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.     

Structural dynamics of the membrane translocation domain of colicin E9 and its interaction with TolB

In order for the 61 kDa colicin E9 protein toxin to enter the cytoplasm of susceptible cells and kill them by hydrolysing their DNA, the colicin must interact with the outer membrane BtuB receptor and Tol translocation pathway of target cells. The translocation function is located in the N-terminal domain of the colicin molecule. (1)H, (1)H-(1)H-(15)N and (1)H-(13)C-(15)N NMR studies of intact colicin E9, its DNase domain, minimal receptor-binding domain and two N-terminal constructs containing the translocation domain showed that the region of the translocation domain that governs the interaction of colicin E9 with TolB is largely unstructured and highly flexible. Of the expected 80 backbone NH resonances of the first 83 residues of intact colicin E9, 61 were identified, with 43 of them being assigned specifically. The absence of secondary structure for these was shown through chemical shift analyses and the lack of long-range NOEs in (1)H-(1)H-(15)N NOESY spectra (tau(m)=200 ms). The enhanced flexibility of the region of the translocation domain containing the TolB box compared to the overall tumbling rate of the protein was identified from the relatively large values of backbone and tryptophan indole (15)N spin-spin relaxation times, and from the negative (1)H-(15)N NOEs of the backbone NH resonances. Variable flexibility of the N-terminal region was revealed by the (15)N T(1)/T(2) ratios, which showed that the C-terminal end of the TolB box and the region immediately following it was motionally constrained compared to other parts of the N terminus. This, together with the observation of inter-residue NOEs involving Ile54, indicated that there was some structural ordering, resulting most probably from the interactions of side-chains. Conformational heterogeneity of parts of the translocation domain was evident from a multiplicity of signals for some of the residues. Im9 binding to colicin E9 had no effect on the chemical shifts or other NMR characteristics of the region of colicin E9 containing the TolB recognition sequence, though the interaction of TolB with intact colicin E9 bound to Im9 did affect resonances from this region. The flexibility of the translocation domain of colicin E9 may be connected with its need to recognise protein partners that assist it in crossing the outer membrane and in the translocation event itself.     

Characterisation of the conformational properties of urea-unfolded Im7: implications for the early stages of protein folding

The colicin immunity protein Im7 folds from its unfolded state in 6 M urea to its native four-helix structure through an on-pathway intermediate that lacks one of the helices of the native structure (helix III). In order to further characterize the folding mechanism of Im7, we have studied the conformational properties of the protein unfolded in 6 M urea in detail using heteronuclear NMR. Triple-resonance experiments with 13C/15N-labelled Im7 in 6 M urea provided almost complete resonance assignments for the backbone nuclei, and measurement of backbone 15N relaxation parameters allowed dynamic ordering of the unfolded polypeptide chain to be investigated. Reduced spectral density mapping and fitting backbone R2 relaxation rates to a polymer dynamics model identified four clusters of interacting residues, each predicted by the average area buried upon folding for each residue. Chemical shift analyses and measurement of NOEs detected with a long mixing-time 1H-1H-15N NOESY-HSQC spectrum confirmed the formation of four clusters. Each cluster of interacting side-chains in urea-unfolded Im7 occurs in a region of the protein that forms a helix in the protein, with the largest clusters being associated with the three long helices that are formed in the on-pathway folding intermediate, whilst the smallest cluster forms a helix only in the native state. NMR studies of a Phe15Ala Im7 variant and a protein in which residues 51-56 are replaced by three glycine residues (H3G3 Im7*), indicated that the clusters do not interact with each other, possibly because they are solvated by urea, as indicated by analysis of NOEs between the protein and the solvent. Based on these data, we suggest that dilution of the chaotrope to initiate refolding will result in collapse of the clusters, leading to the formation of persistent helical structure and the generation of the three-helix folding intermediate.     

