Assessment of protein reorientational diffusion in solution by 13C off-resonance rotating frame spin-lattice relaxation: effect of polydispersity

The 13C off-resonance rotating frame spin-lattice relaxation technique is applicable to the study of protein rotational diffusion behavior in both model in vitro and in vivo systems. The original formalism of James and co-workers [(1978) J. Am. Chem. Soc. 100, 3590-3594] was constrained by the assumption of random isotropic reorientational motion of a monodisperse protein population. Here we extend the formalism to include polydispersity. Application is made to the alkaline pH induced association of lysozyme, lysozyme-bovine serum albumin mixtures, and to the phase separation of lysozyme salt-water mixtures induced by low temperature.     

Assessment of protein rotational diffusion by 13C off-resonance rotating frame spin–lattice relaxation: Effect of backbone and side-chain internal motion

The ¹³C off-resonance rotating frame spin–lattice relaxation technique is applicable to the study of protein rotational diffusion behavior in a variety of experimental situations. The original formalism of James and co-workers (1978) (J. Am. Chem. Soc. Vol. 100, pp. 3590–3594) incorporated random isotropic reorientational motion of a rigid spherical rotor with no provision for backbone or side-chain carbonyl group internal motion. Here we demonstrate that the failure to include such internal motion may lead to erroneous rotational correlation time determinations for overall reorientational motion. The effect becomes severe for protein molecular masses in excess of 100 kD. Inclusion of both backbone and side-chain carbonyl carbon internal motion, using reasonable parameters derived from the literature [R. Levy and M. Karplus (1979), Chemical Physics Letters, Vol. 65, pp. 4–11; G. Careri, P. Fasella, and E. Gratton (1975), Critical Reviews in Biochemistry, Vol. 3, pp. 141–164; G. Lipari, A. Szabo, and R. Levy (1982), Nature, Vol. 300, pp. 197–198], plus corrections for anisotropic tumbling [C. F. Morgan, T. Schleich, G. H. Caines, and D. Michael (1990), Biopolymers, Vol. 29, pp. 469–480] and microscopic viscosity [S. H. Koenig (1980), ACS Symposium. Series, Vol. 127, pp. 157–176], leads to reliable values for the correlation time describing overall protein reorientation up to molecular masses of approximately 1000 kD. © 1993 John Wiley & Sons, Inc.     

Deuterium off-resonance rotating frame spin-lattice relaxation of macromolecular bound ligands.

Deuterated 3-trimethylsilylpropionic acid binding to bovine serum albumin was used as a model system to examine the feasibility and limitations of using the deuterium off-resonance rotating frame spin-lattice relaxation experiment for the study of equilibrium ligand-binding behavior to proteins. The results of this study demonstrate that the rotational-diffusion behavior of the bound species can be monitored directly, i.e., the observed correlation time of the ligand in the presence of a protein is approximately equal to the correlation time of the ligand in the bound state, provided that the fraction of bound ligand is at least 0.20. The presence of local ligand motion and/or chemical exchange contributions to relaxation in the bound state was inferred from the observation that the correlation time of the bound ligand was somewhat smaller than the correlation time characterizing the overall tumbling of the protein. An approximate value for the fraction of bound ligand was obtained from off-resonance relaxation experiments when supplemental spin-lattice or transverse relaxation times were employed in the analysis. Incorporation of local motion effects for the bound species into the theoretical relaxation formalism enabled the evaluation of an order parameter and an effective correlation time, which in conjunction with a wobbling in a cone model, provided additional information about ligand motion in the bound state.     

Hemoglobin aggregation in oxygenated sickle cells studies by carbon-13 rotating frame spin-lattice relaxation in the presence of an off-resonance radiofrequency field

Evidence is presented which shows that hemoglobin S in sickle cells has a tendency to aggregate even in the oxygenated state. The basis for that conclusion is derived from ¹³C-nmr rotating-frame spin–lattice relaxation studies in the presence of an off-resonance radiofrequency field in which the carbonyl resonances of hemoglobins in erythrocytes are examined. The experiments indicate that the rotational correlation time of hemoglobin S in oxygenated sickle cells at 38°C is 130 nsec compared to a value of 95 nsec for hemoglobin A in normal erythrocytes at the same temperature and the same mean cell hemoglobin content.     

