Significance of the four carboxyl terminal amino acid residues of bovine pancreatic ribonuclease A for structural folding

The C-terminal amino acid residues of bovine pancreatic ribonuclease A (RNase A) form a core structure in the initial stage of the folding process that leads to the formation of the tertiary structure. In this paper, roles of the C-terminal four amino acids in the structure, function, and refolding were studied by use of recombinant mutant enzymes in which these residues were deleted or replaced. Purified mutant enzymes were analyzed for their secondary structure, thermal stability, and ability to regenerate from the denatured and reduced state. The C-terminal deleted mutant enzymes showed lower hydrolytic activity for C>p and nearly identical CD spectra compared with the wild-type enzyme. The rate of recovery of activity was significantly different among the C-terminal deleted mutant enzymes when air oxidation was employed in the absence of GSH and GSSG: the rates decreased in the order of des-124-, des-(123-124)-, and des-(122-124)-RNase A. It is noteworthy that the regeneration rates of mutant RNase A in the presence of GSH and GSSG were nearly the same. Des-(121-124)-RNase A failed to recover activity both in the presence and absence of glutathione, due to the mismatched formation of disulfide bonds. The mutant enzyme in which all of the C-terminal four amino acid residues were replaced by alanine residues showed lower hydrolytic activity and an indistinguishable CD spectrum compared with the wild-type enzyme, and also recovered its activity from the denatured and reduced state by air oxidation. The D121 mutant enzymes showed decreased hydrolytic activity and identical CD spectra compared with the wild type. The recovery rates of activity of D121A and D121K were determined to be lower than that of the wild-type enzyme, while the rate of recovery of D121E was comparable to that of the wild type. The C-terminal amino acids play a significant role in the formation of the correct disulfide bonds during the refolding process, and the interaction of amino acid residues and the existence of the main chain around the C-terminal region are both important for achieving the efficient packing of the RNase A molecule.     

Crystallographic studies on the role of the C-terminal segment of human angiogenin in defining enzymatic potency

Human angiogenin (Ang) is an RNase in the pancreatic RNase superfamily that induces angiogenesis. Its catalytic activity is comparatively weak, but nonetheless critical for biological activity. The crystal structure of Ang has shown that enzymatic potency is attenuated in part by the obstructive positioning of Gln117 within the B(1) pyrimidine binding pocket, and that the C-terminal segment of residues 117-123 must reorient for Ang to bind and cleave RNA. The native closed conformation appears to be stabilized by Gln117-Thr44 and Asp116-Ser118 hydrogen bonds, as well as hydrophobic packing of Ile119 and Phe120. Consistent with this view, Q117G, D116H, and I119A/F120A variants are 4-30-fold more active than Ang. Here we have determined crystal structures for these variants to examine the structural basis for the activity increases. In all three cases, the C-terminal segment remains obstructive, demonstrating that none of the residues that has been replaced is essential for maintaining the closed conformation. The Q117G structure shows no changes other than the loss of the side chain of residue 117, whereas those of D116H and I119A/F120A reveal C-terminal perturbations beyond the replacement site, suggesting that the native closed conformation has been destabilized. Thus, the interactions of Gln117 seem to be less important than those of residues 116, 119, and 120 for stabilization. In D116H, His116 does not replicate either of the hydrogen bonds of Asp116 with Ser118 and instead forms a water-mediated interaction with catalytic residue His114; residues 117-121 deviate significantly from their positions in Ang. In I119A/F120A, the segment of residues 117-123 has become highly mobile and all of the interactions thought to position Gln117 have been weakened or lost; the space occupied by Phe120 in Ang is partially filled by Arg101, which has moved several angstroms. A crystal structure was also determined for the deletion mutant des(121-123), which has 10-fold reduced activity toward large substrates. The structure is consistent with the earlier proposal that residues 121-123 form part of a peripheral substrate binding subsite, but also raises the possibility that changes in the position of another residue, Lys82, might be responsible for the decreased activity of this variant.     

The epitopes of apolipoprotein A-I define distinct structural domains including a mobile middle region.

