Fast Assignment of <Superscript>15</Superscript>N-HSQC Peaks using High-Resolution 3D HNcocaNH Experiments with Non-Uniform Sampling

We describe an efficient NMR triple resonance approach for fast assignment of backbone amide resonance peaks in the ¹⁵N-HSQC spectrum. The exceptionally high resolutions achieved in the 3D HncocaNH and hNcocaNH experiments together with non-uniform sampling facilitate error-free sequential connection of backbone amides. Data required for the complete backbone amide assignment of the 56-residue protein GB1 domain were obtained in 14 h. Data analysis was vastly streamlined using a ‘backbone NH walk’ method to determine sequential connectivities without the need for ¹³C chemical shifts comparison. Amino acid residues in the sequentially connected NH chains are classified into two groups by a simple variation of the NMR pulse sequence, and the resulting ‘ZeBra’ stripe patterns are useful for mapping these chains to the protein sequence. In addition to resolving ambiguous assignments derived from conventional backbone experiments, this approach can be employed to rapidly assign small proteins or flexible regions in larger proteins, and to transfer assignments to mutant proteins or proteins in different ligand-binding states.     

An automated system designed for large scale NMR data deposition and annotation: application to over 600 assigned chemical shift data entries to the BioMagResBank from the Riken Structural Genomics/Proteomics Initiative internal database

Biomolecular NMR chemical shift data are key information for the functional analysis of biomolecules and the development of new techniques for NMR studies utilizing chemical shift statistical information. Structural genomics projects are major contributors to the accumulation of protein chemical shift information. The management of the large quantities of NMR data generated by each project in a local database and the transfer of the data to the public databases are still formidable tasks because of the complicated nature of NMR data. Here we report an automated and efficient system developed for the deposition and annotation of a large number of data sets including (1)H, (13)C and (15)N resonance assignments used for the structure determination of proteins. We have demonstrated the feasibility of our system by applying it to over 600 entries from the internal database generated by the RIKEN Structural Genomics/Proteomics Initiative (RSGI) to the public database, BioMagResBank (BMRB). We have assessed the quality of the deposited chemical shifts by comparing them with those predicted from the PDB coordinate entry for the corresponding protein. The same comparison for other matched BMRB/PDB entries deposited from 2001-2011 has been carried out and the results suggest that the RSGI entries greatly improved the quality of the BMRB database. Since the entries include chemical shifts acquired under strikingly similar experimental conditions, these NMR data can be expected to be a promising resource to improve current technologies as well as to develop new NMR methods for protein studies.     

Expression of doubly labeled Saccharomyces cerevisiae iso-1 ferricytochrome c and (1)H, (13)C and (15)N chemical shift assignments by multidimensional NMR

We have expressed [U-(13)C,(15)N]-labeled Saccharomyces cerevisiae iso-1 cytochrome c C102T;K72A in Escherichia coli with a yield of 11 mg/l of growth medium. Nuclear magnetic resonance (NMR) studies were conducted on the Fe(3+) form of the protein. We report herein chemical shift assignments for amide (1)H and (15)N, (13)C(omicron), (13)C(alpha), (13)C(beta), (1)H(alpha) and (1)H(beta) resonances based upon a series of three-dimensional NMR experiments: HNCA, HN(CO)CA, HNCO, HN(CA)CO, HNCACB, HCA(CO)N, HCCH-TOCSY and HBHA(CBCA)NH. An investigation of the chemical shifts of the threonine residues was also made by using density functional theory in order to help solve discrepancies between (15)N chemical shift assignments reported in this study and those reported previously.     