Off-resonance rotating frame spin-lattice NMR relaxation studies of phosphorus metabolite rotational diffusion in bovine lens homogenates

The rotational diffusion behavior of phosphorus metabolites present in calf lens cortical and nuclear homogenates was investigated by the NMR technique of 31P off-resonance rotating frame spin-lattice relaxation as a means of assessing the occurrence and extent of phosphorus metabolite-lens protein interactions. 31P NMR spectra of calf lens homogenates were obtained at 10 and 18 degrees C (below and above the cold cataract phase transition temperature, respectively) at 7.05 T. Effective rotational correlation times (tau 0,eff) for the major phosphorus metabolites present in cortical and nuclear bovine calf lens homogenates were derived from nonlinear least-squares analysis of R vs omega e (spectral intensity ratio vs precessional frequency about the effective field) data with the assumption of isotropic reorientational motion. Intramolecular dipole-dipole (1H-31P, 31P-31P), chemical shift anisotropy (CSA), and solvent (water) translational intermolecular dipole-dipole (1H-31P) relaxation contributions were assumed in the analyses. In those cases where the limiting value of the spectral intensity ratio failed to reach unity at large offset frequency, a modified formalism incorporating chemical exchange mediated saturation transfer between two sites was used. Values of tau 0,eff for phosphorus metabolites present in the cortex varied from a low of ca. 2 ns [L-alpha-glycero-phosphocholine (GPC)] to a high of 12 ns (alpha-ATP) at 10 degrees C, whereas at 18 degrees C the range was from ca. 1 to 9 ns. For the nucleus the tau 0,eff values ranged from ca. 3 ns (GPC) to 41 ns (Pi) at 10 degrees C; at 18 degrees C the corresponding values ranged from 4 to 39 ns. For PME (phosphomonoester; in lens the predominant metabolite is L-alpha-glycerol phosphate) at 18 degrees C evidence was obtained for binding and subsequent exchange with solid like protein domains. The diversity in tau 0,eff values for lenticular phosphorus metabolites is suggestive of differential binding to more slowly tumbling macromolecular species, most likely lens crystallin proteins. Corresponding measurement of tau 0,eff values for the mobile protein fraction present in calf lens cortical and nuclear homogenates at 10 and 18 degrees C, by 13C off-resonance rotating frame spin-lattice relaxation, provided average macromolecular correlation times that were assumed to represent the bound metabolite state. A fast-exchange model (on the T1 time scale), between free and bound forms, was employed in the analysis of the metabolite R vs omega e curves to yield the     

Rotating frame spin-lattice relaxation experiments and the problem of intramolecular motions of peptides in solution

The application of rotating frame spin-lattice relaxtion to the determination of the intramolecular motions in peptides is discussed, and results are presented on the application of ¹³C T_1p to peptide microdynamics in solution. The effective molecular rotational reorientation times at the amide and amino nitrogens may be derived from appropriate data onT_1p of the carbons adjacent to them. We also show by theoretical caculations that ¹H and ¹³C T_1p experiments of suitable ²H and ¹⁵N substituted peptides will allow intramolecular main- and sidechain motions, characterized by times in the range 10^?1–10^?6 sec, to be investigated.     

Pyrrolidine ring conformations in prolyl peptides from 13C spin-lattice relaxation times

A method was developed in the framework of a bistable jump model to obtain the pyrrolidine ring conformations in proline peptides from 13C spin-lattice relaxation times. Equations are presented expressing the ring torsions in terms of the 13C spin-lattice relaxation times of the ring carbons. This method was applied to 26 pyrrolidine ring systems and acceptable conformations were obtained.     

Microsecond time scale dynamics in the RXR DNA-binding domain from a combination of spin-echo and off-resonance rotating frame relaxation measurements

Slow protein dynamics can be studied by 15N spin-echo (CPMG) and off-resonance rotating frame relaxation through the effective field dependence of the exchange-mediated relaxation contribution. It is shown that, by a combination of these complementary techniques, a more extended sampling of the microsecond time scale processes is achieved than by either method alone. 15N R2 and improved off-resonance R1 experiments [Mulder et al. (1998) J. Magn. Reson., 131, 351–357] were applied to the 9- cis-retinoic acid receptor DNA-binding domain and allowed the identification of 14 residues exhibiting microsecond time scale dynamics. Assuming exchange between two conformational substates, average lifetimes ranging from 37 to 416 s, and chemical shift differences of up to 3 ppm were obtained. The largest perturbation of tertiary structure was observed for the second zinc finger region, which was found to be disordered in the solution structure [Holmbeck et al. (1998) J. Mol. Biol., 281, 271–284]. Since this zinc-coordinating domain comprises the principal dimerization interface for RXR in a wide repertoire of complexes with different hormone receptors to their cognate response elements, this finding has important implications for our understanding of nuclear receptor assembly on DNA direct repeats. The flexibility observed for the dimerization domain may explain how RXR, through the ability to adaptively interact with a wide variety of highly homologous partner molecules, demonstrates such a versatile DNA-binding repertoire.     

