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.     

Nonrandom structure in the urea-unfolded Escherichia coli outer membrane protein X (OmpX)

On the basis of sequence-specific resonance assignments for the complete polypeptide backbone and most of the amino acid side chains by heteronuclear nuclear magnetic resonance (NMR) spectroscopy, the urea-unfolded form of the outer membrane protein X (OmpX) from Escherichia coli has been structurally characterized. (1)H-(1)H nuclear Overhauser effects (NOEs), dispersion of the chemical shifts, amide proton chemical shift temperature coefficients, amide proton exchange rates, and (15)N[(1)H]-NOEs show that OmpX in 8 M urea at pH 6.5 is globally unfolded, but adopts local nonrandom conformations in the polypeptide segments of residues 73-82 and 137-145. For these two regions, numerous medium-range and longer-range NOEs were observed, which were used as the input for structure calculations of these polypeptide segments with the program DYANA. The segment 73-82 forms a quite regular helical structure, with only loosely constrained amino acid side chains. In the segment 137-145, the tryptophan residue 140 forms the core of a small hydrophobic cluster. Both nonrandom structures are present with an abundance of about 25% of the protein molecules. The sequence-specific NMR assignment and the physicochemical characterization of urea-denatured OmpX presented in this paper are currently used as a platform for investigations of the folding mechanism of this integral membrane protein.     

Native and non-native secondary structure and dynamics in the pH 4 intermediate of apomyoglobin

The partly folded state of apomyoglobin at pH 4 represents an excellent model for an obligatory kinetic folding intermediate. The structure and dynamics of this intermediate state have been extensively examined using NMR spectroscopy. Secondary chemical shifts, (1)H-(1)H NOEs, and amide proton temperature coefficients have been used to probe residual structure in the intermediate state, and NMR relaxation parameters T(1) and T(2) and ?(1)H?-(15)N NOE have been analyzed using spectral densities to correlate motion of the polypeptide chain with these structural observations. A significant amount of helical structure remains in the pH 4 state, indicated by the secondary chemical shifts of the (13)C(alpha), (13)CO, (1)H(alpha), and (13)C(beta) nuclei, and the boundaries of this helical structure are confirmed by the locations of (1)H-(1)H NOEs. Hydrogen bonding in the structured regions is predominantly native-like according to the amide proton chemical shifts and their temperature dependence. The locations of the A, G, and H helix segments and the C-terminal part of the B helix are similar to those in native apomyoglobin, consistent with the early, complete protection of the amides of residues in these helices in quench-flow experiments. These results confirm the similarity of the equilibrium form of apoMb at pH 4 and the kinetic intermediate observed at short times in the quench-flow experiment. Flexibility in this structured core is severely curtailed compared with the remainder of the protein, as indicated by the analysis of the NMR relaxation parameters. Regions with relatively high values of J(0) and low values of J(750) correspond well with the A, B, G, and H helices, an indication that nanosecond time scale backbone fluctuations in these regions of the sequence are restricted. Other parts of the protein show much greater flexibility and much reduced secondary chemical shifts. Nevertheless, several regions show evidence of the beginnings of helical structure, including stretches encompassing the C helix-CD loop, the boundary of the D and E helices, and the C-terminal half of the E helix. These regions are clearly not well-structured in the pH 4 state, unlike the A, B, G, and H helices, which form a native-like structured core. However, the proximity of this structured core most likely influences the region between the B and F helices, inducing at least transient helical structure.     

Structural and kinetic characterization of early folding events in beta-lactoglobulin

We have defined the structural and dynamic properties of an early folding intermediate of beta-lactoglobulin known to contain non-native alpha-helical structure. The folding of beta-lactoglobulin was monitored over the 100 micros--10 s time range using ultrarapid mixing techniques in conjunction with fluorescence detection and hydrogen exchange labeling probed by heteronuclear NMR. An initial increase in Trp fluorescence with a time constant of 140 micros is attributed to formation of a partially helical compact state. Within 2 ms of refolding, well protected amide protons indicative of stable hydrogen bonded structure were found only in a domain comprising beta-strands F, G and H, and the main alpha-helix, which was thus identified as the folding core of beta-lactoglobulin. At the same time, weak protection (up to approximately 10-fold) of amide protons in a segment spanning residues 12--21 is consistent with formation of marginally stable non-native alpha-helices near the N-terminus. Our results indicate that efficient folding, despite some local non-native structural preferences, is insured by the rapid formation of a native-like alpha/beta core domain.     