RefDB: a database of uniformly referenced protein chemical shifts

RefDB is a secondary database of reference-corrected protein chemical shifts derived from the BioMagResBank (BMRB). The database was assembled by using a recently developed program (SHIFTX) to predict protein ¹H, ¹³C and ¹⁵N chemical shifts from X-ray or NMR coordinate data of previously assigned proteins. The predicted shifts were then compared with the corresponding observed shifts and a variety of statistical evaluations performed. In this way, potential mis-assignments, typographical errors and chemical referencing errors could be identified and, in many cases, corrected. This approach allows for an unbiased, instrument-independent solution to the problem of retrospectively re-referencing published protein chemical shifts. Results from this study indicate that nearly 25% of BMRB entries with ¹³C protein assignments and 27% of BMRB entries with ¹⁵N protein assignments required significant chemical shift reference readjustments. Additionally, nearly 40% of protein entries deposited in the BioMagResBank appear to have at least one assignment error. From this study it evident that protein NMR spectroscopists are increasingly adhering to recommended IUPAC ¹³C and ¹⁵N chemical shift referencing conventions, however, approximately 20% of newly deposited protein entries in the BMRB are still being incorrectly referenced. This is cause for some concern. However, the utilization of RefDB and its companion programs may help mitigate this ongoing problem. RefDB is updated weekly and the database, along with its associated software, is freely available at http://redpoll.pharmacy.ualberta.ca and the BMRB website.     

Parallel acquisition of multi-dimensional spectra in protein NMR

We introduce the use of multiple receivers applied in parallel for simultaneously recording multi-dimensional data sets of proteins in a single experiment. The utility of the approach is established through the introduction of the 2D (15)N,(1)H(N)||(13)CO HSQC experiment in which a pair of two-dimensional (15)N,(1)H(N) and (15)N,(13)CO spectra are recorded. The methodology is further extended to higher dimensionality via the 3D (1)H(N)||(13)CO HNCA in which a pair of data sets recording (13)C(α),(15)N,(1)H(N) and (13)C(α),(15)N,(13)CO chemical shifts are acquired. With the anticipated increases in probe sensitivity it is expected that multiple receiver experiments will become an important approach for efficient recording of NMR data.     

Biophysical characterization of elongin C from Saccharomyces cerevisiae

Elongin C (ELC) is an essential component of the mammalian CBC(VHL) E3 ubiquitin ligase complex. As a step toward understanding the role of ELC in assembly and function of CBC-type ubiquitin ligases, we analyzed the quaternary structure and backbone dynamics of the highly homologous Elc1 protein from Saccharomyces cerevisiae. Analytical ultracentrifugation experiments in conjunction with size exclusion chromatography showed that Elc1 is a nonglobular monomer over a wide range of concentrations. Pronounced line broadening in (1)H,(15)N-HSQC NMR spectra and failure to assign peaks corresponding to the carboxy-terminal helix 4 of Elc1 indicated that helix 4 is conformationally labile. Measurement of (15)N NMR relaxation parameters including T(1), T(2), and the (1)H-(15)N nuclear Overhauser effect revealed (i) surprisingly high flexibility of residues 69-77 in loop 5, and (ii) chemical exchange contributions for a large number of residues throughout the protein. Addition of 2,2,2-trifluoroethanol (TFE) stabilized helix 4 and reduced chemical exchange contributions, suggesting that stabilization of helix 4 suppresses the tendency of Elc1 to undergo conformational exchange on a micro- to millisecond time scale. Binding of a peptide representing the major ELC binding site of the von Hippel-Lindau (VHL) tumor suppressor protein almost completely eliminated chemical exchange processes, but induced substantial conformational changes in Elc1 leading to pronounced rotational anisotropy. These results suggest that elongin C interacts with various target proteins including the VHL protein by an induced fit mechanism involving the conformationally flexible carboxy-terminal helix 4.     