The definition of epitopes on apoA-I provides evidence for a very dense packing of the peptide chain and supports the proposed supersecondary structure, made of repetitive antiparallel helices. Screening with overlapping synthetic hexapeptides shows that only a few epitopes are continuous and that all continuous contact sequences identified within the epitopes coincide or are contiguous to a putative beta-turn, such as residues 1-8, 48-55, 98-104, 118-123, and 135-140. On the N-terminal half of apoA-I we identified 6 overlapping tertiary discontinuous epitopes, uniquely constituted by amino acids and discontinuous sequences on helical segments that are far apart and which define a particular region with a complex tertiary structure. Among these are the epitopes for antibody 5G6, which reacts with residues 45-51, 83-92, 119-126, and 136-143; for A16, which reacts with 14-19, 23-28, and 60-82; and for r-FC1, which reacts with 1-8, 29-35, 78-83, and 98-121. The very far apart discontinuous sequences included in these epitopes can be explained by the predicted turns and coiled domains, and thus provide evidence for such a tertiary structure. Alternatively, these results could also be explained by intermolecular epitopes involving the N-terminal region. In contrast, in the middle of apoA-I, all identified epitopes are shorter and discontinuous within the secondary structure and are constituted by residues forming a beta-turn with all or part of an adjacent alpha-helix. We hypothesize that these multiple epitopes, that are mostly limited to a single helix, reflect the existence of a very mobile domain, possibly with hinged pairs of adjacent alpha-helices. On the C-terminal half of apoA-I, several monoclonal antibodies react with overlapping epitopes located between residues 149 and 186, which probably reflects 2 antiparallel alpha-helices interrupted by a beta-turn.     

Controlled unfolding and refolding of a single sodium-proton antiporter using atomic force microscopy

Single-molecule force-spectroscopy was employed to unfold and refold single sodium-proton antiporters (NhaA) of Escherichia coli from membrane patches. Although transmembrane alpha-helices and extracellular polypeptide loops exhibited sufficient stability to individually establish potential barriers against unfolding, two helices predominantly unfolded pairwise, thereby acting as one structural unit. Many of the potential barriers were detected unfolding NhaA either from the C-terminal or the N-terminal end. It was found that some molecular interactions stabilizing secondary structural elements were directional, while others were not. Additionally, some interactions appeared to occur between the secondary structural elements. After unfolding ten of the 12 helices, the extracted polypeptide was allowed to refold back into the membrane. After five seconds, the refolded polypeptide established all secondary structure elements of the native protein. One helical pair showed a characteristic spring like "snap in" into its folded conformation, while the refolding process of other helices was not detected in particular. Additionally, individual helices required characteristic periods of time to fold. Correlating these results with the primary structure of NhaA allowed us to obtain the first insights into how potential barriers establish and determine the folding kinetics of the secondary structure elements.     

Influence of pH on NMR structure and stability of the human prion protein globular domain

The NMR structure of the globular domain of the human prion protein (hPrP) with residues 121-230 at pH 7.0 shows the same global fold as the previously published structure determined at pH 4.5. It contains three alpha-helices, comprising residues 144-156, 174-194, and 200-228, and a short anti-parallel beta-sheet, comprising residues 128-131 and 161-164. There are slight, strictly localized, conformational changes at neutral pH when compared with acidic solution conditions: helix alpha1 is elongated at the C-terminal end with residues 153-156 forming a 310-helix, and the population of helical structure in the C-terminal two turns of helix alpha 2 is increased. The protonation of His155 and His187 presumably contributes to these structural changes. Thermal unfolding monitored by far UV CD indicates that hPrP-(121-230) is significantly more stable at neutral pH. Measurements of amide proton protection factors map local differences in protein stability within residues 154-157 at the C-terminal end of helix alpha 1 and residues 161-164 of beta-strand 2. These two segments appear to form a separate domain that at acidic pH has a larger tendency to unfold than the overall protein structure. This domain could provide a "starting point" for pH-induced unfolding and thus may be implicated in endosomic PrPC to PrPSc conformational transition resulting in transmissible spongiform encephalopathies.     

