High resolution 1H nuclear magnetic resonance of a transmembrane peptide.

Although the strong 1H-1H dipolar interaction is known to result in severe homogeneous broadening of the 1H nuclear magnetic resonance (NMR) spectra of ordered systems, in the fluid phase of biological and model membranes the rapid, axially symmetric reorientation of the molecules about the local bilayer normal projects the dipolar interaction onto the motional symmetry axis. Because the linewidth then scales as (3 cos2 theta-1)/2, where theta is the angle between the local bilayer normal and the magnetic field, the dipolar broadening has been reduced to an "inhomogeneous" broadening by the rapid axial reorientation. It is then possible to obtain high resolution 1H-NMR spectra of membrane components by using magic angle spinning (MAS). Although the rapid axial reorientation effectively eliminates the homogeneous dipolar broadening, including that due to n = 0 rotational resonances, the linewidths observed in both lipids and peptides are dominated by low frequency motions. For small peptides the most likely slow motions are either a "wobble" or reorientation of the molecular diffusion axis relative to the local bilayer normal, or the reorientation of the local bilayer normal itself through surface undulations or lateral diffusion over the curved surface. These motions render the peptide 1H-NMR lines too broad to be observed at low spinning speeds. However, the linewidths due to these slow motions are very sensitive to spinning rate, so that at higher speeds the lines become readily visible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein Linewidth and Solvent Dynamics in Frozen Solution NMR

Solid-state NMR of proteins in frozen aqueous solution is a potentially powerful technique in structural biology, especially if it is combined with dynamic nuclear polarization signal enhancement strategies. One concern regarding NMR studies of frozen solution protein samples at low temperatures is that they may have poor linewidths, thus preventing high-resolution studies. To learn more about how the solvent shell composition and temperature affects the protein linewidth, we recorded ¹H, ²H, and ¹³C spectra of ubiquitin in frozen water and frozen glycerol-water solutions at different temperatures. We found that the ¹³C protein linewidths generally increase with decreasing temperature. This line broadening was found to be inhomogeneous and independent of proton decoupling. In pure water, we observe an abrupt line broadening with the freezing of the bulk solvent, followed by continuous line broadening at lower temperatures. In frozen glycerol-water, we did not observe an abrupt line broadening and the NMR lines were generally narrower than for pure water at the same temperature. ¹H and ²H measurements characterizing the dynamics of water that is in exchange with the protein showed that the ¹³C line broadening is relatively independent from the arrest of isotropic water motions.     

Dynamics and mechanism of the Tanford transition of bovine beta-lactoglobulin studied using heteronuclear NMR spectroscopy

The Tanford transition is a conformational change of bovine beta-lactoglobulin (betaLG) occurring at around pH 7, identified originally on the basis of optical rotatory dispersion and the accessibility of a thiol group. X-ray analysis has suggested that a conformational change to the EF-loop is responsible for the Tanford transition, with the loop closing the hydrophobic cavity of the beta-barrel of the betaLG molecule below pH 7 and flipping to open the cavity above pH 7. To clarify the dynamics of this conformational change, NMR measurements were made at neutral pH. Since severe signal broadening due to monomer-dimer equilibrium prevented NMR measurements of wild-type betaLG at neutral pH, we searched for optimal sample conditions, finding that a disulfide bond-linked dimer of the mutant A34C gives an HSQC spectrum without signal broadening. The HSQC and CD spectra indicated that in overall conformation A34C is similar to wild-type betaLG, suggesting that the A34C dimer is a good model with which to study the structure and dynamics of the wild-type at neutral pH. The pH-dependent HSQC signal changes and Lipari-Szabo type relaxation analyses of the A34C dimer revealed that the conformational change to the EF-loop occurs above pH 7. We observed two types of motions in the EF-loop region; relatively fast (micro- to milliseconds) and slow (milliseconds or slower) conformational exchanges of the residues located in the hinge and top of the EF-loop regions, respectively. Furthermore, the GH-loop adjacent to the EF-loop exhibited conformational change at a pH slightly lower than that at which the EF-loop motions occurred. From these observations, we propose a three-step mechanism of conformational change in the EF-loop leading to the Tanford transition, in which the GH-loop conformational change, the cleavage of the hydrogen bonds at the hinge, and the flip of the EF-loop occur sequentially.     