ORB, a homology-based program for the prediction of protein NMR chemical shifts

A computer program (ORB) has been developed to predict ¹H,¹³C and ¹⁵N NMR chemical shifts of previouslyunassigned proteins. The program makes use of the information contained in achemical shift database of previously assigned proteins supplemented by astatistically derived averaged chemical shift database in which the shifts arecategorized according to their residue, atom and secondary structure type[Wishart et al. (1991) J. Mol. Biol., 222, 311–333]. The predictionprocess starts with a multiple alignment of all previously assigned proteinswith the unassigned query protein. ORB uses the sequence and secondarystructure alignment program XALIGN for this task [Wishart et al. (1994)CABIOS, 10, 121–132; 687–688]. The prediction algorithm in ORB isbased on a scoring of the known shifts for each sequence. The scores dependon global sequence similarity, local sequence similarity, structuralsimilarity and residue similarity and determine how much weight one particularshift is given in the prediction process. In situations where no applicablepreviously assigned chemical shifts are available, the shifts derived from theaveraged database are used. In addition to supplying the user with predictedchemical shifts, ORB calculates a confidence value for every prediction. Theseconfidence values enable the user to judge which predictions are the mostaccurate and they are particularly useful when ORB is incorporated into acomplete autoassignment package. The usefulness of ORB was tested on threemedium-sized proteins: an interleukin-8 analog, a troponin C synthetic peptideheterodimer and cardiac troponin C. Excellent results are obtained if ORB isable to use the chemical shifts of at least one highly homologous sequence.ORB performs well as long as the sequence identity between proteins with knownchemical shifts and the new sequence is not less than 30%.     

Complete (1)H and (13)C NMR chemical shift assignments of mono-, di-, and trisaccharides as basis for NMR chemical shift predictions of polysaccharides using the computer program casper

The computer program casper uses (1)H and (13)C NMR chemical shift data of mono- to trisaccharides for the prediction of chemical shifts of oligo- and polysaccharides. In order to improve the quality of these predictions the (1)H and (13)C, as well as (31)P when applicable, NMR chemical shifts of 30 mono-, di-, and trisaccharides were assigned. The reducing sugars gave two distinct sets of NMR resonances due to the α- and β-anomeric forms. In total 35 (1)H and (13)C NMR chemical shift data sets were obtained from the oligosaccharides. One- and two-dimensional NMR experiments were used for the chemical shift assignments and special techniques were employed in some cases such as 2D (1)H,(13)C-HSQC Hadamard Transform methodology which was acquired approximately 45 times faster than a regular t(1) incremented (1)H,(13)C-HSQC experiment and a 1D (1)H,(1)H-CSSF-TOCSY experiment which was able to distinguish spin-systems in which the target protons were only 3.3Hz apart. The (1)H NMR chemical shifts were subsequently refined using total line-shape analysis with the PERCH NMR software. The acquired NMR data were then utilized in the casper program (http://www.casper.organ.su.se/casper/) for NMR chemical shift predictions of the O-antigen polysaccharides from Klebsiella O5, Shigella flexneri serotype X, and Salmonella arizonae O62. The data were compared to experimental data of the polysaccharides from the two former strains and the lipopolysaccharide of the latter strain showing excellent agreement between predicted and experimental (1)H and (13)C NMR chemical shifts.     

Quantitative use of chemical shifts for the modeling of protein complexes

Despite recent advances in the modeling of protein-protein complexes by docking, additional information is often required to identify the best solutions. For this purpose, NMR data deliver valuable restraints that can be used in the sampling and/or the scoring stage, like in the data-driven docking approach HADDOCK that can make use of NMR chemical shift perturbation (CSP) data to define the binding site on each protein and drive the docking. We show here that a quantitative use of chemical shifts (CS) in the scoring stage can help to resolve ambiguities. A quantitative CS-RMSD score based on (1) H(α) ,(13) C(α) , and (15) N chemical shifts ranks the best solutions always at the top, as demonstrated on a small benchmark of four complexes. It is implemented in a new docking protocol, CS-HADDOCK, which combines CSP data as ambiguous interaction restraints in the sampling stage with the CS-RMSD score in the final scoring stage. This combination of qualitative and quantitative use of chemical shifts increases the reliability of data-driven docking for the structure determination of complexes from limited NMR data.     

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.     

