Monitoring membrane protein rotational diffusion using time-averaged phosphorescence

Rotational motions of membrane proteins have previously been measured using time-dependent phosphorescence techniques. This paper discusses a method of examining membrane protein mobility at temperatures relevant to biological systems, using a technique similar to steady-state fluorescence. The method is demonstrated using sarcoplasmic reticulum ATPase labelled with erythrosin isothiocyanate, both in its natural condition and crosslinked by incubation with glutaraldehyde. The experimentally-observed dependence of phosphorescence anisotropy on temperature is compared to a calculated anisotropy-temperature curve. Comparison is made between the anisotropy decay curves obtained by time-averaged phosphorescence and steady-state fluorescence.     

Efficient analysis of macromolecular rotational diffusion from heteronuclear relaxation data

A novel program has been developed for the interpretation of ¹⁵N relaxation rates in terms of macromolecular anisotropic rotational diffusion. The program is based on a highly efficient simulated annealing/minimization algorithm, designed specifically to search the parametric space described by the isotropic, axially symmetric and fully anisotropic rotational diffusion tensor models. The high efficiency of this algorithm allows extensive noise-based Monte Carlo error analysis. Relevant statistical tests are systematically applied to provide confidence limits for the proposed tensorial models. The program is illustrated here using the example of the cytochrome c from Rhodobacter capsulatus, a four-helix bundle heme protein, for which data at three different field strengths were independently analysed and compared.     

Anisotropic rotational diffusion in model-free analysis for a ternary DHFR complex

Model-free analysis has been extensively used to extract information on motions in proteins over a wide range of timescales from NMR relaxation data. We present a detailed analysis of the effects of rotational anisotropy on the model-free analysis of a ternary complex for dihydrofolate reductase (DHFR). Our findings show that the small degree of anisotropy exhibited by DHFR (D_||/D=1.18) introduces erroneous motional models, mostly exchange terms, to over 50% of the NH spins analyzed when isotropic tumbling is assumed. Moreover, there is a systematic change in S², as large as 0.08 for some residues. The significant effects of anisotropic rotational diffusion on model-free motional parameters are in marked contrast to previous studies and are accentuated by lowering of the effective correlation time using isotropic tumbling methods. This is caused by the preponderance of NH vectors aligned perpendicular to the principal diffusion tensor axis and is readily detected because of the high quality of the relaxation data. A novel procedure, COPED (COmparison of Predicted and Experimental Diffusion tensors) is presented for distinguishing genuine motions from the effects of anisotropy by comparing experimental relaxation data and data predicted from hydrodynamic analyses. The procedure shows excellent agreement with the slow motions detected from the axially symmetric model-free analysis and represents an independent procedure for determining rotational diffusion and slow motions that can confirm or refute established procedures that rely on relaxation data. Our findings show that neglect of even small degrees of rotational diffusion anisotropy can introduce significant errors in model-free analysis when the data is of high quality. These errors can hinder our understanding of the role of internal motions in protein function.     

Decreased rotational diffusion of band 3 in melanesian ovalocytes from Papua,New Guinea

Summary Melanesian ovalocytes from Papua New Guinea have an N-terminal extension of the band 3 polypeptide (Jones, G.L., Edmunson. H.M., Wesche, D., Saul, A. 1990.Biochim. Biophys. Acta 1096:33–40). The ovalocytes showed a threefold increase in shear elastic modulus as determined by micropipette aspiration measurements of membrane rigidity. Time-resolved phosphorescence anisotropy has been used to study the rotational freedom of band 3 in membranes prepared from ovalocytes. The ovalocytic polymorphism was found to be associated with a marked decrease in the rotational mobility of band 3. This may indicate participation of band 3 in large homoaggregates or in complexes with other proteins at the cytoplasmic surface. There was no morphological clustering of band 3 detectable by immunofluorescence microscopy.     

Analysis of peptide rotational diffusion by homonuclear NMR

The analysis of the rotational diffusion of a molecule using homonuclear NMR is investigated. The homonuclear longitudinal and transverse cross-relaxation rates, which can be quantitatively measured using off-Resonance Rotating frame nuclear Overhauser Effect Spectroscopy (ROESY), are used to build a distribution, which exhibits a solid-state-like pattern characteristic of the diffusion tensor. The distributions of the antimicrobial peptide ranalexin in water and in 30% of trifluoracetic acid (TFE) are compared, and the peptide rotational diffusion is shown to be more isotropic in water than in 30% TFE. This difference is further supported by the analysis of NMR ranalexin conformers in 30% TFE, and by the analysis of a molecular dynamics simulation of peptide in water.     