Conformation of the ATP binding peptide in actin revealed by proton NMR spectroscopy

The actin peptide 106-124 exists in a completely conserved region of the sequence and binds strongly to both ATP and tripolyphosphate. Binding particularly affects residues 116 and 118 and generally affects the two segments 115-118 and 121-124 [Barden, J. A., & Kemp, B. E. (1987) Biochemistry 26, 1471-1478]. One-dimensional nuclear Overhauser enhancement difference spectroscopy was used to detect molecular interactions between both adjacent and nonadjacent residues. The N-terminal segment 106-112 was found to be largely extended. A sharp bend was detected between Pro-112 and Lys-113. The triphosphate moiety binds to the strongly hydrophilic central segment of the peptide. Evidence was obtained for a reverse turn involving residues 121-124. Amide proton temperature coefficients and coupling constants provide evidence for a type I beta-turn. A model of the ATP binding site is proposed together with its relationship to other parts of the actin structure and to the phalloidin binding site.     

The isolated C-terminal (F2) fragment of the Escherichia coli tryptophan synthase beta 2-subunit folds into a stable, organized nonnative conformation

Proteolysis of the beta 2-subunit of Escherichia coli tryptophan synthase by the endoproteinase Glu C from Staphylococcus aureus V8 yields a peptide, F2, corresponding to the C-terminal 101 residues of the beta-chain. The conformation and stability of isolated F2 in phosphate buffer at pH 7.8 (where native beta 2 is stable) have been investigated. Circular dichroism spectra in the far-UV showed the presence of large amounts of secondary structure (19% alpha-helices, 34% extended beta-structures). Circular dichroism spectra in the near-UV and sedimentation velocity studies indicated an open globular structure with the aromatic side chains in a symmetric (or disordered) environment. NMR spectra and rates of amide proton exchange showed that F2 fluctuates rapidly between several conformations. The thermal denaturation of F2 observed by the loss of far-UV circular dichroism with increasing temperature appeared noncooperative, and indicates a high thermal stability (Tm = 70 degrees C). Differential scanning microcalorimetry confirmed the absence of cooperativity and indicated a very low value for the calorimetric enthalpy of denaturation (delta H = 17 kJ/mol). All these properties were compatible with a molten globule. However, the low sedimentation coefficient of F2 suggested a very hydrated and/or expanded structure, and the secondary structure content of isolated F2 (see above) differed widely from that reported in the literature for F2 within the context of native beta 2 (49% alpha-helices and 13% extended beta-structures). Thus, neither the secondary nor the tertiary structure of isolated F2 resembled those of native F2. In this respect, isolated F2 is not a "molten globule".(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA-induced partial unfolding of prion protein leads to its polymerisation to amyloid

The full-length mouse recombinant prion protein (23-231 amino acid residues) contains all of its structural elements viz. three alpha-helices and a short two-stranded antiparallel beta-sheet in its C-terminal fragment comprising 121-231 amino acid residues. The incubated mixture of this prion protein fragment and nucleic acid results in the formation of amyloid fibres evidenced from electron microscopy, birefringence and fluorescence of the fibre bound Congo Red and Thioflavin T dyes, respectively. The secondary structure of the amyloid formed in nucleic acid solution is similar to the in vivo isolated prion protein 27-30 amyloid but unlike in it, a hydrophobic milieu is absent in the 121-231 amyloid. Thermal denaturation study demonstrates a partial unfolding of the protein fragment in nucleic acid solution. We propose that nucleic acid catalyses unfolding of prion protein helix 1 followed by a nucleation-dependent polymerisation of the protein to amyloid.     

Variation of amino acid properties in protein secondary structures, alpha-helices and beta-strands

A study was made on the physical, chemical, energetic, conformational, geometric, and dynamic property potentials of amino acid residues in protein secondary structures: alpha-helix and beta-strand. Property patterns were obtained by computing the average property values for specified residue units partitioned longitudinally and transversely about the chain. It was found that in alpha-helices with not more than 15 residues, there exist longitudinally opposing portions, one characteristically higher in average property potentials than the other. The helical chain, in general, acquires either an increasing or decreasing average potential in the N-terminal to C-terminal direction. The sequence-wise and surface-wise variations of property potentials in the elements of beta-structure also revealed such general patterns. Possible wrong predictions in statistical methods of one secondary structural class over the other are pointed out.     