3d Trosy-HncaCodedcb and Trosy-HncaCodedco Experiments: Triple Resonance nmr Experiments With two Sequential Connectivity Pathways and High Sensitivity

The concept of chemical shift-coding monitors chemical shifts in multi-dimensional NMR experiments without additional polarization transfer elements and evolution periods. The chemical shifts are coded in the line-shape of the cross-peak through an apparent scalar coupling dependent upon the chemical shift. This concept is applied to the three-dimensional triple-resonance experiment HNCA adding the information of (13)C(beta) or (13)C' chemical shifts. On average, the proposed TROSY-HNCA(coded)CB experiment is a factor of 2 less sensitive than the HNCA experiment. However, it contains correlations via the chemical shifts of both (13)C(alpha) and (13)C(beta), and provides up to three times higher resolution along the (13)C(alpha) chemical shift axis. Thus, it dramatically reduces ambiguities in linking the spin systems of adjacent residues in the protein sequence during the sequential assignment. The TROSY-HNCA(coded)CO experiment which is conceptually similar contains correlations via the chemical shifts of (13)C(alpha) and (13)C' without major signal losses. The proposed triple resonance experiments are applied to a approximately 70% (2)H, approximately 85% (13)C,(15)N labeled protein with a molecular weight of 25 kDa.     

Structural analysis of nanoscale self-assembled discoidal lipid bilayers by solid-state NMR spectroscopy

Nanodiscs are an example of discoidal nanoscale self-assembled lipid/protein particles similar to nascent high-density lipoproteins, which reduce the risk of coronary artery disease. The major protein component of high-density lipoproteins is human apolipoprotein A-I, and the corresponding protein component of Nanodiscs is membrane scaffold protein 1 (MSP1), a 200-residue lipid-binding domain of human apolipoprotein A-I. Here we present magic-angle spinning (MAS) solid-state NMR studies of uniformly (13)C,(15)N-labeled MSP1 in polyethylene glycol precipitated Nanodiscs. Two-dimensional MAS (13)C-(13)C correlation spectra show excellent microscopic order of MSP1 in precipitated Nanodiscs. Secondary isotropic chemical shifts throughout the protein are consistent with a predominantly helical structure. Moreover, the backbone conformations of prolines derived from their (13)C chemical shifts are consistent with the molecular belt model but not the picket fence model of lipid-bound MSP1. Overall comparison of experimental spectra and (13)C chemical shifts predicted from several structural models also favors the belt model. Our study thus supports the belt model of Nanodisc structure and demonstrates the utility of MAS NMR to study the structure of high molecular weight lipid-protein complexes.     

A 1H NMR determination of the solution conformation of a synthetic peptide analogue of calcium-binding site III of rabbit skeletal troponin C

NMR techniques have been used to determine the structure in solution of acetyl (Asp 105) skeletal troponin C (103-115) amide, one of a series of synthetic peptide analogues of calcium-binding site III of rabbit skeletal troponin C [Marsden et al. (1988) Biochemistry 27, 4198-4206]. The NMR measurements include 1H-1H nuclear Overhauser enhancements and gadolinium-induced 1H relaxation measurements. The former yield short-range internuclear distances (less than 4 A); the latter, once properly corrected for chemical exchange, yield longer range metal to proton distances (5-10 A). These measurements were then used as pseudo potential energy restraints in energy minimization and molecular dynamics calculations to determine the solution structure. Further information was provided by NMR coupling constants, amide proton exchange rates, and the temperature dependences of amide proton chemical shifts. The solution structure of the peptide analogue is very similar to that of the calcium-binding loop in the protein, the root-mean-square deviation between the backbone atoms being approximately 1.1 A.     

