NMR Relaxation Studies Research Articles

Crystallographic and nuclear magnetic resonance evaluation of the impact of peptide binding to the second PDZ domain of protein tyrosine phosphatase 1E.

PDZ (PSD95/Discs large/ZO-1) domains are ubiquitous protein interaction motifs found in scaffolding proteins involved in signal transduction. Despite the fact that many PDZ domains show a limited tendency to undergo structural change, the PDZ family has been associated with long-range communication and allostery. One of the PDZ domains studied most in terms of structure and biophysical properties is the second PDZ ("PDZ2") domain from protein tyrosine phosphatase 1E (PTP1E, also known as PTPL1). Previously, we showed through NMR relaxation studies that binding of the RA-GEF2 C-terminal peptide substrate results in long-range propagation of side-chain dynamic changes in human PDZ2 [Fuentes, E. J., et al. (2004) J. Mol. Biol. 335, 1105-1115]. Here, we present the first X-ray crystal structures of PDZ2 in the absence and presence of RA-GEF2 ligand, determined to resolutions of 1.65 and 1.3 Å, respectively. These structures deviate somewhat from previously determined NMR structures and indicate that very minor structural changes in PDZ2 accompany peptide binding. NMR residual dipolar couplings confirm the crystal structures to be accurate models of the time-averaged atomic coordinates of PDZ2. The impact on side-chain dynamics was further tested with a C-terminal peptide from APC, which showed results nearly identical to those of RA-GEF2. Thus, allosteric transmission in PDZ2 induced by peptide binding is conveyed purely and robustly by dynamics. (15)N relaxation dispersion measurements did not detect appreciable populations of a kinetic structural intermediate. Collectively, for ligand binding to PDZ2, these data support a lock-and-key binding model from a structural perspective and an allosteric model from a dynamical perspective, which together suggest a complex energy landscape for functional transitions within the ensemble.

NMR Studies into the Potential Interactions of Fullerene C60 with Tetraphenylporphyrin and Some of Its Derivatives

1H NMR relaxation studies were employed to investigate potential interactions between C60 and tetraphenylporphyrin, H2[TPP], and parasubstituted tetraphenylporphyrins, H2[(p-X)4TPP], where X = CN and OCH3 in solution. The substituted porphyrins provided a means by which to investigate the role that electronic effects play in the interaction process. A comparison of the relaxation rates, R1, and correlation times, τC, of the pyrrole and phenyl hydrogens in these complexes, without and with the presence of C60, revealed that the introduction of C60 into solution did not have a noticeable effect on R1 and τC of these protons in H2[TPP], indicating the absence of long-term intermolecular interaction at either of these two sites. A similar analysis of the two protons in the other two substituted tetraphenylporphyrin analogs revealed slower molecular dynamics indicating the presence of intermolecular interactions. Stronger interactions were observed in H2[(p-OCH3)4TPP] indicating that the electron-donating abilities of the -OCH3 group promote the interaction process. Our results indicate that it is very likely that enhanced selectivity in the chemical purifications of fullerenes and metallofullerenes can be achieved by employing tetraphenylporphyrin-silica stationary phases which have been modified with electron-donating groups.

Structural Features of Cytochromes P450 and Ligands that Affect Drug Metabolism as Revealed by X-Ray Crystallography and NMR

Cytochromes P450 (P450s) play a major role in the clearance of drugs, toxins, and environmental pollutants. Additionally, metabolism by P450s can result in toxic or carcinogenic products. The metabolism of pharmaceuticals by P450s is a major concern during the design of new drug candidates. Determining the interactions between P450s and compounds of very diverse structures is complicated by the variability in P450-ligand interactions. Understanding the protein structural elements and the chemical attributes of ligands that dictate their orientation in the P450 active site will aid in the development of effective and safe therapeutic agents. The goal of this review is to describe P450-ligand interactions from two perspectives. The first is the various structural elements that microsomal P450s have at their disposal to assume the different conformations observed in X-ray crystal structures. The second is P450-ligand dynamics analyzed by NMR relaxation studies.

