine clearance assays and formation of reconstituted high-density lipoproteins. Furthermore, the monomeric and wild-type apoE proteins display an identical LDL receptor binding activity. Availability of this monomeric, biologically active, full-length apoE allows us to collect high quality NMR data for structural determination. Our initial NMR data of lipid-free apoE demonstrates that the N-terminal domain in the full-length apoE adopts a nearly identical structure as the isolated N-terminal domain, whereas the C-terminal domain appears to become more structured than the isolated C-terminal domain fragment, suggesting a weak domain-domain interaction. This interaction is confirmed by NMR examination of a segmental labeled apoE, in which the N-terminal domain is deuterated and the C-terminal domain is double-labeled. NMR titration experiments further suggest that the hinge region (residues 192-215) that connects apoE's N- and C-terminal domains may play an important role in mediating this domain-domain interaction.     

Different effects of trifluoroethanol and glycerol on the stability of tropomyosin helices and the head-to-tail complex

Tropomyosin (Tm) is a dimeric coiled-coil protein, composed of 284 amino acids (410 A), that forms linear homopolymers through head-to-tail interactions at low ionic strength. The head-to-tail complex involves the overlap of approximately nine N-terminal residues of one molecule with nine C-terminal residues of another Tm molecule. In this study, we investigate the influence of 2,2,2-trifluoroethanol (TFE) and glycerol on the stability of recombinant Tm fragments (ASTm1-142, Tm143-284(5OHW269)) and of the dimeric head-to-tail complex formed by the association of these two fragments. The C-terminal fragment (Tm143-284(5OHW269)) contains a 5-hydroxytryptophan (5OHW) probe at position 269 whose fluorescence is sensitive to the head-to-tail interaction and allows us to accompany titrations of Tm143-284(5OHW269) with ASTm1-142 to calculate the dissociation constant (Kd) and the interaction energy at TFE and glycerol concentrations between 0% and 15%. We observe that TFE, but not glycerol, reduces the stability of the head-to-tail complex. Thermal denaturation experiments also showed that the head-to-tail complex increases the overall conformational stability of the Tm fragments. Urea and thermal denaturation assays demonstrated that both TFE and glycerol increase the stability of the isolated N- and C-terminal fragments; however, only TFE caused a significant reduction in the cooperativity of unfolding these fragments. Our results show that these two cosolvents stabilize the structures of individual Tm fragments in different manners and that these differences may be related to their opposing effects on head-to-tail complex formation.     

Role of side-chains in the cooperative beta-hairpin folding of the short C-terminal fragment derived from streptococcal protein G

A short C-terminal fragment of immunoglobulin-binding domain of streptococcal protein G is known to form nativelike beta-hairpin at physiological conditions. To understand the cooperative folding of the short peptide, eight Ala-substituted mutants of the fragment were investigated with respect to their structural stabilities by analyzing temperature dependence of NMR signals. On comparison of the obtained thermodynamic parameters, we found that the nonpolar residues Tyr45 and Phe52 and the polar residues Asp46 and Thr49 are crucial for the beta-hairpin folding. The results suggest a strong interaction between the nonpolar side chains that participates in a putative hydrophobic cluster and that the polar side chains form a fairly rigid conformation around the loop (46-51). We also investigated the complex formation of the mutants with N-terminal fragment at the variety of temperature to get their thermal unfolding profiles and found that the mutations on the residues Asp46 and Thr49 largely destabilized the complexes, while substitution of Asp47 slightly stabilized the complex. From these results, we deduced that both the hydrophobic cluster formation and the rigidity of the loop (46-51) cooperatively stabilize the beta-hairpin structure of the fragment. These interactions which form a stable beta-hairpin may be the initial structural scaffold which is important in the early folding events of the whole domain.     

Stabilization of helical structure in two 17-residue amphipathic analogues of the C-terminal peptide of cytochrome C

The conformations of two 17-residue peptide analogues derived from the C-terminal sequence of pigeon cytochrome c (native sequence = KAERADLIAYLKQATAK) were examined in aqueous and lipid environments by CD spectroscopy. The two analogues, KKLLKKLIAYLKQATAK (K peptide) and EELLEELIAYLKQATAK (E peptide), were made amphipathic with respect to helical segregation by substituting a 6-residue sequence at the N-terminus of the native peptide. Their structures were compared to the native peptide under aqueous conditions of varying pH and temperature, and in the presence of liposomes composed of phosphatidylcholine and phosphatidylserine in the ratio of 9:1. The results indicated that the native peptide remains unstructured under all the conditions examined even though this region of the native molecule is surface exposed and helical. The E peptide, however, was helical under aqueous conditions at 25 degrees C from pH 2-10 with a maximum helicity at pH 4 (54% helix from analysis of CD data). The ellipticity of the E peptide at pH 4 and 8 was concentration dependent, indicating an aggregation phenomenon. In studies in which the CD spectrum was measured at different temperatures, the E peptide became more helical at lower temperatures at pH 4 but not at pH 8. Upon interaction with a lipid membrane in the form of liposomes, there appeared to be a slight destabilization in the structure of the E peptide. The K peptide in an aqueous environment behaved like the native peptide in that it was structureless at all pHs and temperatures examined. In the presence of liposomes, however, this peptide had a high helical content (75% helix from analysis of CD data). These findings suggest that while stabilization of the helix dipole with negative charges at the N-terminus are important in inducing helical conformation in the E peptide, hydrophobic interactions created during aggregation appear to provide the principal stabilizing force. The results with the K peptide demonstrate that the positive N-terminal sequence of this peptide is able to interact with the negatively charged head groups in the phospholipid membrane in such a fashion as to stabilize a helical structure that is not apparent in an aqueous environment alone.     