The conformation of oxytocin in dimethylsulfoxide as revealed by carbon-13 spin-lattice relaxation times

Information was obtained on rates of overall molecular reorientation and segmental motion of amino acid sidechains of oxytocin in dimethylsulfoxide by determination of spin-lattice relaxation times (T₁) at 25 MHz for carbon-13 in natural abundance in the hormone. The T₁ values of the α-carbons of amino acid residues located in the 20-membered ring of oxytocin are all about 50 msec. The overall correlation time for the hormone backbone was estimated to be 8.8 × 10^?10 sec. The sidechains of Tyr, Ile and Gln undergo segmental motion with respect to the backbone of the ring. The T₁ value of the α-carbon of the Leu residue is greater than for any α-carbon in the ring, indicating an increased mobility of the backbone of the C-terminal acyclic peptide as compared to the ring. The β- and γ-carbons of the Pro residue undergo an exo-endo interconversion with regard to the plane formed by α-carbon, δ-carbon and N atom of the Pro pyrollidine ring. These data are discussed in light of results from other experimental and theoretical studies, including carbon-13 spin-lattice relaxation times for oxytocin in aqueous solution.     

Conformational flexibility of luteinizing hormone-releasing hormone in aqueous solution. A carbon-13 spin-lattice relaxation time study.

The carbon-13 nuclear magnetic resonance (13C NMR) spectra of luteinizing hormone-releasing hormone (LH-RH) and lower homologous peptides have been assigned in aqueous solutions at various pH values. 13C spin-lattice relaxation times (T1) have been measured for all proton-bearing carbons at 25.2 and 67.9 MHz. From the T1 data the rates of overall molecular motion and intramolecular motion of side chains have been estimated. LH-RH is a flexible molecule in solution, having segmental motion along the backbone as well as in the nonaromatic side chains.     

Rotational diffusion tensor of nucleic acids from 13C NMR relaxation

Rotational diffusion properties have been derived for the DNA dodecamer d(CGCGAATTCGCG)₂ from ¹³C R₁ and R₁ measurements on the C₁, C₃, and C₄ carbons in samples uniformly enriched in ¹³C. The narrow range of C-H bond vector orientations relative to the DNA axis make the analysis particularly sensitive to small structural deviations. As a result, the R₁/R₁ ratios are found to fit poorly to the crystal structures of this dodecamer, but well to a recent solution NMR structure, determined in liquid crystalline media, even though globally the structures are quite similar. A fit of the R₁/R₁ ratios to the solution structure is optimal for an axially symmetric rotational diffusion model, with a diffusion anisotropy, D_||/D, of 2.1±0.4, and an overall rotational correlation time, (2D_||+4D)^–1, of 3.35 ns at 35 °C in D₂O, in excellent agreement with values obtained from hydrodynamic modeling.     

A 13C NMR spin-lattice relaxation study of the interaction of myelin proteins with lipid vesicles

Natural abundance 13C nuclear magnetic spin-lattice relaxation times have been measured for bovine brain phosphatidylserine vesicles with and without bound proteins. The relaxation times were lower than published values for the corresponding nuclei in egg phosphatidylcholine, but showed the same trend, with relaxation times increasing along the acyl chains away from the polar headgroup. These times were inversely related to the degree of saturation of the lipid. Cytochrome c caused insignificant changes in the lipid acyl chain relaxation rates but reduced the resonance intensities, in agreement with Brown and Wüthrich (Biochem. Biophys. Acta 468 (1977) 389). In contrast, the basic protein from bovine myelin did not affect the intensities but reduced the relaxation times for 13C nuclei near the bilayer centre, and for nuclei near carbon-carbon double bonds. These proteins also dramatically broadened the serine headgroup carboxyl resonance. It appears, in accord with other recent evidence, that the basic protein does penetrate the hydrophobic region of the bilayer (possibly to the centre), producing quantitatively similar changes in the relaxation rates to proteolipid protein, an integral membrane protein.     