NMR Relaxation and Self-Diffusion Study at High and Low Magnetic Fields of Ionic Association in Protic Ionic Liquids

NMR relaxation and diffusion characterization of several protic ionic liquids at high and low magnetic fields are reported. The dynamics of cations and anions were similar at both frequencies, with similar trends and magnitudes for a fixed component paired with oppositely charged species. An Arrhenius relationship was displayed between the molecular motion and the glass transition temperature. The diffusion of ions showed a strong degree of ion correlation between cation and anion, and Arrhenius plots of relaxation and diffusion indicated that the ions diffused as a pair. At high field diffusion was dominated by mobile species that followed Stokes-Einstein behavior. Conversely, diffusion observed at low field emphasized relatively immobile species that displayed fractional Stokes-Einstein behavior. No evidence was found to indicate the influence of magnetic field on structural and dynamic properties of the studied ILs; however, variation between diffusion coefficients at different magnetic fields indicated dynamic heterogeneities (or temporal aggregates) within the ionic liquid.

Determination of the antisymmetric part of the chemical shift anisotropy tensor via spin relaxation in nuclear magnetic resonance

Relaxation processes induced by the antisymmetric part of the chemical shift anisotropy tensor (henceforth called anti-CSA) are usually neglected in NMR relaxation studies. It is shown here that anti-CSA components contribute to longitudinal relaxation rates of the indole (15)N nucleus in tryptophan in solution at different magnetic fields and temperatures. To determine the parameters of several models for rotational diffusion and internal dynamics, we measured the longitudinal relaxation rates R(1)=1/T(1) of (15)N, the (15)N-(1)H dipole-dipole (DD) cross-relaxation rates (Overhauser effects), and the cross-correlated CSA/DD relaxation rates involving the second-rank symmetric part of the CSA tensor of (15)N at four magnetic fields B(0)=9.4, 14.1, 18.8, and 22.3 T (400, 600, 800, and 950 MHz for protons) over a temperature range of 270<T<310 K. A good agreement between experimental and theoretical rates can only be obtained if the CSA tensor is assumed to comprise first-rank antisymmetric (anti-CSA) components. The magnitude of the hitherto neglected antisymmetric components is of the order of 10% of the CSA.

NMR Relaxation Studies of an RNA-Binding Segment of the Rous Sarcoma Virus Gag Polyprotein in Free and Bound States: A Model for Autoinhibition of Assembly

Assembly of retrovirus particles is promoted by interaction of the Gag polyprotein with RNA. Nonspecific RNA association with the nucleocapsid domain (NC) of Gag induces the dimerization of Gag through protein-protein contacts in the capsid domain (CA), followed by higher order assembly to form the immature virus particle. NMR relaxation studies were conducted to investigate the initial steps of Rous sarcoma virus (RSV) assembly by examining the association with nucleic acid of a fragment of Gag comprising the C-terminal domain of CA (CTD) postulated to mediate Gag dimerization, the spacer region between CA and NC (SP), and NC. This fragment, CTD-SP-NC (residues 394-577), spans the critical SP region and allows assessment of this key Gag-nucleic acid interaction in the context of the Gag polyprotein rather than the isolated domains. Main-chain amide relaxation of CTD-SP-NC was measured in the absence and presence of (GT)(4), an 8-mer DNA oligonucleotide that binds tightly to the polyprotein but is too short to promote Gag dimerization. The results show that the CTD and NC domains tumble independently. In contrast, the two zinc finger domains within NC are rotationally coupled in both the unbound and bound states, even though only the first zinc finger appears to make direct contact with (GT)(4). In addition, the NMR data indicate that SP and flanking residues undergo a conformational exchange process that is slowed in the presence of (GT)(4). This region around SP where relaxation is strongly affected by (GT)(4) binding is nearly identical to the assembly domain defined previously by mutagenesis studies. Other changes in relaxation induced by (GT)(4) implicate conformational perturbations of helices 1 and 4 in CTD. On the basis of the combined data, we propose a model for the promotion of Gag dimerization by RNA association in which NC-RNA binding disrupts an assembly inhibitory, intramolecular interaction involving SP and CTD. Disruption of this intramolecular interaction is proposed to enhance the accessibility of the Gag dimer contact surface and release the assembly domain to promote intermolecular oligomerization.