Deletion of Phenylalanine 508 in the First Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Increases Conformational Exchange and Inhibits Dimerization

Deletion of Phe-508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) results in destabilization of the domain, intramolecular interactions involving the domain, and the entire channel. The destabilization caused by F508del manifests itself in defective channel processing and channel gating defects. Here, we present NMR studies of the effect of F508del and the I539T stabilizing mutation on NBD1 dynamics, with a view to understanding these changes in stability. Qualitatively, F508del NMR spectra exhibit significantly more peak broadening than WT spectra due to the enhanced intermediate time scale (millisecond to microsecond) motions in the mutant. Unexpectedly, studies of fast (nanosecond to picosecond) motions revealed that F508del NBD1 tumbles more rapidly in solution than WT NBD1. Whereas F508del tumbles at a rate nearly consistent with the monomeric state, the WT protein tumbles significantly more slowly. Paramagnetic relaxation enhancement experiments confirm that NBD1 homodimerizes in solution in the expected head-to-tail orientation. NMR spectra of WT NBD1 reveal significant concentration-dependent chemical shift perturbations consistent with NBD1 dimerization. Chemical shift analysis suggests that the more rapid tumbling of F508del is the result of an impaired ability to dimerize. Based on previously published crystal structures and NMR spectra of various NBD1 mutants, we propose that deletion of Phe-508 affects Q-loop conformational sampling in a manner that inhibits dimerization. These results provide a potential mechanism for inhibition of channel opening by F508del and support the dimer interface as a target for cystic fibrosis therapeutics.     

Hydration dependent dynamics in RNA

The essential role played by local and collective motions in RNA function has led to a growing interest in the characterization of RNA dynamics. Recent investigations have revealed that even relatively simple RNAs experience complex motions over multiple time scales covering the entire ms–ps motional range. In this work, we use deuterium solid-state NMR to systematically investigate motions in HIV-1 TAR RNA as a function of hydration. We probe dynamics at three uridine residues in different structural environments ranging from helical to completely unrestrained. We observe distinct and substantial changes in ²H solid-state relaxation times and lineshapes at each site as hydration levels increase. By comparing solid-state and solution state ¹³C relaxation measurements, we establish that ns–μs motions that may be indicative of collective dynamics suddenly arise in the RNA as hydration reaches a critical point coincident with the onset of bulk hydration. Beyond that point, we observe smaller changes in relaxation rates and lineshapes in these highly hydrated solid samples, compared to the dramatic activation of motion occurring at moderate hydration.     

Toward the solution structure of human insulin: sequential 2D 1H NMR assignment of a des-pentapeptide analogue and comparison with crystal structure

2D 1H NMR studies are presented of des-pentapeptide-insulin, an analogue of human insulin lacking the C-terminal five residues of the B chain. Removal of these residues, which are not required for function, is shown to reduce conformational broadening previously described in the spectrum of intact insulin [Weiss et al. (1989) Biochemistry 28, 9855-9873]. This difference presumably reflects more rapid internal motions in the fragment, which lead to more complete averaging of chemical shifts on the NMR time scale. Sequential 1H NMR assignment and preliminary structural analysis demonstrate retention in solution of the three alpha-helices observed in the crystal state and the relative orientation of the receptor-binding surfaces. These studies provide a foundation for determining the solution structure of insulin.     