Molecular mechanism underlying the thermal stability and pH-induced unfolding of CHABII

The 37-residue alpha/beta protein CHABII was previously demonstrated to undergo a gradual pH-induced unfolding. It has been shown that even at pH 4.0 CHABII still retained a highly native-like secondary structure and tertiary topology although its tight side-chain packing was severely disrupted, typical of the molten globule state. Here, we have expressed and refolded the recombinant proteins of CHABII and its mutant [Phe21]-CHABII, and subsequently conducted extensive CD and NMR characterizations. The results indicated: (1) replacement of His21 by Phe in [Phe21]-CHABII eliminated the pH-induced unfolding from pH 6.5 to 4.0, indicating that His21 was responsible for the observed pH-induced unfolding of CHABII. Further examinations revealed that although the pH-induced unfolding of CHABII was also triggered by the protonation of the His residue as previously uncovered for apomyoglobin, their molecular mechanisms are different. (2) Monitoring the pH-induced unfolding by 1H-15N HSQC spectroscopy allowed us to visualize the gradual development of the CHABII molten globule. At pH 4.0, the HSQC spectrum of CHABII was poorly dispersed with dispersions of approximately 1 ppm over proton dimension and 10 ppm over 15N dimension, characteristic of severely or even "completely unfolded" proteins. One the other hand, unambiguous assignments of the NOESY spectra of CHABII led to the identification of the persistent medium and long-range NOEs at pH 4.0, which define a highly native-like secondary structure and tertiary packing. This implies that the degree of the native-like topology might be underestimated in the previous characterization of partially folded and even completely unfolded proteins. (3) Replacement of His21 by Phe with higher side-chain hydrophobicity only caused a minor structural rearrangement but considerably enhanced the packing interaction of the hydrophobic core, as evident from a dramatic increase in NOE contacts in [Phe21]-CHABII. The enhancement led to an increase of the thermal stability of [Phe21]-CHABII by approximately 17 deg. C.     

Local structural preferences and dynamics restrictions in the urea-denatured state of SUMO-1: NMR characterization

We have investigated by multidimensional NMR the structural and dynamic characteristics of the urea-denatured state of activated SUMO-1, a 97-residue protein belonging to the growing family of ubiquitin-like proteins involved in post-translational modifications. Complete backbone amide and 15N resonance assignments were obtained in the denatured state by using HNN and HN(C)N experiments. These enabled other proton assignments from TOCSY-HSQC spectra. Secondary Halpha chemical shifts and 1H-1H NOE indicate that the protein chain in the denatured state has structural preferences in the broad beta-domain for many residues. Several of these are seen to populate the (phi,psi) space belonging to polyproline II structure. Although there is no evidence for any persistent structures, many contiguous stretches of three or more residues exhibit structural propensities suggesting possibilities of short-range transient structure formation. The hetero-nuclear 1H-15N NOEs are extremely weak for most residues, except for a few at the C-terminal, and the 15N relaxation rates show sequence-wise variation. Some of the regions of slow motions coincide with those of structural preferences and these are interspersed by highly flexible residues. The implications of these observations for the early folding events starting from the urea-denatured state of activated SUMO-1 have been discussed.     

Unusually slow denaturation and refolding processes of pyrrolidone carboxyl peptidase from a hyperthermophile are highly cooperative: real-time NMR studies

The refolding rate of heat-denatured cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from Pyrococcus furiosus has been reported to be unusually slow under some conditions. To elucidate the structural basis of the unusually slow kinetics of the protein, the denaturation and refolding processes of the PCP-0SH were investigated using a real-time 2D (1)H-(15)N HSQC and CD experiments. At 2 M urea denaturation of the PCP-0SH in the acidic region, all of the native peaks in the 2D HSQC spectrum completely disappeared. The conformation of the PCP-0SH just after removal of 6 M GuHCl could be observed as a stable intermediate (D(1) state) in 2D HSQC and CD experiments, which is similar to a molten globule structure. The D(1) state of the PCP-0SH, which is the initial state of refolding, corresponded to the state at 2 M urea and seemed to be the denatured state in equilibrium with the native state under the physiological conditions. The refolding of PCP-0SH from the D(1) state to the native state could be observed to be highly cooperative without any intermediates between them, even if the refolding rate was quite slow. In the higher concentration of denaturants, PCP-0SH showed HSQC and CD spectra characteristic of completely unfolded proteins called the D(2) state. The unusually slow refolding rate was discussed as originating in the conformations in the transition state and/or the retardation of reorganization in an ensemble of nonrandom denatured structures in the D(1) state.     