High Resolution Field Cycling for Analysis of Lipid Dynamics in Membranes

Phospholipid dynamics often play a role in binding of peripheral membrane proteins. Whether there are discrete phospholipid binding sites on the proteins is often difficult to assess unless binding is quite tight. Yet for many proteins that bind reversibly to membranes, weaker interactions with specific phospholipids can have important functional consequences. For example, phosphatidylinositol‐specific phospholipase C (PI‐PLC) enzymes can be activated by nonsubstrate phospholipids with the extent of activation tuned by the lipid composition. Activity of the Bacillus thuringiensis PI‐PLC is significantly enhanced by a small amount of phosphatidylcholine (PC) in the target membrane. Whether (and where) there is a discrete site for an activator PC molecule or whether lipid mixtures alter bilayer properties is hard to distinguish. High resolution 31P and 13C NMR relaxation studies from 0.005 to 11.7 T can be used to explore interactions of such a peripheral membrane protein with mixed phospholipid vesicles as well as with small molecules. If spin‐labeled proteins are used, estimates of site‐off rates and phospholipid binding site location can be made. With this novel approach for PI‐PLC, we see that (i) discrete complexes for each type of phospholipid on the enzyme with lifetimes around a microsecond must exist; (ii) the discrete site for a substrate analog is spatially different from that for activator PC; and (iii) modeling coupled with the NMR results suggests a PC site, near a series of tyrosine residues, that would aid in anchoring the protein to membranes. The high resolution field cycling method illustrated here may be extended to a wide range of proteins that reversibly bind to membranes to explore differential interactions with phospholipid components as well as other intermolecular interactions.

NMR relaxation study of the local environments and phase transitions of CsCr(SO4)2·12H2O and CsAl(SO4)2·12H2O single crystals

Abstract The physical properties and phase-transition mechanisms of CsCr(SO4)2·12H2O and CsAl(SO4)2·12H2O single crystals were investigated, with the phase transitions of both crystals being determined from DSC and NMR data. The relaxation times of the 27Al and 133Cs nuclei in the two crystals undergo significant changes at the temperature lower than the phase transition temperature TC. The variations in the temperature dependence of the spin-lattice relaxation time T1 near TC are related to the changes in the symmetry of the octahedra of water molecules surrounding Al3+ and Cs+. The Cs relaxation time in CsCr(SO4)2·12H2O is faster than that in CsAl(SO4)2·12H2O at 300 K; the spin-lattice relaxation time is inversely proportional to the square of the magnetic moment of the paramagnetic ion. From these results, it can be inferred that the different trivalent cation of Cr and Al are responsible for the different spin-lattice relaxation times in the two crystals.

Inhomogeneity in the drying process of gelatin film formation: NMR microscopy and relaxation study

Gelatin film formation was investigated in situ during evaporation of the solvent. Gelatin was dissolved in deuterium oxide (D 2O) at 50 °C followed by casting. The drying process of the gelatin–D 2O solution system was observed using a single-sided NMR scanner by application of the Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence at different layer positions. The echo decays were analysed and spin–spin relaxation times ( T 2) were obtained for each layer. It was found that the drying process for the film formation occurs in a stratified fashion.

Dynamics of Retinal Studied by 2H NMR Relaxation Underlie Multiscale Conformational Changes in Rhodopsin Activation

The activation mechanism of the G protein-coupled receptor rhodopsin has been investigated through solid-state 2H NMR relaxation studies. Rhodopsin was regenerated with retinal 2H-labeled at the C5-, C9-, and C13-methyl groups and was recombined into POPC (dark state and Meta I) or POPC:DOPE (Meta II) [1-4]. The 2H NMR relaxation rates of Zeeman order (R1Z) or quadrupolar order (R1Q) were measured from −30 to −150°C in the dark, Meta I, and Meta II states. Relaxation data were interpreted in terms of axial rotation and off-axial motion of the methyl groups and revealed interactions between the retinal cofactor and the rhodopsin binding pocket. Relaxation rates show considerable mobility of the retinal ligand in the dark state despite the complete absence of basal activity. 2H NMR data indicate that the retinal C9-methyl group does not change its orientation upon photoisomerization. Rather it acts as a hinge for light-induced rotation of the C13-methyl toward the second extracellular loop E2. The C13-methyl rotation leads to a displacement of the E2 loop that is observed already in the Meta I state. Short T1Z relaxation times for the C5-methyl indicate that the β-ionone ring adopts a twisted 6-s-cis conformation, and remains in its hydrophobic binding pocket up to the Meta II state. An activation mechanism is proposed whereby the photonic energy is channeled by 11-cis to trans isomerization of retinal against the E2 loop by the C9- and C13-methyl groups, and toward helices H5 and H3 by the β-ionone ring and C5-methyl group.[1] G.F.J. Salgado et al. (2004) Biochemistry43, 12819. [2] G.F.J. Salgado et al. (2006) JACS128, 11067. [3] A.V. Struts et al. (2007) JMB372, 50. [4] M.F. Brown et al. (2009) Photochem. Photobiol.85, 442.