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol.

We have examined the proteolysis of bovine pancreatic ribonuclease A (RNase) by thermolysin when dissolved in aqueous buffer, pH 7.0, in the presence of 50% (v/v) trifluoroethanol (TFE). Under these solvent conditions, RNase acquires a conformational state characterized by an enhanced content of secondary structure (helix) and reduced tertiary structure, as given by CD measurements. It was found that the TFE-resistant thermolysin, despite its broad substrate specificity, selectively cleaves the 124-residue chain of RNase in its TFE state (20-42 degrees C, 6-24 h) at peptide bond Asn 34-Leu 35, followed by a slower cleavage at peptide bond Thr 45-Phe 46. In the absence of TFE, native RNase is resistant to proteolysis by thermolysin. Two nicked RNase species, resulting from cleavages at one or two peptide bonds and thus constituted by two (1-34 and 35-124) (RNase Th1) or three (1-34, 35-45 and 46-124) (RNase Th2) fragments linked covalently by the four disulfide bonds of the protein, were isolated to homogeneity by chromatography and characterized. CD measurements provided evidence that RNase Th1 maintains the overall conformational features of the native protein, but shows a reduced thermal stability with respect to that of the intact species (-delta Tm 16 degrees C); RNase Th2 instead is fully unfolded at room temperature. That the structure of RNase Th1 is closely similar to that of the intact protein was confirmed unambiguously by two-dimensional NMR measurements. Structural differences between the two protein species are located only at the level of the chain segment 30-41, i.e., at residues nearby the cleaved Asn 34-Leu 35 peptide bond. RNase Th1 retained about 20% of the catalytic activity of the native enzyme, whereas RNase Th2 was inactive. The 31-39 segment of the polypeptide chain in native RNase forms an exposed and highly flexible loop, whereas the 41-48 region forms a beta-strand secondary structure containing active site residues. Thus, the conformational, stability, and functional properties of nicked RNase Th1 and Th2 are in line with the concept that proteins appear to tolerate extensive structural variations only at their flexible or loose parts exposed to solvent. We discuss the conformational features of RNase in its TFE-state that likely dictate the selective proteolysis phenomenon by thermolysin.     

Crystal structure of Escherichia coli ketopantoate reductase at 1.7 A resolution and insight into the enzyme mechanism.

Ketopantoate reductase (KPR, EC 1.1.1.169) catalyzes the NADPH-dependent reduction of ketopantoate to pantoate on the pantothenate (vitamin B(5)) biosynthetic pathway. The Escherichia coli panE gene encoding KPR was cloned and expressed at high levels as the native and selenomethionine-substituted (SeMet) proteins. Both native and SeMet recombinant proteins were purified by three chromatographic steps, to yield pure proteins. The wild-type enzyme was found to have a K(M)(NADPH) of 20 microM, a K(M)(ketopantoate) of 60 microM, and a k(cat) of 40 s(-1). Regular prismatic KPR crystals were prepared using the hanging drop technique. They belonged to the tetragonal space group P4(2)2(1)2, with cell parameters: a = b = 103.7 A and c = 55.7 A, accommodating one enzyme molecule per asymmetric unit. The structure of KPR was determined by the multiwavelength anomalous dispersion method using the SeMet protein, for which data were collected to 2.3 A resolution. The native data were collected to 1.7 A resolution and used to refine the final structure. The secondary structure comprises 12 alpha-helices, three 3(10)-helices, and 11 beta-strands. The enzyme is monomeric and has two domains separated by a cleft. The N-terminal domain has an alphabeta-fold of the Rossmann type. The C-terminal domain (residues 170-291) is composed of eight alpha-helices. KPR is shown to be a member of the 6-phosphogluconate dehydrogenase C-terminal domain-like superfamily. A model for the ternary enzyme-NADPH-ketopantoate ternary complex provides a rationale for kinetic data reported for specific site-directed mutants.     