Equilibrium and pressure-jump relaxation studies of the conformational transitions of P13MTCP1

The conformational transitions of a small oncogene product, p13(MTCP1), have been studied by high-pressure fluorescence of the intrinsic tryptophan emission and high-pressure 1D and 2D 1H-15N NMR. While the unfolding transition monitored by fluorescence is cooperative, two kinds of NMR spectral changes were observed, depending on the pressure range. Below approximately 200 MPa, pressure caused continuous, non-linear shifts of many of the 15N and 1H signals, suggesting the presence of an alternate folded conformer(s) in rapid equilibrium (tau相似文献   

Compact molten globule-like state of hUBF HMG Box1 at extremely low pH

Using far and near-UV CD, ANS fluorescence and 2D NMR spectroscopy, an acid-induced partly folded state (A state) at extremely low pH for hUBF HMG Box1 was identified and characterized. As compared to the native state (N), the A state has similar secondary structure, less compact pack with larger amounts of exposed hydrophobic surface, and narrower chemical shift dispersion in (1)H-(15)N HSQC spectrum, which implies that it is a molten globule (MG)-like species. On the other hand, substantial tertiary contacts and cooperative thermal denaturing transition indicate that the A state is closer-relative to the classic MG-to the native folded state. In addition, when the solution pH is adjusted to neutrality, the protein in the A state refolds to the native state easily. All these data suggest that the A state of hUBF HMG Box1 could represent a potential folding intermediate on protein folding pathway.     

Time-dependent changes in the denatured state(s) influence the folding mechanism of an all beta-sheet protein

Newt fibroblast growth factor (nFGF-1) is an approximately 15-kDa all beta-sheet protein devoid of disulfide bonds. Urea-induced equilibrium unfolding of nFGF-1, monitored by steady state fluorescence and far-UV circular dichroism spectroscopy, is cooperative with no detectable intermediate(s). Urea-induced unfolding of nFGF-1 is reversible, but the percentage of the protein recovered in the native state depends on the time of incubation of the protein in the denaturant. The yield of the protein in the native state decreases with the increase in time of incubation in the denaturant. The failure of the protein to refold to its native state is not due to trivial chemical reactions that could possibly occur upon prolonged incubation in the denaturant. (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra, limited proteolytic digestion, and fluorescence data suggest that the misfolded state(s) of nFGF-1 has structural features resembling that of the denatured state(s). GroEL, in the presence of ATP, is observed to rescue the protein from being trapped in the misfolded state(s). (1)H-(15)N HSQC data of nFGF-1, acquired in the denatured state(s) (in 8 m urea), suggest that the protein undergoes subtle time-dependent structural changes in the denaturant. To our knowledge, this report for the first time demonstrates that the commitment to adapt unproductive pathways leading to protein misfolding/aggregation occurs in the denatured state ensemble.     

Characterization of the structure and dynamics of a near-native equilibrium intermediate in the unfolding pathway of an all beta-barrel protein

The structure and dynamics of equilibrium intermediate in the unfolding pathway of the human acidic fibroblast growth factor (hFGF-1) are investigated using a variety of biophysical techniques including multidimensional NMR spectroscopy. Guanidinium hydrochloride (GdnHCl)-induced unfolding of hFGF-1 proceeds with the accumulation of a stable intermediate state. The transition from the intermediate state to the unfolded state(s) is cooperative without the accumulation of additional intermediate(s). The intermediate state induced maximally in 0.96 m GdnHCl is found to be obligatory in the folding/unfolding pathway of hFGF-1. Most of the native tertiary structure interactions are preserved in the intermediate state. (1)H-(15)N chemical shift perturbation data suggest that the residues in the C-terminal segment including those located in the beta-strands IX, X, and XI undergo the most discernible structural change(s) in the intermediate state in 0.96 m GdnHCl. hFGF-1 in the intermediate state (0.96 m GdnHCl) does not bind to its ligand, sucrose octasulfate. Limited proteolytic digestion experiments and hydrogen-deuterium exchange monitored by (15)N heteronuclear single quantum coherence (HSQC) spectra show that the conformational flexibility of the protein in the intermediate state is significantly higher than in the native conformation. (15)N spin relaxation experiments show that many residues located in beta-strands IX, X, and XI exhibit conformational motions in the micro- to millisecond time scale. Analysis of (15)N relaxation data in conjunction with the amide proton exchange kinetics suggests that residues in the beta-strands II, VIII, and XII possibly constitute the stability core of the protein in the near-native intermediate state.     