Cyclic peptides. XVIII. 13C spin-lattice relaxation times of (X-pro-Y)2 cyclic hexapeptides

¹³C spin-lattice relaxation times (T₁'s) of four cyclic hexapeptides of sequence, (X-L -Pro-Y)₂, are reported. The T₁'s of the protonated carbons, which undergo dipolar relaxation, are interpreted qualitatively in terms of the overall tumbling motion of the molecule and in terms of internal motion. It is found that three of the cyclic hexapeptides, those which adopt all-trans β-conformers, tumble isotropically and appear to lack internal motion in the peptide backbone. The method of Torchia and Lyerla was applied to these compounds in order to compare the mobility of the proline rings. The results show that the sequence and particular type of β-turn present affect the internal motion of the Pro ring. Data on a fourth cyclic hexapeptide, which occurs in a conformation with two-cis X-Pro bonds, suggests that internal motion of the backbone contributes an additional frequency component to the motion of the Y residue α-carbons. A consideration of the mobility of the proline rings in the conformer with two-cis peptide bonds revealed that they are significantly more rigid in the two-cis structure than in the all-trans.     

Lateral diffusion of the phospholipid molecule in dipalmitoylphosphatidylcholine bilayers. An investigation using nuclear spin--lattice relaxation in the rotating frame

Measurements of the proton NMR spin--lattice relaxation time in the rotating frame (T 1rho) have permitted the explicit determination of the lateral diffusion coefficient of phospholipid molecules in the lamellar mesophase of dipalmitoylphosphatidylcholine at temperatures above the phase-transition temperature. The experimentally observed temperature and frequency dependence of T 1rho for the dipalmitoylphosphatidylcholine protons suggest that intermolecular dipole--dipole relaxation contributions are important. Proton T 1rho experiments involving dilution with deuterated dipalmitoylphosphatidylcholine support the premise that intermolecular dipolar interactions are significant and, concomitantly, that lateral diffusion is the motion modulating that interaction. The lateral diffusion coefficient is determined directly from the dependence of the rotating frame spin--lattice relaxation rate (1/T 1rho) on the strength of the applied radiofrequency field in the spin-locking experiment. A series of experiments with varying concentrations of dipalmitoylphosphatidylcholine in the lamellar mesophase indicates that the lateral diffusion coefficient varies as a function of phospholipid concentration.     

Water in agarose gels studied by nuclear magnetic resonance relaxation in the rotating frame.

The dependence of the spin-lattice relaxation time in the rotating frame (T1rho) on radio frequency (RF) field strength and temperature has been studied for agarose gels in order to investigate molecular motion. The results indicate the presence of slow motions with a correlation time of ca. 5-10(-6) s at room temperature. This interaction is responsible for the short spin-spin relaxation times (T2) for water protons in agarose gels and is ascribed to firmly bound water. The fraction of bound water is estimated to about 0.003 for a 7.3% agarose gel. The motion of the more mobile protons in agarose-water systems can not be characterized by single correlation time. This fraction is presumably composed of water in different motional states and some of the agarose hydroxyl protons. Higher mobilities are the most common.     

A 13C spin-lattice relaxation study of dipeptides containing glycine and proline: mobility of the cyclic proline side chain.

Molecular dynamics of the cyclic dipeptides cyclo(Gly-L -Pro), cyclo-(L-Pro-L -Pro), and cyclo(L-Pro-D-Pro) and the linear dipeptides L-Pro-Gly and cis and trans Gly-L -Pro were studied in neutral aqueous solution by ¹³C nuclear magnetic resonance. Spinlattice relaxation times (T₁) were determined for each individual carbon atom. The correlation times, τ, were derived from a semiquantitative analysis of the T₁ data. The correlation times of the proline ring carbons, β, γ, and δ suggest that the cyclic dipeptides have more restriction of conformational freedom in the proline ring than the linear dipeptides. This effect is most pronounced on the γ carbon.     

Intramolecular microdynamical and conformational parameters of peptides from 1H and 13C NMR spin-lattice relaxation. Tetragastrin.

Measurements of 1H and 13C spin-lattice relaxation and 13C nuclear Overhauser enhancement factors are reported for dimethyl sulfoxide solutions of tetragastrin, a pharmacologically active tetrapeptide. The use of the dipolar formalism for predicting 1H and quaternary 13C relaxation rates is discussed. Furthermore, the prospect is opened for the use of quaternary 13C and 1H relaxation times to obtain information on the peptide torsion angles phi, psi, and chi in a way supplementing NMR coupling constant methods presently in use.