The physical basis of model-free analysis of NMR relaxation data from proteins and complex fluids

NMR relaxation experiments have provided a wealth of information about molecular motions in macromolecules and ordered fluids. Even though a rigorous theory of spin relaxation is available, the complexity of the investigated systems often makes the interpretation of limited datasets challenging and ambiguous. To allow physically meaningful information to be extracted from the data without commitment to detailed dynamical models, several versions of a model-free (MF) approach to data analysis have been developed. During the past 2 decades, the MF approach has been used in the vast majority of all NMR relaxation studies of internal motions in proteins and other macromolecules, and it has also played an important role in studies of colloidal systems. Although the MF approach has been almost universally adopted, substantial disagreement remains about its physical foundations and range of validity. It is our aim here to clarify these issues. To this end, we first present rigorous derivations of the three well-known MF formulas for the time correlation function relevant for isotropic solutions. These derivations are more general than the original ones, thereby substantially extending the range of validity of the MF approach. We point out several common misconceptions and explain the physical significance of the approximations involved. In particular, we discuss symmetry requirements and the dynamical decoupling approximation that plays a key role in the MF approach. We also derive a new MF formula, applicable to anisotropic fluids and solids, including microcrystalline protein samples. The so-called slowly relaxing local structure (SRLS) model has been advanced as an alternative to the MF approach that does not require dynamical decoupling of internal and global motions. To resolve the existing controversy about the relative merits of the SRLS model and the MF approach, we formulate and solve a planar version of the SRLS model. The analytical solution of this model reveals the unphysical consequences of the symmetrical two-body Smoluchowski equation as applied to protein dynamics, thus refuting the widely held belief that the SRLS model is more accurate than the MF approach. The different results obtained by analyzing data with these two approaches therefore do not indicate the importance of dynamical coupling between internal and global motions. Finally, we explore the two principal mechanisms of dynamical coupling in proteins: torque-mediated and friction-mediated coupling. We argue by way of specific analytically solvable models that torque-mediated coupling (which the SRLS model attempts to capture) is unimportant because the relatively slow internal motions that might couple to the global motion tend to be intermittent (jumplike) in character, whereas friction-mediated coupling (which neither the SRLS model nor the MF approach incorporates) may be important for proteins with unstructured parts or flexibly connected domains.

NMR control of aging and durability of hardened cement pastes

Abstract We report on proton NMR relaxation of hardened and aged grey and white CEM I paste with several controlled degree of relative humidity. A proton NMR relaxation study of these two CEM I cement pastes shows very different pore size hierarchies for these two materials. The good sensitivity of the proposed method for analysing the evolution of the pore size dependencies under drying and wetting history of the material could be very useful for studying the durability of building material under severe conditions of use.