The effects of rigid motions on elastic network model force constants

Elastic network models provide an efficient way to quickly calculate protein global dynamics from experimentally determined structures. The model's single parameter, its force constant, determines the physical extent of equilibrium fluctuations. The values of force constants can be calculated by fitting to experimental data, but the results depend on the type of experimental data used. Here, we investigate the differences between calculated values of force constants and data from NMR and X-ray structures. We find that X-ray B factors carry the signature of rigid-body motions, to the extent that B factors can be almost entirely accounted for by rigid motions alone. When fitting to more refined anisotropic temperature factors, the contributions of rigid motions are significantly reduced, indicating that the large contribution of rigid motions to B factors is a result of over-fitting. No correlation is found between force constants fit to NMR data and those fit to X-ray data, possibly due to the inability of NMR data to accurately capture protein dynamics.     

Dynamic pictures of membrane proteins in two-dimensional crystal, lipid bilayer and detergent as revealed by site-directed solid-state 13C NMR

We have compared site-directed 13C solid-state NMR spectra of [3-13C]Ala- and/or [1-13C]Val-labeled membrane proteins, including bacteriorhodopsin (bR), pharaonis phoborhodopin (ppR), its cognate transducer (pHtrII) and Escherichia coli diacylglycerol kinase (DGK), in two-dimensional (2D) crystal, lipid bilayers, and detergent. Restricted fluctuation motions of these membrane proteins due to oligomerization of bR by specific protein-protein interactions in the 2D crystalline lattice or protein complex between ppR and pHtrII provide the most favorable environment to yield well-resolved, fully visible 13C NMR signals for [3-13C]Ala-labeled proteins. In contrast, several signals from such membrane proteins were broadened or lost owing to interference of inherent fluctuation frequencies (10(4)-10(5)Hz) with frequency of either proton decoupling or magic angle spinning, if their 13C NMR spectra were recorded as a monomer in lipid bilayers at ambient temperature. The presence of such protein dynamics is essential for the respective proteins to achieve their own biological functions. Finally, spectral broadening found for bR and DGK in detergents were discussed.     

Nanometer propagation of millisecond motions in V-type allostery

Imidazole glycerol phosphate synthase (IGPS) is a V-type allosteric enzyme, which is catalytically inactive for glutamine hydrolysis until the allosteric effector, N'-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) binds 30?? away. In the apo state, NMR relaxation dispersion experiments indicate the absence of millisecond (ms) timescale motions. Binding of the PRFAR to form the active ternary complex is endothermic with a large positive entropy change. In addition, there is a protein wide enhancement of conformational motions in the ternary complex, which connect the two active sites. NMR chemical shift changes and acrylamide quenching experiments suggest that little in the way of structural changes accompany these motions. The data indicate that enzyme activation in the ternary complex is primarily due to an enhancement of ms motions that allows formation of a population of enzymatically active conformers.     

NMR characterization of the pH 4 beta-intermediate of the prion protein: the N-terminal half of the protein remains unstructured and retains a high degree of flexibility

Prion diseases are associated with the misfolding of the PrP (prion protein) from a largely alpha-helical isoform to a beta-sheet-rich oligomer. CD has shown that lowering the pH to 4 under mildly denaturing conditions causes recombinant PrP to convert from an alpha-helical protein into one that contains a high proportion of beta-sheet-like conformation. In the present study, we characterize this soluble pH 4 folding intermediate using NMR. (15)N-HSQC (heteronuclear single-quantum correlation) studies with mPrP (mouse PrP)-(23-231) show that a total of 150 dispersed amide signals are resolved in the native form, whereas only 65 amide signals with little chemical shift dispersion are observable in the pH 4 form. Three-dimensional (15)N-HSQC-TOCSY and NOESY spectra indicate that the observable residues are all assigned to amino acids in the N-terminus: residues 23-118. (15)N transverse relaxation measurements indicate that these N-terminal residues are highly flexible with additional fast motions. These observations are confirmed via the use of truncated mPrP-(112-231), which shows only 16 (15)N-HSQC amide peaks at pH 4. The loss of signals from the C-terminus can be attributed to line broadening due to an increase in the molecular size of the oligomer or exchange broadening in a molten-globule state.     

Backbone dynamics of azurin in solution: slow conformational change associated with deprotonation of histidine 35.