Structural and dynamic characterization of the acid-unfolded state of hUBF HMG box 1 provides clues for the early events in protein folding

To understand the events that occur in the early stages of the folding of hUBF HMG box 1, we characterized its pH 2.1 unfolded state in detail with NMR. Through a triple resonance strategy, the assignments of complete backbone and some side chains were achieved. Then, significant conformational information was extracted from secondary chemical shifts, interresidual (1)H-(1)H NOEs, (3)J(HNHA) coupling constants, amide proton temperature coefficients, and (15)N relaxation data. The secondary chemical shifts for (13)CA, (13)CB, (13)CO, (1)HA, and (1)HN indicate that the residues between 64 and 78 exhibit a substantial preference for helical structure in the acid-unfolded state, which is also evidenced by the relatively more negative deviations of (3)J(HNHA) and amide proton temperature coefficients from their corresponding random-coil values and particularly confirmed by the strongest sequential d(NN)(i, i + 1) proton NOEs along the region. Following this region until residue 82 is a segment that tends to form a turn-like structure, which is unstable and exchanges between alternative states. In addition, some evidences imply that the regions 18-28 and 38-43 also possess propensities for helical structure but to a different less degree than the region 64-78. The polypeptide backbone dynamics investigated using reduced spectral density function shows apparent motional restrictions in residual structural regions and to less extent at some hydrophobic residues. On the basis of the results presented herein, we propose a potential protein-folding pathway on which these residual structures play a role of initiation site in the early folding stages.     

Apparent local stability of the secondary structure of Azotobacter vinelandii holoflavodoxin II as probed by hydrogen exchange: implications for redox potential regulation and flavodoxin folding.

As a first step to determine the folding pathway of a protein with an alpha/beta doubly wound topology, the 1H, 13C, and 15N backbone chemical shifts of Azotobacter vinelandii holoflavodoxin II (179 residues) have been determined using multidimensional NMR spectroscopy. Its secondary structure is shown to contain a five-stranded parallel beta-sheet (beta2-beta1-beta3-beta4-beta5) and five alpha-helices. Exchange rates for the individual amide protons of holoflavodoxin were determined using the hydrogen exchange method. The amide protons of 65 residues distributed throughout the structure of holoflavodoxin exchange slowly at pH* 6.2 [kex < 10(-5) s(-1)] and can be used as probes in future folding studies. Measured exchange rates relate to apparent local free energies for transient opening. We propose that the amide protons in the core of holoflavodoxin only exchange by global unfolding of the apo state of the protein. The results obtained are discussed with respect to their implications for flavodoxin folding and for modulation of the flavin redox potential by the apoprotein. We do not find any evidence that A. vinelandii holoflavodoxin II is divided into two subdomains based on its amide proton exchange rates, as opposed to what is found for the structurally but not sequentially homologous alpha/beta doubly wound protein Che Y.     

Role of disulfide bridges in the folding, structure and biological activity of omega-conotoxin GVIA.