Local interactions and the role of the 6-120 disulfide bond in alpha-lactalbumin: implications for formation of the molten globule state

Molten globule states are partially folded states of proteins which are compact and contain a high degree of secondary structure but which lack many of the fixed tertiary interactions associated with the native state. A set of peptides has been prepared in order to probe the role of local interactions in the vicinity of the Cys(6)-Cys(120) disulfide bond in stabilizing the molten globule state of human alpha-lactalbumin. Peptides derived from the N-terminal and C-terminal regions of human alpha-lactalbumin have been analyzed using nuclear magnetic resonance, circular dichroism, fluorescence spectroscopy and sedimentation equilibrium experiments. A peptide corresponding to the first helical region in the native protein, residues 1-13, is only slightly helical in isolation. Extending the peptide to include residues 14-18 results in a modest increase in helicity. A peptide derived from the C-terminal 12 residues, residues 112-123, is predominantly unstructured. Crosslinking the N- and C-terminal peptides by the native disulfide bond results in almost no increase in structure and there is no evidence for any significant cooperative structure formation over the range of pH 2.2-11.7. These results demonstrate that there is very little enhancement of local structure due to the formation of the Cys(6)-Cys(120) disulfide bond. This is in striking contrast to peptides derived from the region of the Cys(28)-Cys(111) disulfide.     

Secondary structure and structure-activity relationships of peptides corresponding to the subunit interface of herpes simplex virus DNA polymerase

The interaction of the catalytic subunit of herpes simplex virus DNA polymerase with the processivity subunit, UL42, is essential for viral replication and is thus a potential target for antiviral drug discovery. We have previously reported that a peptide analogous to the C-terminal 36 residues of the catalytic subunit, which are necessary and sufficient for its interaction with UL42, forms a monomeric structure with partial alpha-helical character. This peptide and one analogous to the C-terminal 18 residues specifically inhibit UL42-dependent long chain DNA synthesis. Using multidimensional (1)H nuclear magnetic resonance spectroscopy, we have found that the 36-residue peptide contains partially ordered N- and C-terminal alpha-helices separated by a less ordered region. A series of "alanine scan" peptides derived from the C-terminal 18 residues of the catalytic subunit were tested for their ability to inhibit long-chain DNA synthesis and by circular dichroism for secondary structure. The results identify structural aspects and specific side chains that appear to be crucial for interacting with UL42. These findings may aid in the rational design of new drugs for the treatment of herpesvirus infections.     

Identification of amino acids involved in protein structural uniqueness: implication for de novo protein design

Structural uniqueness is characteristic of native proteins and is essential to express their biological functions. The major factors that bring about the uniqueness are specific interactions between hydrophobic residues and their unique packing in the protein core. To find the origin of the uniqueness in their amino acid sequences, we analyzed the distribution of the side chain rotational isomers (rotamers) of hydrophobic amino acids in protein tertiary structures and derived deltaS(contact), the conformational-entropy changes of side chains by residue-residue contacts in each secondary structure. The deltaS(contact) values indicate distinct tendencies of the residue pairs to restrict side chain conformation by inter-residue contacts. Of the hydrophobic residues in alpha-helices, aliphatic residues (Leu, Val, Ile) strongly restrict the side chain conformations of each other. In beta-sheets, Met is most strongly restricted by contact with Ile, whereas Leu, Val and Ile are less affected by other residues in contact than those in alpha-helices. In designed and native protein variants, deltaS(contact) was found to correlate with the folding-unfolding cooperativity. Thus, it can be used as a specificity parameter for designing artificial proteins with a unique structure.     