15N relaxation measurements have been performed on the type Iota blue copper protein azurin from Pseudomonas aeruginosa. The relaxation times show that one loop (residues 103-108) and one turn (residues 74-77) display fast internal motions. The rest of the protein is rigid with an average order parameter S(2) of 0.85 +/- 0. 05. The copper binding site shows the same degree of rigidity even though is it composed of several loops and lies outside the beta-sheet sandwich. Substantial exchange broadening was found for a number of residues surrounding the side chain of His-35. The average exchange rate has been determined from NMR exchange spectroscopy experiments and is 45 +/- 6 s(-)(1) at 41 degrees C. The exchange broadening is caused by the protonation/deprotonation equilibrium of His-35. The NMR results indicate that the two structures of azurin observed by X-ray diffraction of crystals at pH 5.5 and 9.0 [Nar, H., Messerschmidt, A., Huber, R., Van de Kamp, M., Canters, G. W. (1991) J. Mol. Biol. 221, 765-772] are present in solution and that they interconvert slowly.     

Magic angle spinning carbon-13 NMR of tobacco mosaic virus. An application of the high-resolution solid-state NMR spectroscopy to very large biological systems.

Magic angle spinning 13C NMR was used to study tobacco mosaic virus (TMV) in solution. Well-resolved 13C NMR spectra were obtained, in which several carbon resonances of amino acids of the TMV coat protein subunits that are not observable by conventional high-resolution NMR spectroscopy can be designed. RNA resonance were absent, however, in the magic angle spinning 13C NMR spectra. Since three different binding sites are available for each nucleotide of the RNA, this is probably due to a line broadening caused by distributions of isotropic chemical shift values. In 13C-enriched TM 13C-13C dipolar interactions also gave rise to line broadening. By suitable pulse techniques that discriminate carbon resonances on the basis of their T1 and T1 rho values, it was possible to select particular groups of carbon nuclei with characteristic motional properties. Magic angle spinning 13C NMR spectra obtained with these pulse techniques are extremely well resolved.     

Changes in structure and dynamics of the Fv fragment of a catalytic antibody upon binding of inhibitor

Binding of the product inhibitor p-nitrophenol to the monoclonal esterolytic antibody NPN43C9 has been investigated by performing NMR spectroscopy of the heterodimeric variable-domain fragment (Fv) of the antibody in the presence and absence of inhibitor. Structural information from changes in chemical shift upon binding has been related to the changes in local dynamics in the active site of the catalytic antibody using NMR relaxation measurements. Significant changes in the chemical shifts of the backbone resonances upon binding extend beyond the immediate vicinity of the antigen binding site into the interface between the two associated polypeptides that form the Fv heterodimer, a possible indication that the binding of ligand causes a change in the relative orientations of the component light (V(L)) and heavy (V(H)) chain polypeptides. Significant differences in backbone dynamics were observed between the free Fv and the complex with p-nitrophenol. A number of resonances, including almost all of the third hypervariable loop of the light chain (L3), were greatly broadened in the free form of the protein. Other residues in the antigen-binding site showed less broadening of resonances, but still required exchange terms (R(ex)) in the model-free dynamics analysis, consistent with motion on a slow timescale in the active site region of the free Fv. Binding of p-nitrophenol caused these resonances to sharpen, but some R(ex) terms are still required in the analysis of the backbone dynamics. We conclude that the slow timescale motions in the antigen-binding site are very different in the bound and free forms of the Fv, presumably due to the damping of large-amplitude motions by the bound inhibitor.     