Omega-Conotoxin GVIA (GVIA), an N-type calcium channel blocker from the cone shell Conus geographus, is a 27 residue polypeptide cross-linked by three disulfide bonds. Here, we report the synthesis, structural analysis by (1)H NMR and bioassay of analogues of GVIA with disulfide bridge deletions and N- and C-terminal truncations. Two analogues that retain the crucial Lys-2 and Tyr-13 residues in loops constrained by two native disulfide bridges were synthesised using orthogonal protection of cysteine residues. In the first analogue, the Cys-15-Cys-26 disulfide bridge was deleted (by replacing the appropriate Cys residues with Ser), while in the second, this disulfide bridge and the eight C-terminal residues were deleted. No activity was detected for either analogue in a rat vas deferens assay, which measures N-type calcium channel activity in sympathetic nerve, and NMR studies showed that this was due to a gross loss of secondary and tertiary structure. Five inactive analogues that were synthesised without orthogonal protection of Cys residues as part of a previous study (Flinn et al. (1995) J. Pept. Sci. 1, 379-384) were also investigated. Three had single disulfide deletions (via Ser substitutions) and two had N- or C-terminal deletions in addition to the disulfide deletion. Peptide mapping and NMR analyses demonstrated that at least four of these analogues had non-native disulfide pairings, which presumably accounts for their lack of activity. The NMR studies also showed that all five analogues had substantially altered tertiary structures, although the backbone chemical shifts and nuclear Overhauser enhancements (NOEs) implied that native-like turn structures persisted in some of these analogues despite the non-native disulfide pairings. This work demonstrates the importance of the disulfides in omega-conotoxin folding and shows that the Cys-15-Cys-26 disulfide is essential for activity in GVIA. The NMR analyses also emphasise that backbone chemical shifts and short- and medium-range NOEs are dictated largely by local secondary structure elements and are not necessarily reliable monitors of the tertiary fold.     

Temperature dependence of NMR chemical shifts: Tracking and statistical analysis

Isotropic chemical shifts measured by solution nuclear magnetic resonance (NMR) spectroscopy offer extensive insights into protein structure and dynamics. Temperature dependences add a valuable dimension; notably, the temperature dependences of amide proton chemical shifts are valuable probes of hydrogen bonding, temperature‐dependent loss of structure, and exchange between distinct protein conformations. Accordingly, their uses include structural analysis of both folded and disordered proteins, and determination of the effects of mutations, binding, or solution conditions on protein energetics. Fundamentally, these temperature dependences result from changes in the local magnetic environments of nuclei, but correlations with global thermodynamic parameters measured via calorimetric methods have been observed. Although the temperature dependences of amide proton and nitrogen chemical shifts are often well approximated by a linear model, deviations from linearity are also observed and may be interpreted as evidence of fast exchange between distinct conformational states. Here, we describe computational methods, accessible via the Shift‐T web server, including an automated tracking algorithm that propagates initial (single temperature) ¹H? ¹⁵N cross peak assignments to spectra collected over a range of temperatures. Amide proton and nitrogen temperature coefficients (slopes determined by fitting chemical shift vs. temperature data to a linear model) are subsequently calculated. Also included are methods for the detection of systematic, statistically significant deviation from linearity (curvature) in the temperature dependences of amide proton chemical shifts. The use and utility of these methods are illustrated by example, and the Shift‐T web server is freely available at http://meieringlab.uwaterloo.ca/shiftt .     

NMR elucidation of early folding hierarchy in HIV-1 protease

Folding studies on proteases by the conventional hydrogen exchange experiments are severely hampered because of interference from the autolytic reaction in the interpretation of the exchange data. We report here NMR identification of the hierarchy of early conformational transitions (folding propensities) in HIV-1 protease by systematic monitoring of the changes in the state of the protein as it is subjected to different degrees of denaturation by guanidine hydrochloride. Secondary chemical shifts, HN-Halpha coupling constants, 1H-15N nuclear Overhauser effects, and 15N transverse relaxation parameters have been used to report on the residual structural propensities, motional restrictions, conformational transitions, etc., and the data suggest that even under the strongest denaturing conditions (6 m guanidine) hydrophobic clusters as well as different native and non-native secondary structural elements are transiently formed. These constitute the folding nuclei, which include residues spanning the active site, the hinge region, and the dimerization domain. Interestingly, the proline residues influence the structural propensities, and the small amino acids, Gly and Ala, enhance the flexibility of the protein. On reducing the denaturing conditions, partially folded forms appear. The residues showing high folding propensities are contiguous along the sequence at many locations or are in close proximity on the native protein structure, suggesting a certain degree of local cooperativity in the conformational transitions. The dimerization domain, the flaps, and their hinges seem to exhibit the highest folding propensities. The data suggest that even the early folding events may involve many states near the surface of the folding funnel.     