A 3-disulfide mutant of mouse prion protein expression, oxidative folding, reductive unfolding, conformational stability, aggregation and isomerization

The structure of wild-type mouse prion protein mPrP(23-231) consists of two distinctive segments with approximately equal size, a disordered and flexible N-terminal domain encompassing residues 23-124 and a largely structured C-terminal domain containing about 40% of helical structure and stabilized by one disulfide bond (Cys(178)-Cys(213)). We have expressed a mPrP mutant with 4 Ala/Ser-->Cys replacements, two each at the N-(Cys(36), Cys(112)) and C-(Cys(134), Cys(169)) domains. Our specific aims are to study the interaction between N- and C-domains of mPrP during the oxidative folding and to produce stabilized isomers of mPrP for further analysis. Oxidative folding of fully reduced mutant, mPrP(6C), generates one predominant 3-disulfide isomer, designated as N-mPrP(3SS), which comprises the native disulfide (Cys(178)-Cys(213)) and two non-native disulfide bonds (Cys(36)-Cys(134) and Cys(112)-Cys(169)) that covalently connect the N- and C-domains. In comparison to wild-type mPrP(23-231), N-mPrP(3SS) exhibits an indistinguishable CD spectra, a similar conformational stability in the absence of thiol and a reduced ability to aggregate. In the presence of thiol catalyst and denaturant, N-mPrP(3SS) unfolds and generates diverse isomers that are amenable to further isolation, structural and functional analysis.     

Unusual property of prion protein unfolding in neutral salt solution

The unfolding of cellular prion protein and its refolding to the scrapie isoform are related to prion diseases. Studies in the literature have shown that structures of proteins, either acidic or basic, are stabilized against denaturation by certain neutral salts, for example, sulfate and fluoride. Contrary to these observations, the full-length recombinant prion protein (amino acid residues 23-231) is denatured by these protein structure stabilizing salts. Under identical concentrations of salts, the structure of the sheep prion protein, which contains a greater number of glycine groups in the N-terminal unstructured segment than the mouse protein, becomes more destabilized. In contrast to the full-length protein, the C-terminal 121-231 prion protein fragment, consisting of all the structural elements of the protein, viz., three alpha-helices and two short beta-strands, is stabilized against denaturation by these salts. We suggest that an increase in the concentration of the anions on the surface of the prion protein molecule due to their preferential interaction with the glycine residues in the N-terminal segment destabilizes the structure of the prion protein by perturbing the prion helix 1 which is the most soluble of all the protein alpha-helices reported so far in the literature. The present results could be relevant to explain the observed structural conversion of the prion protein by anionic nucleic acids and sulfated glycosaminoglycans.     

Detection and characterization of an early folding intermediate of T4 lysozyme using pulsed hydrogen exchange and two-dimensional NMR.

Two-dimensional 1H-15N NMR techniques combined with pulsed hydrogen-deuterium exchange have been used to characterize the folding pathway of T4 lysozyme. In the unfolded state, there is little differential protection of the various amides from hydrogen exchange. In the native folded structure, 84 amides of the 164 residues are sufficiently spectrally resolved and protected from solvent exchange to serve as probes of the folding pathway. These probes are located in both the N-terminal and C-terminal domains of the native folded structure of the protein. The studies described here show that at least one intermediate is formed early during refolding at low denaturant concentrations. This intermediate (or intermediates) forms very rapidly (within the 10-ms temporal resolution of our mixing device) under the conditions used and is completed at least 10 times faster than the overall folding event. The intermediate(s) protect(s) from exchange a subset of amides in the N-terminal and C-terminal regions of the protein. In the final folded states these protected regions correspond to two alpha-helices and a beta-sheet region. These amides are protected from exchange by factors between 20 and 200 as compared to the fully unfolded protein. Protection of this magnitude is consistent with the formation of somewhat exposed secondary structure in these regions and could represent a "molten globule"-like or a "framework"-like structure for the intermediate(s) in which specific parts of the sequence form isolated secondary structures that are not stabilized by extensive tertiary interactions.     