Polypeptide backbone resonance assignments and secondary structure of Bacillus subtilis enzyme IIIglc determined by two-dimensional and three-dimensional heteronuclear NMR spectroscopy

The enzyme IIIglc-like domain of Bacillus subtilis IIglc (IIIglc, 162 residues, 17.4 kDa) has been cloned and overexpressed in Escherichia coli. Sequence-specific assignment of the backbone 1H and 15N resonances has been carried out with a combination of homonuclear and heteronuclear two-dimensional and heteronuclear three-dimensional (3D) NMR spectroscopy. Amide proton solvent exchange rate constants have been determined from a series of 1H-15N heteronuclear single-quantum coherence (HSQC) spectra acquired following dissolution of the protein in D2O. Major structural features of IIIglc have been inferred from the pattern of short-, medium- and long-range NOEs in 3D heteronuclear 1H nuclear Overhauser effect 1H-15N multiple-quantum coherence (3D NOESY-HMQC) spectra, together with the exchange rate constants. IIIglc contains three antiparallel beta-sheets comprised of eight, three, and two beta-strands. In addition, five beta-bulges were identified. No evidence of regular helical structure was found. The N-terminal 15 residues of the protein appear disordered, which is consistent with their being part of the Q-linker that connects the C-terminal enzyme IIIglc-like domain to the membrane-bound IIglc domain. Significantly, two histidine residues, His 68 and His 83, which are important for phosphotransferase function, are found from NOE measurements to be in close proximity at the ends of adjacent strands in the major beta-sheet.     

Insights into signal transduction revealed by the low resolution structure of the FixJ response regulator

P19(INK4d) is a tumor suppressing protein and belongs to a family of cyclin D-dependent kinase inhibitors of CDK4 and CDK6, which play a key role in human cell cycle control. P19 comprises ten alpha-helices arranged sequentially in five ankyrin repeats forming an elongated structure. This rather simple topology, combined with its physiological function, makes p19 an interesting model protein for folding studies. Urea-induced unfolding transitions monitored by far-UV CD and phenylalanine fluorescence coincide and suggest a two-state mechanism for equilibrium unfolding. Unfolding of p19 followed by 2D (1)H-(15)N HSQC spectra revealed a third species at moderate urea concentrations with a maximum population of about 30 % near 3.2 M urea. It shows poor chemical shift dispersion, but cross-peaks emerge for some residues that are distinct from the native or unfolded state. This equilibrium intermediate either arises only at high protein concentrations (as in the NMR experiment) or has similar optical properties to the unfolded state. Stopped-flow far-UV CD experiments at various urea concentrations revealed that alpha-helical structure is formed in three phases, of which only the fastest phase (10 s(-1)) depends upon the urea concentration. The kinetic of the slowest phase (0.017 s(-1)) can be resolved by 1D real-time NMR and accelerated by cyclophilin. It is limited in rate by prolyl isomerization, and native-like ordered structure cannot form prior to this isomerization. The two fast phases lead to 83 % native protein within the dead time of the NMR experiment. In contrast to p16(INK4a), which exhibits only a marginal stability and high unfolding rates, p19 shows the expected stability for a protein of this size with a clear kinetic barrier between the unfolded and folded state. Therefore, p19 might complement the function of less stable INK4 inhibitors in cell cycle control under unfavorable conditions.     

Backbone dynamics of the Bacillus subtilis glucose permease IIA domain determined from 15N NMR relaxation measurements.

The backbone dynamics of the uniformly 15N-labeled IIA domain of the glucose permease of Bacillus subtilis have been characterized using inverse-detected two-dimensional 1H-15N NMR spectroscopy. Longitudinal (T1) and transverse (T2) 15N relaxation time constants and steady-state (1H)-15N NOEs were measured, at a spectrometer proton frequency of 500 MHz, for 137 (91%) of the 151 protonated backbone nitrogens. These data were analyzed by using a model-free dynamics formalism to determine the generalized order parameter (S2), the effective correlation time for internal motions (tau e), and 15N exchange broadening contributions (Rex) for each residue, as well as the overall molecular rotational correlation time (tau m). The T1 and T2 values for most residues were in the ranges 0.45-0.55 and 0.11-0.15 s, respectively; however, a small number of residues exhibited significantly slower relaxation. Similarly, (1H)-15N NOE values for most residues were in the range 0.72-0.80, but a few residues had much smaller positive NOEs and some exhibited negative NOEs. The molecular rotational correlation time was 6.24 +/- 0.01 ns; most residues had order parameters in the range 0.75-0.90 and tau e values of less than ca. 25 ps. Residues found to be more mobile than the average were concentrated in three areas: the N-terminal residues (1-13), which were observed to be highly disordered; the loop from P25 to D41, the apex of which is situated adjacent to the active site and may have a role in binding to other proteins; and the region from A146 to S149. All mobile residues occurred in regions close to termini, in loops, or in irregular secondary structure.     