Resonance assignment and structural analysis of acid denatured E. coli [U-15N]-glutaredoxin 3

Virtually complete sequence specific ¹H and ¹⁵N resonance assignments are presented for acid denatured reduced E. coli glutaredoxin 3. The sequential resonance assignments of the backbone rely on the combined use of 3D F₁-decoupled ROESY-¹⁵N-HSQC and 3D ¹⁵N-HSQC-(TOCSY-NOESY)-¹⁵N-HSQC using a single uniformly ¹⁵N labelled protein sample. The sidechain resonances were assigned from a 3D TOCSY-¹⁵N-HSQC and a homonouclear TOCSY spectrum. The presented assignment strategy works in the absence of chemical exchange peaks with signals from the native conformation and without ¹³C/¹⁵N double labelling. Chemical shifts, ³J(H, NH) coupling constants and NOEs indicate extensive conformational averaging of both backbone and side chains in agreement with a random coil conformation. The only secondary structure element persisting at pH 3.5 appears to be a short helical segment comprising residues 37 to 40.Abbreviations HSQC heteronuclear single quantum coherence - NMR nuclear magnetic resonance - NOE nuclear Overhauser effect - NOESY two-dimensional NOE spectroscopy - ROE nuclear Overhauser effect in the rotating frame - ROESY two-dimensional ROE spectroscopy - TOCSY total correlation spectroscopy - TPPI time proportional phase incrementation Correspondence to: G. Otting     

The dilated cardiomyopathy G159D mutation in cardiac troponin C weakens the anchoring interaction with troponin I

NMR spectroscopy has been employed to elucidate the molecular consequences of the DCM G159D mutation on the structure and dynamics of troponin C, and its interaction with troponin I (TnI). Since the molecular effects of human mutations are often subtle, all NMR experiments were conducted as direct side-by-side comparisons of the wild-type C-domain of troponin C (cCTnC) and the mutant protein, G159D. With the mutation, the affinity toward the anchoring region of cTnI (cTnI 34-71) was reduced ( K D = 3.0 +/- 0.6 microM) compared to that of the wild type ( K D < 1 microM). Overall, the structure and dynamics of the G159D.cTnI 34-71 complex were very similar to those of the cCTnC.cTnI 34-71 complex. There were, however, significant changes in the (1)H, (13)C, and (15)N NMR chemical shifts, especially for the residues in direct contact with cTnI 34-71, and the changes in NOE connectivity patterns between the G159D.cTnI 34-71 and cCTnC.cTnI 34-71 complexes. Thus, the most parsimonious hypothesis is that the development of disease results from the poor anchoring of cTnI to cCTnC, with the resulting increase in the level of acto-myosin inhibition in agreement with physiological data. Another possibility is that long-range electrostatic interactions affect the binding of the inhibitory and switch regions of cTnI (cTnI 128-147 and cTnI 147-163) and/or the cardiac specific N-terminus of cTnI (cTnI 1-29) to the N-domain of cTnC. These important interactions are all spatially close in the X-ray structure of the cardiac TnC core.     

Anti-HIV and anti-tumor protein MAP30, a 30 kDa single-strand type-I RIP, shares similar secondary structure and beta-sheet topology with the A chain of ricin, a type-II RIP

MAP30 is a 30 kDa single-stranded, type-I ribosome inactivating protein (RIP) possessing anti-tumor and anti-HIV activities. It binds both ribosomal RNA and the HIV-1 long-terminal repeat DNA. To understand the structural basis for MAP30 activities, we undertook the study of MAP30 by solution NMR spectroscopy. We report nearly complete 1H, 13C, and 15N chemical shift assignments of its 263 amino acids. Based upon an analysis of secondary 13C chemical shifts, 3J(HNHA) coupling constants, hydrogen exchange data, and nuclear Overhauser effect patterns, we find that the secondary structure and beta-sheet topology of MAP30 are very similar to those of the ricin A chain, a subunit of the well-known type-II RIP, even though two proteins display distinct activities. We therefore suggest that MAP30 and ricin A chain share a similar three-dimensional fold, and that the reported functional differences between two proteins arise primarily from differences in local three-dimensional structure and other structural properties such as surface electrostatic potentials.     