Active site dynamics in NADH oxidase from Thermus thermophilus studied by NMR spin relaxation

We have characterized the backbone dynamics of NADH oxidase from Thermus thermophilus (NOX) using a recently-developed suite of NMR experiments designed to isolate exchange broadening, together with (15)N R (1), R (1ρ ), and {(1)H}-(15)N steady-state NOE relaxation measurements performed at 11.7 and 18.8 T. NOX is a 54?kDa homodimeric enzyme that belongs to a family of structurally homologous flavin reductases and nitroreductases with many potential biotechnology applications. Prior studies have suggested that flexibility is involved in the catalytic mechanism of the enzyme. The active site residue W47 was previously identified as being particularly important, as its level of solvent exposure correlates with enzyme activity, and it was observed to undergo "gating" motions in computer simulations. The NMR data are consistent with these findings. Signals from W47 are dynamically broadened beyond detection and several other residues in the active site have significant R ( ex ) contributions to transverse relaxation rates. In addition, the backbone of S193, whose side chain hydroxyl proton hydrogen bonds directly with the FMN cofactor, exhibits extensive mobility on the ns-ps timescale. We hypothesize that these motions may facilitate structural rearrangements of the active site that allow NOX to accept both FMN and FAD as cofactors.     

Solution NMR and computer simulation studies of active site loop motion in triosephosphate isomerase

Solution NMR spin relaxation experiments and classical MD simulations are used to study the dynamics of triosephosphate isomerase (TIM) in complex with glycerol 3-phosphate (G3P). Three regions in TIM exhibit conformational transitions on the micros-ms time scale as detected by chemical exchange broadening effects in NMR spectroscopy: residue Lys 84 on helix C, located at the dimeric interface; active site loop 6; and helix G. The results indicate that the conformational exchange process affecting the residues of loop 6 is the correlated opening and closing of the loop. Distinct processes are responsible for the chemical exchange linebroadening observed in the other regions of TIM. MD simulations confirm that motions of individual residues within the active site loop are correlated and suggest that the chemical exchange processes observed for residues in helix G arise from transitions between 3(10)- and alpha-helical structures. The results of the joint NMR and MD study provide global insight into the role of conformational dynamic processes in the function of TIM.     

Thermal broadening of the Soret band in heme complexes and in heme-proteins: role of iron dynamics

We report the thermal broadening of the Soret band in heme-CO, heme-OH and protoporphyrin IX in the temperature range 300–20 K. For protoporphyrin IX the temperature dependent Gaussian line broadening follows the behavior predicted by the harmonic approximation in the entire temperature range investigated. In contrast, for heme-CO and heme-OH the harmonic behavior is obeyed only up to about 180 K and an anomalous line broadening increase is observed at higher temperatures. This effect is attributed to the onset of anharmonic motions of the iron atom with respect to the porphyrin plane. Comparison with previously reported analogous data for heme proteins enables us to suggest that the onset of substate interconversions in these latter systems can be reflected in motions of the iron atom with respect to the porphyrin plane. Correspondence to: L. Cordone     

NMR study on the major mite allergen Der f 2: its refined tertiary structure, epitopes for monoclonal antibodies and characteristics shared by ML protein group members

Group 2 major mite allergens Der f 2 and Der p 2 are classified into the recently identified group of MD-2-related lipid-recognition (ML) proteins, but the ligands and biological functions of these allergens are unknown. We have obtained a high-quality NMR structure for Der f 2, and found that it is more similar to the crystal structure of NPC2, a distant homologue, than to that of Der p 2, in terms of the separation and angle between the two major beta-sheets. This made us propose that ML proteins undergo clamshell-like motions that change the sizes of ligand-binding spaces inside their immunoglobulin-fold beta-sandwich to accommodate lipid molecules. This type of motion in lipopolysaccaride recognition of MD-2 is suggested to be likely as well by structural models. We also report the applicability of NMR differential exchange broadening experiments for complexes of intact monoclonal antibodies and antigens; using this technique, we have detected the conformational epitopes for monoclonal antibodies 15E11 and 13A4 as two separate surface patches.     