A combined molecular dynamic simulation and urea [formula omitted] NMR relaxation study of the urea–lysozyme system

Urea in the lysozyme solvation shell has been studied by utilizing a combination of urea N 14 , water O 17 NMR relaxation experiments and a molecular dynamics simulation of the urea–lysozyme system. Samples with lysozyme in the native fold in water as well as in 3 M urea have been studied, as well as denatured lysozyme in a 8.5 M urea solvent. The spin relaxation rates of the samples with folded protein show a clear field dependence, which is consistent with a few urea molecules having long residence times on the protein surface and preferentially located in pockets and grooves on the protein. By combining the 3 M urea NMR relaxation data and data from the MD simulation, a full parameter set of the relaxation model is found which can successfully predict the experimental relaxation rates of the 3 M urea sample. However, in the parameter fitting it is evident that the rotational dynamics of urea in the MD simulation is slightly too fast to be consistent with the NMR relaxation rates, perhaps a result of the fast dynamics of the TIP3P water model. The relaxation rates of urea in the proximity of the unfolded lysozyme lack field dependence, which can be interpreted as a loss of pockets and grooves on the denatured protein. The extracted model parameters from the 3 M sample are adjusted and tested on a simple model of the unfolded protein sample and are seen to be in agreement with the relaxation rates. It is shown that the combination of NMR relaxation and MD simulations can be used to create a microscopic picture of the solvent at the protein interface, which can be used for example in the study of chemical denaturation.

Carbon-13 NMR Relaxation Study of the Internal Dynamics in Cyclodextrins in Isotropic Solution

(13)C nuclear spin relaxation processes in seven cyclodextrins (from six-membered alpha to twelve-membered eta) were investigated in (2)H(2)O solution at multiple magnetic fields. Detailed analysis of (13)C longitudinal relaxation in laboratory and rotating frames and (13)C{(1)H} nuclear Overhauser enhancement in these molecules yielded their rotational diffusion tensors and a semiquantitative picture of their internal dynamics.

Effect of Gel Firmness at Cutting Time, pH, and Temperature on Rennet Coagulation and Syneresis: An in situ 1H NMR Relaxation Study

The objective of this study was to monitor rennet-induced milk gel formation and mechanically induced gel syneresis in situ by low-field NMR. pH, temperature, and gel firmness at cutting time were varied in a factorial design. The new curve-fitting method Doubleslicing revealed that during coagulation two proton populations with distinct transverse relaxation times (T2,1=181, T2,2=465 ms) were present in fractions (f1=98.9%, f2=1.1%). Mechanical cutting of the gel in the NMR tube induced macrosyneresis, which led to the appearance of an additional proton population (T2,3=1500-2200 ms) identified as whey. On the basis of NMR quantification of whey water the syneresis rate was calculated and found to be significantly dependent on pH and temperature.

From the SmA to the Hexatic, Including the SmC*, SmC*A and SmC*re Phases: A 2H NMR Relaxation Study

The molecular dynamics of a ferroelectric liquid crystal, denoted ZLL 7/*, is investigated by means of (2)H NMR relaxation. The spin-lattice (T(1Q) and T(1Z)) and spin-spin (T(2)) relaxation times of two isotopomers of ZLL 7/*, labeled on the phenyl and biphenyl fragments, are measured and their behavior upon passing from the SmA to the hexatic phase, through the ferroelectric SmC*, antiferroelectric SmC*(A), and re-entrant ferroelectric SmC*(re) phases, is discussed. A comparison between the measured T(2) and T(2)*, directly related to the experimental linewidth, provides information on the heterogeneity of the system, thus allowing confirmation of previous hypotheses concerning the structural and ordering properties of the SmC*(A) and SmC*(re) phases. The possibility to look at different sites of the core of the ZLL 7/* smectogen reveals a peculiar sensitivity of the phenyl moiety with respect to the biphenyl fragment, which may be justified by its vicinity to the chiral centers. Interestingly, the trend of the longitudinal relaxation times is characterized by a minimum that corresponds to the SmC*(A) and SmC*(re) phases, which is reproducible for the two isotopomers and at several Larmor frequencies. A quantitative analysis of T(1Q) and T(1Z) is performed in the SmA and SmC* phases, for which the narrowing regime approximation is valid. A multifrequency approach is applied to self-consistently determine the diffusion coefficients for the overall molecular motions, namely spinning and tumbling, and the internal rotations around the para axes of the phenyl and biphenyl fragments. The effect of the magnetic field in unwinding the helical structure of the SmC* phase (for H>9 T) allows observation of a sensitive change in the rotational diffusion coefficients in the frustrated unwound SmC* phase with respect to the SmC* phase.

Deuterium spin probes of backbone order in proteins: 2H NMR relaxation study of deuterated carbon alpha sites.