Backbone structure confirmation and side chain conformation refinement of a bradykinin mimic BKM-824 by comparing calculated (1)H, (13)C and (19)F chemical shifts with experiment

Calculated and experimental (1)H, (13)C and (19)F chemical shifts were compared in BKM-824, a cyclic bradykinin antagonist mimic, c[Ava(1)-Igl(2)-Ser(3)-DF5F(4)-Oic(5)-Arg(6)] (Ava=5-aminovaleric acid, Igl=alpha-(2-indanyl)glycine, DF5F=pentafluorophenylalanine, Oic=(2S,3aS,7aS)-octahydroindole-2-carboxylic acid). The conformation of BKM-824 has been studied earlier by NMR spectroscopy (M. Miskolzie et al., J. Biomolec. Struct. Dyn. 17, 947-955 (2000)). All NMR structures have qualitatively the same backbone structure but there is considerable variation in the side chain conformations. We have carried out quantum mechanical optimization for three representative NMR structures at the B3LYP/6-31G* level, constraining the backbone dihedral angles at their NMR structure values, followed by NMR chemical shift calculations at the optimized structures with the 6-311G** basis set. There is an intramolecular hydrogen bond at Ser(3) in the optimized structures. The experimental (13)C chemical shifts at five C(alpha) positions as well as at the Cbeta, Cgamma and Cdelta position of Ava(1), which forms part of the backbone, are well reproduced by the calculations, confirming the NMR backbone structure. A comparison between the calculated and experimental H(beta) chemical shifts in Igl(2) shows that the dominant conformation at this residue is gauche. Changes of proton chemical shifts with the scan of the chi(1) angle in DF5F(4) suggest that chi(1)180 degrees. The calculated (1)H and (13)C chemical shifts are in good agreement with experiment at the rigid residue Oic(5). None of the models gives accurate results for Arg(6), presumably because of its positive charge. Our study indicates that calculated NMR shifts can be used as additional constraints in conjunction with NMR data to determine protein conformations. However, to be computationally effective, a database of chemical shifts in small peptide fragments should be precalculated.     

Combined solid state and solution NMR studies of α,ɛ-<Superscript>15</Superscript>N labeled bovine rhodopsin

Rhodopsin is the visual pigment of the vertebrate rod photoreceptor cell and is the only member of the G protein coupled receptor family for which a crystal structure is available. Towards the study of dynamics in rhodopsin, we report NMR-spectroscopic investigations of α,ɛ-¹⁵N-tryptophan labeled rhodopsin in detergent micelles and reconstituted in phospholipids. Using a combination of solid state ¹³C,¹⁵N-REDOR and HETCOR experiments of all possible ¹³C′ _i-1 carbonyl/¹⁵N _i -tryptophan isotope labeled amide pairs, and H/D exchange ¹H,¹⁵N-HSQC experiments conducted in solution, we assigned chemical shifts to all five rhodopsin tryptophan backbone ¹⁵N nuclei and partially to their bound protons. ¹H,¹⁵N chemical shift assignment was achieved for indole side chains of Trp35^1.30 and Trp175^4.65. ¹⁵N chemical shifts were found to be similar when comparing those obtained in the native like reconstituted lipid environment and those obtained in detergent micelles for all tryptophans except Trp175^4.65 at the membrane interface. The results suggest that the integrated solution and solid state NMR approach presented provides highly complementary information in the study of structure and dynamics of large membrane proteins like rhodopsin.     

Toward the quantum chemical calculation of nuclear magnetic resonance chemical shifts of proteins

Despite the many protein structures solved successfully by nuclear magnetic resonance (NMR) spectroscopy, quality control of NMR structures is still by far not as well established and standardized as in crystallography. Therefore, there is still the need for new, independent, and unbiased evaluation tools to identify problematic parts and in the best case also to give guidelines that how to fix them. We present here, quantum chemical calculations of NMR chemical shifts for many proteins based on our fragment-based quantum chemical method: the adjustable density matrix assembler (ADMA). These results show that (13)C chemical shifts of reasonable accuracy can be obtained that can already provide a powerful measure for the structure validation. (1)H and even more (15)N chemical shifts deviate more strongly from experiment due to the insufficient treatment of solvent effects and conformational averaging.     

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.