Relationship between nuclear magnetic resonance chemical shift and protein secondary structure

An analysis of the 1H nuclear magnetic resonance chemical shift assignments and secondary structure designations for over 70 proteins has revealed some very strong and unexpected relationships. Similar studies, performed on smaller databases, for 13C and 15N chemical shifts reveal equally strong correlations to protein secondary structure. Among the more interesting results to emerge from this work is the finding that all 20 naturally occurring amino acids experience a mean alpha-1H upfield shift of 0.39 parts per million (from the random coil value) when placed in a helical configuration. In a like manner, the alpha-1H chemical shift is found to move downfield by an average of 0.37 parts per million when the residue is placed in a beta-strand or extended configuration. Similar changes are also found for amide 1H, carbonyl 13C, alpha-13C and amide 15N chemical shifts. Other relationships between chemical shift and protein conformation are also uncovered; in particular, a correlation between helix dipole effects and amide proton chemical shifts as well as a relationship between alpha-proton chemical shifts and main-chain flexibility. Additionally, useful relationships between alpha-proton chemical shifts and backbone dihedral angles as well as correlations between amide proton chemical shifts and hydrogen bond effects are demonstrated.     

An efficient high-throughput resonance assignment procedure for structural genomics and protein folding research by NMR.

Sequence specific resonance assignment is the primary requirement for all investigations of proteins by NMR methods. In the present postgenomic era where structural genomics and protein folding have occupied the center stage of NMR research, there is a high demand on the speed of resonance assignment, whereas the presently available methods based either on NOESY or on some triple-resonance experiments are rather slow. They also have limited success with unfolded proteins because of the lack of NOEs, and poor dispersion of amide and carbon chemical shifts. This paper describes an efficient approach to rapid resonance assignment that is suitable for both folded and unfolded proteins, making use of the triple-resonance experiments described recently [HNN and HN(C)N]. It has three underlying principles. First, the experiments exploit the (15)N chemical shift dispersions which are generally very good for both folded and unfolded proteins, along two of the three dimensions; second, they directly display sequential amide and (15)N correlations along the polypeptide chain, and third, the sign patterns of the diagonal and the sequential peaks originating from any residue are dependent on the nature of the adjacent residues, especially the glycines and the prolines. These lead to so-called "triplet fixed points" which serve as starting points and/or check points during the course of sequential walks, and explicit side chains assignment becomes less crucial for unambiguous backbone assignment. These features significantly enhance the speed of data analysis, reduce the amount of experimentation required, and thus result in a substantially faster and unambiguous assignment. Following the amide and (15)N assignments, the other proton and carbon assignments can be obtained in a straightforward manner, from the well-established three-dimensional triple-resonance experiments. We have successfully tested the new approach with different proteins in the molecular mass range of 10-22 kDa, and for illustration, we present here the backbone results on the HIV-1 protease-tethered dimer (molecular mass approximately 22 kDa), both in the folded and in the unfolded forms, the two ends of the folding funnel. We believe that the new assignment approach will be of great value for both structural genomics and protein folding research by NMR.     

Differences between the electronic environments of reduced and oxidized Escherichia coli DsbA inferred from heteronuclear magnetic resonance spectroscopy.

DsbA is the strongest protein disulfide oxidant yet known and is involved in catalyzing protein folding in the bacterial periplasm. Its strong oxidizing power has been attributed to the lowered pKa of its reactive active site cysteine and to the difference in thermodynamic stability between the oxidized and the reduced form. However, no structural data are available for the reduced state. Therefore, an NMR study of DsbA in its two redox states was undertaken. We report here the backbone 1HN, 15N, 13C(alpha) 13CO, 1H(alpha), and 13Cbeta NMR assignments for both oxidized and reduced Escherichia coli DsbA (189 residues). Ninety-nine percent of the frequencies were assigned using a combination of triple (1H-13C-15N) and double resonance (1H-15N or 1H-13C) experiments. Secondary structures were established using the CSI (Chemical Shift Index) method, NOE connectivity patterns, 3(J)H(N)H(alpha) and amide proton exchange data. Comparison of chemical shifts for both forms reveals four regions of the protein, which undergo some changes in the electronic environment. These regions are around the active site (residues 26 to 43), around His60 and Pro 151, and also around Gln97. Both the number and the amplitude of observed chemical shift variations are more substantial in DsbA than in E. coli thioredoxin. Large 13C(alpha) chemical shift variations for residues of the active site and residues Phe28, Tyr34, Phe36, Ile42, Ser43, and Lys98 suggest that the backbone conformation of these residues is affected upon reduction.     