(2)H spin relaxation NMR experiments to study the dynamics of deuterated backbone alpha-positions, D(alpha), are developed. To date, solution-state (2)H relaxation measurements in proteins have been confined to side-chain deuterons-primarily (13)CH(2)D or (13)CHD(2) methyl groups. It is shown that quantification of (2)H relaxation rates at D(alpha) backbone positions and the derivation of associated order parameters of C(alpha)-D(alpha) bond vector motions in small [U-(15)N,(13)C,(2)H]-labeled proteins is feasible with reasonable accuracy. The utility of the developed methodology is demonstrated on a pair of proteins-ubiquitin (8.5 kDa) at 10, 27, and 40 degrees C, and a variant of GB1 (6.5 kDa) at 22 degrees C. In both proteins, the D(alpha)-derived parameters of the global rotational diffusion tensor are in good agreement with those obtained from (15)N relaxation rates. Semiquantitative solution-state NMR measurements yield an average value of the quadrupolar coupling constant, QCC, for D(alpha) sites in proteins equal to 174 kHz. Using a uniform value of QCC for all D(alpha) sites, we show that C(alpha)-D(alpha) bond vectors are motionally distinct from the backbone amide N-H bond vectors, with (2)H-derived squared order parameters of C(alpha)-D(alpha) bond vector motions, S(2)(CalphaDalpha), on average slightly higher than their N-H amides counterparts, S(2)(NH). For ubiquitin, the (2)H-derived backbone mobility compares well with that found in a 1-mus molecular dynamics simulation.

Enzymology with a Spin-Labeled Phospholipase C: Soluble Substrate Binding by 31P NMR from 0.005 to 11.7 T

31P NMR relaxation studies from 0.005 to 11.7 T are used to monitor water-soluble inositol 1,2-(cyclic) phosphate (cIP) binding to phosphatidylinositol-specific phospholipase C spin-labeled at H82C, a position near the active site of the enzyme, and to determine how activating phosphatidylcholine (PC) molecules affect this interaction. We show that, in the absence of an interface, cIP binding to the protein is not rate-limiting, and that lower activation by PC vesicles as opposed to micelles is likely due to hindered product release. The methodology is general and could be used for determining distances in other weakly binding small molecule ligand-protein interactions.

A NMR Relaxation Study for the Interactions of some 1-alkyl-3-methylimidazolium Ionic Liquids with Acetone

Abstract 13C spin-lattice relaxation times of room temperature ionic liquids [C4mim][PF6], [C6mim][PF6], [C8mim][PF6],[C6mim][BF4] and [C6mim]Br in acetone-d6 have been determined at various acetone-d6 mole fractions. It was shown that the spin-lattice relaxation time increased with increasing acetone-d6 mole fractions, and was influenced not only by the type of the anions but also by the length of alkyl chain on the imidazolium cations. The results suggested that interactions between the cation and the anion of ionic liquids were weakened and the ionic motion of ionic liquids was enhanced with the addition of acetone-d6.

Water-Based Latex Dispersions. 5: NMR Relaxation Studies of Deuterium Labeled Nonylphenol Ethoxylate

The dynamics and conformation of the nonionic surfactant NP100 (nonylphenol ethoxylate with an average of 84 oxyethylene units), adsorbed on colloidal silica and on polystyrene latex particles, have been studied by the nuclear magnetic resonance relaxation technique. To obtain specific information about the spin relaxation from the methylene groups situated at the hydroxyl-terminal end of the polyoxyethylene (POE) chain, this part was labeled with deuterium in a two-step synthesis. Spin relaxation studies of the labeled part of the adsorbed surfactant (2H relaxation) suggested that the dynamics was slower in comparison with the spin relaxation of the surfactant in solution. Also, it was seen that the dynamics was slower on the silica surface than on the polystyrene surface. This was in agreement with previous studies on the average proton spin relaxation of the whole POE chain of the surfactant on silica and on polystyrene surfaces. The correlation time constant for the slow motions of the POE chain was calculated to be as large as 7.4 ms for NP100 on the polystyrene particles. Also this was in good agreement with studies made on the exchange dynamics of the surfactant in different dispersions, where it was found that the exchange between the solution and the particles was slow.