Sequence-specific 1H NMR assignments and secondary structure in solution of Escherichia coli trp repressor

Sequence-specific 1H NMR assignments are reported for the active L-tryptophan-bound form of Escherichia coli trp repressor. The repressor is a symmetric dimer of 107 residues per monomer; thus at 25 kDa, this is the largest protein for which such detailed sequence-specific assignments have been made. At this molecular mass the broad line widths of the NMR resonances preclude the use of assignment methods based on 1H-1H scalar coupling. Our assignment strategy centers on two-dimensional nuclear Overhauser spectroscopy (NOESY) of a series of selectively deuterated repressor analogues. A new methodology was developed for analysis of the spectra on the basis of the effects of selective deuteration on cross-peak intensities in the NOESY spectra. A total of 90% of the backbone amide protons have been assigned, and 70% of the alpha and side-chain proton resonances are assigned. The local secondary structure was calculated from sequential and medium-range backbone NOEs with the double-iterated Kalman filter method [Altman, R. B., & Jardetzky, O. (1989) Methods Enzymol. 177, 218-246]. The secondary structure agrees with that of the crystal structure [Schevitz, R., Otwinowski, Z., Joachimiak, A., Lawson, C. L., & Sigler, P. B. (1985) Nature 317, 782], except that the solution state is somewhat more disordered in the DNA binding region and in the N-terminal region of the first alpha-helix. Since the repressor is a symmetric dimer, long-range intersubunit NOEs were distinguished from intrasubunit interactions by formation of heterodimers between two appropriate selectively deuterated proteins and comparison of the resulting NOESY spectrum with that of each selectively deuterated homodimer. Thus, from spectra of three heterodimers, long-range NOEs between eight pairs of residues were identified as intersubunit NOEs, and two additional long-range intrasubunits NOEs were assigned.     

Monitoring the early steps of unfolding of dicalcium and mono-Ce3+-substituted forms of P43M calbindin D9k

Early steps of unfolding of P43M Calbindin D(9k) have been evaluated by NMR spectroscopy on the native dicalcium and on the paramagnetic monocerium-substituted derivative. Although at 2 M GdmHCl the protein core maintains its overall folding and structure, amide (15)N R(2) measurements and cross correlation rates between N-H dipole-dipole relaxation and (15)N CSA relaxation reveal a closer and stronger packing of the hydrophobic interactions in the protein as a response to the presence of denaturing agents in solution. A complete reorientation of the Met43 side chain toward the hydrophobic core is accomplished by the disappearance of the millisecond dynamics observed on the native form of Calbindin D(9k), while cross correlation rates provide evidence that the two-way hydrogen bond between Leu23 and Val61 is broken or substantially weakened. The substitution of the calcium ion in site II with the paramagnetic Ce(3+) ion allowed us to obtain a number of long-range nonconventional constraints, namely, pseudocontact shifts, which were used, together with the NOEs collected on the native state, to monitor subtle structural variations occurring in the non-native state of the protein. Although the average rmsd between the structures of native and non-native states is small (0.48 A), structural rearrangements could be reliably identified. Our results provide unprecedented information about the behavior of Calbindin D(9k) during the early steps of unfolding. Furthermore, they constitute strong evidence of the efficiency of paramagnetism-based constraints in monitoring subtle structural changes that are beyond the sensitivity of an approach based only on NOE.     

Heteronuclear three-dimensional NMR spectroscopy of a partially denatured protein: The A-state of human ubiquitin