Binding of glucose to the D-galactose/D-glucose-binding protein from Escherichia coli restores the native protein secondary structure and thermostability that are lost upon calcium depletion

The effect of the depletion of calcium on the structure and thermal stability of the D-galactose/D-glucose-binding protein (GGBP) from Escherichia coli was studied by fluorescence spectroscopy and Fourier-transform infrared spectroscopy. The calcium-depleted protein (GGBP-Ca) was also studied in the presence of glucose (GGBP-Ca/Glc). The results show that calcium depletion has a small effect on the secondary structure of GGBP, and, in particular it affects a population of alpha-helices with a low exposure to solvent. Alternatively, glucose-binding to GGBP-Ca eliminates the effect induced by calcium depletion by restoring a secondary structure similar to that of the native protein. In addition, the infrared and fluorescence data obtained reveal that calcium depletion markedly reduces the thermal stability of GGBP. In particular, the spectroscopic experiments show that the depletion of calcium mainly affects the stability of the C-terminal domain of the protein. However, the binding of glucose restores the thermal stability of GGBP-Ca. The thermostability of GGBP and GGBP-Ca was also studied by molecular dynamics simulations. The simulation data support the spectroscopic results. New insights into the role of calcium in the thermal stability of GGBP contribute to a better understanding of the protein function and constitute important information for the development of biotechnological applications of this protein. Mutations and/or labelling of amino acid residues located in the protein C-terminal domain may affect the stability of the whole protein structure.     

Relationship between sequence determinants of stability for two natural homologous proteins with different folds

In the Cro protein family, an evolutionary change in secondary structure has converted an alpha-helical fold to a mixture of alpha-helix and beta-sheet. P22 Cro and lambda Cro represent the ancestral all-alpha and descendant alpha+beta folds, respectively. The major structural differences between these proteins are at the C-terminal end of the domain (residues 34-56), where two alpha-helices in P22 Cro align with two beta-strands in lambda Cro. We sought to assess the possibility that smooth evolutionary transitions could have converted the all-alpha structure to the alpha+beta structure through sequences that could adopt both folds. First, we used scanning mutagenesis to identify and compare patterns of key stabilizing residues in the C-terminal regions of both P22 Cro and lambda Cro. These patterns exhibited little similarity to each other, with structurally important residues in the two proteins most often occurring at different sequence positions. Second, "hybrid scanning" studies, involving replacement of each wild-type residue in P22 Cro with the aligned wild-type residue in lambda Cro and vice versa, revealed five or six residues in each protein that strongly destabilized the other. These results suggest that key stability determinants for each Cro fold are quite different and that the P22 Cro sequence strongly favors the all-alpha structure while the lambda Cro sequence strongly favors the alpha+beta structure. Nonetheless, we were able to design a "structurally ambivalent" sequence fragment (SASF1), which corresponded to residues 39-56 and simultaneously incorporated most key stabilizing residues for both P22 Cro and lambda Cro. NMR experiments showed SASF1 to stably fold as a beta-hairpin when incorporated into the lambda Cro sequence but as a pair of alpha-helices when incorporated into P22 Cro.     

Evidence for partial secondary structure formation in the transition state for arc repressor refolding and dimerization

Structure formation and dimerization are concerted processes in the refolding of Arc repressor. The integrity of secondary structure in the transition state of Arc refolding has been investigated here by determining the changes in equilibrium stability and refolding/unfolding kinetics for a set of Ala --> Gly mutations at residues that are solvent-exposed in the native Arc dimer. At some sites, reduced stability was caused primarily by faster unfolding, indicating that secondary structure at these positions is largely absent in the transition state. However, most of the Ala --> Gly substitutions in the alpha-helices of Arc and a triple mutant in the beta-sheet also resulted in decreased refolding rates, in some cases, accounting for the major fraction of thermodynamic destabilization. Overall, these results suggest that some regions of native secondary structure are present but incompletely formed in the transition state of Arc refolding and dimerization. Consolidation of this secondary structure, like close packing of the hydrophobic core, seems to occur later in the folding process. On average, Phi(F) values for the Ala --> Gly mutations were significantly larger than Phi(F) values previously determined for alanine-substitution mutants, suggesting that backbone interactions in the transition state may be stronger than side chain interactions. Mutations causing significant reductions in the Arc refolding rate were found to cluster in the central turn of alpha-helix A and in the first two turns of alpha-helix B. In the Arc dimer, these elements pack together in a compact structure, which might serve as nucleus for further folding.