A graphical method for the analysis of anisotropic rotational diffusion in proteins

A graphical method for the analysis of relaxation data is presented. It allows a fast estimation of the range of values of the components of the axially symmetric rotational diffusion tensor that are compatible with the experimental relaxation data. The graphical method clearly shows the contribution of different experimental relaxation parameters to the measured anisotropy. In particular, for proteins with moderate anisotropy, data from at least two N-H bonds forming angles close to 0° and 90° with respect to the principal axis of the rotational diffusional tensor are needed. For very anisotropic systems, combination of different relaxation parameters from a single residue is enough to characterize the local anisotropy.     

The size of the lactose permease derived from rotational diffusion measurements.

The lactose permease of Escherichia coli was labeled with eosinyl-maleimide, reconstituted into vesicles of dimyristoylphosphatidylcholine and subjected to time-dependent phosphorescence anisotropy measurements in order to determine the rotational diffusion coefficient. By comparison with bacteriorhodopsin, the diffusion coefficient is evaluated in terms of an effective radius of the lactose permease in the plane of the membrane. This radius amounts to 20 +/- 2 A which implies that the lactose permease is a monomer. The monomeric state is maintained in the presence of a membrane potential.     

Characterization of the overall and local dynamics of a protein with intermediate rotational anisotropy: Differentiating between conformational exchange and anisotropic diffusion in the B3 domain of protein G

Because the overall tumbling provides a major contribution to protein spectral densities measured in solution, the choice of a proper model for this motion is critical for accurate analysis of protein dynamics. Here we study the overall and backbone dynamics of the B3 domain of protein G using ¹⁵N relaxation measurements and show that the picture of local motions is markedly dependent on the model of overall tumbling. The main difference is in the interpretation of the elevated R ₂ values in the -helix: the isotropic model results in conformational exchange throughout the entire helix, whereas no exchange is predicted by anisotropic models that place the longitudinal axis of diffusion tensor almost parallel to the helix axis. Due to small size (fast tumbling) of the protein, the T ₁ values have low sensitivity to NH bond orientation. The diffusion tensor derived from orientation dependence of R ₂/R ₁ is anisotropic (D _par/D _perp=1.4), with a small rhombic component. In order to distinguish the correct picture of motion, we apply model-independent methods that are sensitive to conformational exchange and do not require knowledge of protein structure or assumptions about its dynamics. A comparison of the CSA/dipolar cross-correlation rate constants with ¹⁵N relaxation rates and the estimation of R _ex terms from relaxation data at 9.4 and 14.1 T indicate no conformational exchange in the helix, in support of the anisotropic models. The experimentally derived diffusion tensor is in excellent agreement with theoretical predictions from hydrodynamic calculations; a detailed comparison with various hydrodynamic models revealed optimal parameters for hydrodynamic calculations.     

Fluorescence correlation spectroscopy in the nanosecond time range: rotational diffusion of bovine carbonic anhydrase B

A fluorescence correlation experiment for measurement of rotational diffusion in the nanosecond time scale is described. Using this method, the rotational diffusion coefficient of bovine carbonic anhydrase B labelled with tetramethylrhodamine isothiocyanate was estimated to be D _r=(1.14±0.15)×10⁷ s^-1 at 22°C. The experiment is based on a cw argon ion laser, a microfluorimeter with local solution flow inside the sample cell, and two photon detectors. The fluorescence intensity autocorrelation function in the nanosecond time range is computed with the help of a time-to-amplitude converter and a multichannel pulse-amplitude analyser.     

Observing membrane protein diffusion at subnanometer resolution

Single sodium-driven rotors from a bacterial ATP synthase were embedded into a lipid membrane and observed in buffer solution at subnanometer resolution using atomic force microscopy (AFM). Time-lapse AFM topographs show the movement of single proteins within the membrane. Subsequent analysis of their individual trajectories, in consideration of the environment surrounding the moving protein, allow principal modes of the protein motion to be distinguished. Within one trajectory, individual proteins can undergo movements assigned to free as well as to obstacled diffusion. The diffusion constants of these two modes of motion are considerably different. Without the structural information about the membrane environment restricting the moving proteins, it would not be possible to reveal insight into these mechanisms. The high-resolution AFM topographs suggest that, in future studies, such data revealed under various physiological conditions will provide novel insights into molecular mechanisms that drive membrane protein assembly and supply excellent boundary conditions to model protein-protein arrangements.     