Substrate-dependent millisecond domain motions in DNA polymerase β

DNA polymerase β (Pol β) is a 39-kDa enzyme that performs the vital cellular function of repairing damaged DNA. Mutations in Pol β have been linked to various cancers, and these mutations are further correlated with altered Pol β enzymatic activity. The fidelity of correct nucleotide incorporation into damaged DNA is essential for Pol β repair function, and several studies have implicated conformational changes in Pol β as a determinant of this repair fidelity. In this work, the rate constants for domain motions in Pol β have been determined by solution NMR relaxation dispersion for the apo and substrate-bound, binary forms of Pol β. In apo Pol β, molecular motions, primarily isolated to the DNA lyase domain, are observed to occur at 1400 s(-1). Additional analysis suggests that these motions allow apo Pol β to sample a conformation similar to the gapped DNA-substrate-bound form. Upon binding DNA, these lyase domain motions are significantly quenched, whereas evidence for conformational motions in the polymerase domain becomes apparent. These NMR studies suggest an alteration in the dynamic landscape of Pol β due to substrate binding. Moreover, a number of the flexible residues identified in this work are also the location of residues, which upon mutation lead to cancer phenotypes in vivo, which may be due to the intimate role of protein motions in Pol β fidelity.     

NMR solution structure of the angiostatic peptide anginex

Anginex, a designed peptide 33mer, is known to function both as an antiangiogenic and bactericidal agent. Solving the NMR solution structure of the peptide is key to understand better its structure-activity relationships and to design more bioactive peptides and peptide mimetics. However, structure elucidation of anginex has been elusive due to subunit exchange-induced resonance broadening. Here, we found that performing NMR structural studies in a micellar environment abolishes exchange broadening and allows the structure of anginex to be determined. Anginex folds in an amphipathic, three-stranded antiparallel beta-sheet conformation with functionally key hydrophobic residues lying on one face of the beta-sheet and positively charged, mostly lysine residues, lying on the opposite face. Structural comparison is made with a homologous, yet relatively inactive peptide, betapep-28. These results contribute to the design of peptidomimetics of anginex for therapeutic use against angiogenically-related diseases like cancer, as well as infectious diseases.     

Molecular motions and thermotropic phase behavior of cholesteryl esters with triolein

The phase behavior of cholesteryl esters with triglyceride has been characterized by differential scanning calorimetry (DSC), light microscopy, and polarizing light microscopy (PLM). Temperature-dependent molecular motions determined by 13C NMR spectroscopy were correlated with thermotropic phase behavior. Two systems, cholesteryl oleate (CO) and a 3/1 w/w mixture of cholesteryl linoleate (CL) and CO, were examined in the presence of small amounts of triolein (TO). Both systems exhibited metastable cholesteric and smectic (or only smectic) phases. Increasing amounts of TO progressively lowered the liquid-crystalline phase transition temperatures and eventually abolished the cholesteric phase, but at differing amounts of TO for the two systems (between 4% and 5% with CL/CO and between 7% and 10% with CO). DSC and PLM showed a progressive broadening of the phase transitions as well as an overlapping of the temperature ranges of the cholesteric and smectic phases. At greater than or equal to 4% TO, a separate isotropic liquid phase coexisted with liquid-crystalline phases. 13C NMR spectroscopy was used to monitor the molecular motions of the cholesteryl ester steroid ring and acyl chain in liquid and liquid-crystalline phases. In the liquid phase, no significant changes in fatty acyl motions, as reflected in spin-lattice relaxation time (T1) and nuclear Overhauser enhancement (NOE) values, were found on addition of TO. The line width (v 1/2) of the steroid ring resonances increased markedly near (1-5 degrees C above) the isotropic liquid----liquid-crystal phase transition temperature (TLC). However, the C3/C6 v 1/2 ratio at 1 degree C above TLC was greater for mixtures exhibiting an isotropic----cholesteric transition than for mixtures exhibiting an isotropic----smectic transition. Rotational correlation times calculated for motions about the long molecular axis and the nonunique axis showed (i) that the ring motions became more anisotropic as TLC was approached and (ii) that the motions were more anisotropic at TLC + 1 degree C for systems exhibiting a cholesteric phase than for systems exhibiting only a smectic phase. 13C line widths in spectra of the cholesteryl ester liquid-crystalline phases suggested that TO perturbed the cholesteryl ester intermolecular interactions and increased the rates of cholesteryl ester molecular motions relative to neat esters.