Detection and structure determination of an equilibrium unfolding intermediate of Rd-apocytochrome b562: native fold with non-native hydrophobic interactions

The absence of detectable kinetic and equilibrium folding intermediates by optical probes is commonly taken to indicate that protein folding is a two-state process. However, for some small proteins with apparent two-state behavior, unfolding intermediates have been identified in native-state hydrogen exchange or kinetic unfolding experiments monitored by nuclear magnetic resonance. Rd-apocytochrome b(562), a four-helix bundle, is one such protein. Here, we found another unfolding intermediate for Rd-apocytochrome b(562). It is based on a cooperative transition of (15)N chemical shifts of amide protons as a function of urea concentrations before the global unfolding. We have solved the high-resolution structure of the protein at 2.8 M urea, which is after this cooperative transition but before the global unfolding. All four helices remained intact, but a number of hydrophobic core residues repacked. This intermediate provides a possible structural interpretation for the kinetic unfolding intermediates observed using nuclear magnetic resonance methods for several proteins and has important implications for theoretical studies of protein folding.     

NMR analysis of a kinetically trapped intermediate of a disulfide-deficient mutant of the starch-binding domain of glucoamylase

A thermally unfolded disulfide-deficient mutant of the starch-binding domain of glucoamylase refolds into a kinetically trapped metastable intermediate when subjected to a rapid lowering of temperature. We attempted to characterise this intermediate using multidimensional NMR spectroscopy. The ¹H-¹⁵N heteronuclear single quantum coherence spectrum after a rapid temperature decrease (the spectrum of the intermediate) showed good chemical shift dispersion but was significantly different from that of the native state, suggesting that the intermediate adopts a nonnative but well-structured conformation. Large chemical shift changes for the backbone amide protons between the native and the intermediate states were observed for residues in the β-sheet consisting of strands 2, 3, 5, 6, and 7 as well as in the C-terminal region. These residues were found to be in close proximity to aromatic residues, suggesting that the chemical shift changes are mainly due to ring current shifts caused by the aromatic residues. The two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy experiments showed that the intermediate contained substantial, native-like NOE connectivities, although there were fewer cross peaks in the spectrum of the intermediate compared with that of the native state. It was also shown that there were native-like interresidue NOEs for residues buried in the protein, whereas many of the NOE cross peaks were lost for the residues involved in a surface-exposed aromatic cluster. These results suggest that, in the intermediate, the aromatic cluster at the surface is structurally less organised, whereas the interior of the protein has relatively rigid, native-like side-chain packing.     

Exploring the Minimally Frustrated Energy Landscape of Unfolded ACBP

The unfolded state of globular proteins is not well described by a simple statistical coil due to residual structural features, such as secondary structure or transiently formed long-range contacts. The principle of minimal frustration predicts that the unfolded ensemble is biased toward productive regions in the conformational space determined by the native structure. Transient long-range contacts, both native-like and non-native-like, have previously been shown to be present in the unfolded state of the four-helix-bundle protein acyl co-enzyme binding protein (ACBP) as seen from both perturbations in nuclear magnetic resonance (NMR) chemical shifts and structural ensembles generated from NMR paramagnetic relaxation data. To study the nature of the contacts in detail, we used paramagnetic NMR relaxation enhancements, in combination with single-point mutations, to obtain distance constraints for the acid-unfolded ensemble of ACBP. We show that, even in the acid-unfolded state, long-range contacts are specific in nature and single-point mutations affect the free-energy landscape of the unfolded protein. Using this approach, we were able to map out concerted, interconnected, and productive long-range contacts. The correlation between the native-state stability and compactness of the denatured state provides further evidence for native-like contact formation in the denatured state. Overall, these results imply that, even in the earliest stages of folding, ACBP dynamics are governed by native-like contacts on a minimally frustrated energy landscape.     