Summary Human ubiquitin is a 76-residue protein that serves as a protein degradation signal when conjugated to another protein. Ubiquitin has been shown to exist in at least three states: native (N-state), unfolded (U-state), and, when dissolved in 60% methanol:40% water at pH 2.0, partially folded (A-state). If the A-state represents an intermediate in the folding pathway of ubiquitin, comparison of the known structure of the N-state with that of the A-state may lead to an understanding of the folding pathway. Insights into the structural basis for ubiquitin's role in protein degradation may also be obtained. To this end we determined the secondary structure of the A-state using heteronuclear three-dimensional NMR spectroscopy of uniformly ¹⁵N-enriched ubiquitin. Sequence-specific ¹H and ¹⁵N resonance assignments were made for more than 90% of the residues in the A-state. The assignments were made by concerted analysis of three-dimensional ¹H-¹⁵N NOESY-HMQC and TOCSY-HMQC data sets. Because of ¹H chemical shift degeneracies, the increased resolution provided by the ¹⁵N dimension was critical. Analysis of short- and long-range NOEs indicated that only the first two strands of -sheet, comprising residues 2–17, remain in the A-state, compared to five strands in the N-state. NOEs indicative of an -helix, comprising residues 25–33, were also identified. These residues were also helical in the N-state. In the N-state, residues in this helix were in contact with residues from the first two strands of -sheet. It is likely, therefore, that residues 1–33 comprise a folded domain in the A-state of ubiquitin. On the basis of ¹H chemical shifts and weak short-range NOEs, residues 34–76 do not adopt a rigid secondary structure but favor a helical conformation. This observation may be related to the helix-inducing effects of the methanol present. The secondary structure presented here differs from and is more thorough than that determined previously by two-dimensional ¹H methods [Harding et al. (1991) Biochemistry, 30, 3120–3128].     

The secondary structure of the colicin E3 immunity protein as studied by 1H-1H and 1H-15N two-dimensional NMR spectroscopy.

By performing 1H-1H and 1H-15N two-dimensional (2D) nuclear magnetic resonance (NMR) experiments, the complete sequence-specific resonance assignment was determined for the colicin E3 immunity protein (84 residues; ImmE3), which binds to colicin E3 and inhibits its RNase activity. First, the fingerprint region of the spectrum was analyzed by homonuclear 1H-1H HOHAHA and NOESY methods. For the identification of overlapping resonances, heteronuclear 1H-15N (HMQC-HOHAHA, HMQC-NOESY) experiments were performed, so that the complete 1H and 15N resonance assignments were provided. Then the secondary structure of ImmE3 was determined by examination of characteristic patterns of sequential backbone proton NOEs in combination with measurement of exchange rates of amide protons and 3JHN alpha coupling constants. From these results, it was concluded that ImmE3 contains a four-stranded antiparallel beta-sheet (residues 2-10, 19-22, 47-49, and 71-79) and a short alpha-helix (residues 31-36).     

A designed apoplastocyanin variant that shows reversible folding

Plastocyanin, like many other metalloproteins, does not undergo reversible folding, which is thought to be due to an irreversible conformational change in the copper-binding site. Moreover, apoplastocyanin's ability to adopt a native tertiary structure is highly salt-dependent, and even in high salt, it has an irreversible thermal denaturation. Here, we report a designed apoplastocyanin variant, PCV, that is well folded and has reversible folding in both high and low salt conditions. This variant provides a tractable model for understanding and designing protein beta-sheets.     

BetaCore,a designed water soluble four-stranded antiparallel beta-sheet protein

BetaCore is a designed approximately 50-residue protein in which two BPTI-derived core modules, CM I and CM II, are connected by a 22-atom cross-link. At low temperature and pH 3, homo- and heteronuclear NMR data report a dominant folded ('f') conformation with well-dispersed chemical shifts, i, i+1 periodicity, numerous long-range NOEs, and slowed amide hydrogen isotope exchange patterns that is a four-stranded antiparallel beta-sheet with nonsymmetrical and specific association of CM I and CM II. BetaCore 'f' conformations undergo reversible, global, moderately cooperative, non-two-state thermal transitions to an equilibrium ensemble of unfolded 'u' conformations. There is a significant energy barrier between 'f' and 'u' conformations. This is the first designed four-stranded antiparallel beta-sheet that folds in water.     

A gradual disruption of tight side-chain packing: 2D 1H-NMR characterization of acid-induced unfolding of CHABII

Little is known about the mechanism of the transition between native proteins and partially folded intermediates. Complete assignments of 2D 1H-NOESY spectra of CHABII at 5 degrees C, pH 6.3, 5.5, 4.6 and 4.0, reveal that lowering of pH results in an extensive but gradual disappearance of NOEs, implying a gradual disruption of tight side-chain packing. Moreover, a tertiary packing core is identified at 5 degrees C and pH 4.0, characterized by persistent long-range NOEs. Thus, we suggest that severe disruption of tight side-chain packing of CHABII can occur at a stage where its secondary structure and tertiary topology remain highly native-like.