Molecular dynamics simulations of the native and partially folded states of ubiquitin: influence of methanol cosolvent, pH, and temperature on the protein structure and dynamics

A series of explicit-solvent molecular dynamics simulations of the protein ubiquitin are reported, which investigate the effect of environmental factors (presence of methanol cosolvent in the aqueous solution, neutral or low pH value, room or elevated temperature) on the structure, stability, and dynamics of the protein. The simulations are initiated either from the native structure of the protein or from a model of a partially folded state (A-state) that is known to exist at low pH in methanol-water mixtures. The main results of the simulations are: (1) The ubiquitin native structure is remarkably stable at neutral pH in water; (2) the addition of the methanol cosolvent enhances the stability of the secondary structure but weakens tertiary interactions within the protein; (3) this influence of methanol on the protein structure is enhanced at low pH, while the effect of lowering the pH in pure water is limited; and (4) the A-state of ubiquitin can be described as a set of relatively rigid secondary structure elements (a native-like beta-sheet and native-like alpha-helix plus two nonnative alpha-helices) connected by flexible linkers.     

The solution structure of photosystem I accessory protein E from the cyanobacterium Nostoc sp. strain PCC 8009.

PsaE is a small basic subunit located on the stromal (cytoplasmic) side of photosystem I. In cyanobacteria, this subunit participates in cyclic electron transport and modulates the interactions of the complex with soluble ferredoxin. The PsaE protein isolated from the cyanobacterium Synechococcus sp. strain PCC 7002 adopts the beta topology of an SH3 domain, with five beta strands (betaA through betaE) and a turn of 3(10) helix between strands betaD and betaE [Falzone, C. J., Kao, Y.-H., Zhao, J., Bryant, D. A., and Lecomte, J. T. J. (1994) Biochemistry 33, 6052-6062]. The primary structure of the PsaE protein is strongly conserved across all oxygen-evolving photosynthetic organisms. However, variability in loop lengths, as well as N- or C-terminal extensions, suggests that the structure of a second representative PsaE subunit would be useful to characterize the interactions among photosystem I polypeptides. In this work, the solution structure of PsaE from the cyanobacterium Nostoc sp. strain PCC 8009 was determined by NMR methods. Compared to PsaE from Synechococcus sp. strain PCC 7002, this PsaE has a seven-residue deletion in the loop connecting strands betaC and betaD, and an eight-residue C-terminal extension. Angular and distance restraints derived from homonuclear and heteronuclear NMR experiments were used to calculate structures by a distance-geometry/simulated-annealing protocol. A family of 20 structures (rmsd of 0.24 A in the regular secondary structure) is presented. Differences between the two cyanobacterial proteins are mostly confined to the CD loop region; the C-terminal extension is disordered. The thermodynamic stability of Nostoc sp. strain PCC 8009 PsaE toward urea denaturation was measured by circular dichroism and fluorescence spectroscopy, and thermal denaturation was monitored by UV absorption spectroscopy. Chemical and thermal denaturation curves are modeled satisfactorily with two-state processes. The DeltaG degrees of unfolding at room temperature is 12.4 +/- 0.3 kJ mol(-1) (pH 5), and the thermal transition midpoint is 59 +/- 1 degrees C (pH 7). Interactions with other proteins in the photosystem I complex may aid in maintaining PsaE in its native state under physiological conditions.     

Structural dissection of alkaline-denatured pepsin

It has been established in a number of studies that the alkaline-denatured state of pepsin (the I(P) state) is composed of a compact C-terminal lobe and a largely unstructured N-terminal lobe. In the present study, we have investigated the residual structure in the I(P) state in more detail, using limited proteolysis to isolate and characterize a tightly folded core region from this partially denatured pepsin. The isolated core region corresponds to the 141 C-terminal residues of the pepsin molecule, which in the fully native state forms one of the two lobes of the structure. A comparative study using NMR and CD spectroscopy has revealed, however, that the N-terminal lobe contributes a substantial amount of additional residual structure to the I(P) state of pepsin. CD spectra indicate in addition that significant nonnative alpha-helical structure is present in the C-terminal lobe of the structure when the N-terminal lobe of pepsin is either unfolded or removed by proteolysis. This study demonstrates that the structure of pepsin in the I(P) state is significantly more complex than that of a fully folded C-terminal lobe connected to an unstructured N-terminal lobe.