NMR characterization of residual structure in the denatured state of protein L

Triple-resonance NMR experiments were used to assign the (13)C(alpha), (13)C(beta), (15)N and NH resonances for all the residues in the denatured state of a destabilized protein L variant in 2 M guanidine. The chemical shifts of most resonances were very close to their random coil values. Significant deviations were observed for G22, L38 and K39; increasing the denaturant concentration shifted the chemical shifts of these residues towards theory random coil values. Medium-range nuclear Overhauser enhancements were detected in segments corresponding to the turn between the first two strands, the end of the second strand through the turn between the second strand and the helix, and the turn between the helix and the third strand in 3D H(1), N(15)-HSQC-NOESY-HSQC experiments on perdeuterated samples. Longer-range interactions were probed by measuring the paramagnetic relaxation enhancement produced by nitroxide spin labels introduced via cysteine residues at five sites around the molecule. Damped oscillations in the magnitude of the paramagnetic relaxation enhancement as a function of distance along the sequence suggested native-like chain reversals in the same three turn regions. The more extensive interactions within the region corresponding to the first beta-turn than in the region corresponding to the second beta-turn suggests that the asymmetry in the folding reaction evident in previous studies of the protein L folding transition state is already established in the denatured state.     

NMR structural characterization and computational predictions of the major intermediate in oxidative folding of leech carboxypeptidase inhibitor

The III-A intermediate constitutes the major rate-determining step in the oxidative folding of leech carboxypeptidase inhibitor (LCI). In this work, III-A has been directly purified from the folding reaction and structurally characterized by NMR spectroscopy. This species, containing three native disulfides, displays a highly native-like structure; however, it lacks some secondary structure elements, making it more flexible than native LCI. III-A represents a structurally determined example of a disulfide-insecure intermediate; direct oxidation of this species to the fully native protein seems to be restricted by the burial of its two free cysteine residues inside a native-like structure. We also show that theoretical approaches based on topological constraints predict with good accuracy the presence of this folding intermediate. Overall, the derived results suggest that, as it occurs with non-disulfide bonded proteins, native-like interactions between segments of secondary structure rather than the crosslinking of disulfide bonds direct the folding of LCI.     

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.     

Conformational plasticity of the influenza A M2 transmembrane helix in lipid bilayers under varying pH, drug binding, and membrane thickness

Membrane proteins change their conformations to respond to environmental cues, thus conformational plasticity is important for function. The influenza A M2 protein forms an acid-activated proton channel important for the virus lifecycle. Here we have used solid-state NMR spectroscopy to examine the conformational plasticity of membrane-bound transmembrane domain of M2 (M2TM). (13)C and (15)N chemical shifts indicate coupled conformational changes of several pore-facing residues due to changes in bilayer thickness, drug binding, and pH. The structural changes are attributed to the formation of a well-defined helical kink at G34 in the drug-bound state and in thick lipid bilayers, nonideal backbone conformation of the secondary-gate residue V27 in the presence of drug, and nonideal conformation of the proton-sensing residue H37 at high pH. The chemical shifts constrained the (?, ψ) torsion angles for three "basis" states, the equilibrium among which explains the multiple resonances per site in the NMR spectra under different combinations of bilayer thickness, drug binding, and pH conditions. Thus, conformational plasticity is important for the proton conduction and inhibition of M2TM. The study illustrates the utility of NMR chemical shifts for probing the structural plasticity and folding of membrane proteins.     

Assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin

As a necessary first step in the use of heteronuclear correlated spectra to obtain high resolution solution structures of the protein, assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin (Mr 12,000) uniformly labeled with 15N has been performed. The 15N chemical shifts of backbone amide nitrogen atoms have been determined for both oxidation states of thioredoxin using 15N-1H correlated and two-dimensional heteronuclear single-quantum coherence (HSQC) TOCSY and NOESY spectra. The backbone assignments are complete, except for the proline imide nitrogen resonances and include Gly33, whose amide proton resonance is difficult to observe in homonuclear 1H spectra. The differences in the 15N chemical shift between oxidized and reduced thioredoxin, which occur mainly in the vicinity of the two active site cysteines, including residues distant in the amino acid sequence which form a hydrophobic surface close to the active site, are consistent with the differences observed for proton chemical shifts in earlier work on thioredoxin.