The smoking epidemic across generations,genders, and educational groups: A matter of diffusion of innovations

This study determines whether the temporal variations in smoking habits across generations and genders and among groups with differing levels of education fit the pattern proposed by the theory of the diffusion of innovations (TDI) (Rogers, 2003). We focus on the Italian case and employ a pseudo-panel derived from repeated cross-sections of the annual household survey, “Aspects of Daily Life,” that was part of the Multipurpose Survey carried out by the Italian National Statistical Office (ISTAT) for the period 1997 to 2012. The results confirm Rogers’ TDI and show that smoking prevalence has declined over time and across age cohorts: Younger men of all educational levels and women with higher education are less likely to smoke than are those in other cohorts, while less-educated women who entered the smoking-diffusion process later than others are more likely to smoke. Hence, socio-economic differences in smoking continue to persist, especially for women. According to Rogers’ TDI, smoking prevalence is expected to continue to decline, particularly among little-educated women.     

Mobility of NH bonds in DNA-binding protein HU of shape Bacillus stearothermophilus from reduced spectral density mapping analysis at multiple NMR fields

The dynamics of the backbone NH bonds of protein HU from Bacillus stearothermophilus (HUBst) have been characterized using measurements of cross-relaxation, longitudinal and transverse relaxation rates at 11.7, 14.1 and 17.6 T. Linear regression of the values with the squared Larmor frequency _N ² has revealed global exchange processes, which contributed on the order of 0.5–5.0 s^-1to the transverse relaxation rate. Subsequently, the experimental values were corrected for these exchange contributions. A reduced spectral density mapping procedure has been employed with the experimental relaxation rates and seven values of the spectral density function J() have been extracted. These spectral densities have been fitted within the framework of the model-free approach. The densities agree well with an axially symmetric rotational diffusion tensor with a diffusion anisotropy D_/D_ of 1.15, indicating that the flexible arms of HUBst do not significantly contribute to the rotational diffusion. The overall correlation time is 8.9 ± 0.6 ns/rad. The fast internal motions of most of the NH bonds in the core display order parameters ranging between 0.74 and 0.83 and internal correlation times between 1 and 20 ps. For the residues in the DNA-binding -arms, an extended version of the model function has been used. The slow internal motions show correlation times of 1–2 ns. The concomitant order parameters (0.3–0.6) are lower than those observed on the fast time scale, indicating that the flexibility of the -arms is mainly determined by the slower internal motions. A substantial decrease of the generalized order parameters in the -arms starting at residues Arg⁵⁵ and Ser⁷⁴, opposite on both strands of the -ribbon arms, has been explained as a hinge motion. A comparison of the order parameters for free and DNA-bound protein has demonstrated that the slow hinge motions largely disappear when HU binds DNA.     

Optimisation of NMR dynamic models II. A new methodology for the dual optimisation of the model-free parameters and the Brownian rotational diffusion tensor

Finding the dynamics of an entire macromolecule is a complex problem as the model-free parameter values are intricately linked to the Brownian rotational diffusion of the molecule, mathematically through the autocorrelation function of the motion and statistically through model selection. The solution to this problem was formulated using set theory as an element of the universal set —the union of all model-free spaces (d’Auvergne EJ and Gooley PR (2007) Mol BioSyst 3(7), 483–494). The current procedure commonly used to find the universal solution is to initially estimate the diffusion tensor parameters, to optimise the model-free parameters of numerous models, and then to choose the best model via model selection. The global model is then optimised and the procedure repeated until convergence. In this paper a new methodology is presented which takes a different approach to this diffusion seeded model-free paradigm. Rather than starting with the diffusion tensor this iterative protocol begins by optimising the model-free parameters in the absence of any global model parameters, selecting between all the model-free models, and finally optimising the diffusion tensor. The new model-free optimisation protocol will be validated using synthetic data from Schurr JM et al. (1994) J Magn Reson B 105(3), 211–224 and the relaxation data of the bacteriorhodopsin (1–36)BR fragment from Orekhov VY (1999) J Biomol NMR 14(4), 345–356. To demonstrate the importance of this new procedure the NMR relaxation data of the Olfactory Marker Protein (OMP) of Gitti R et al. (2005) Biochem 44(28), 9673–9679 is reanalysed. The result is that the dynamics for certain secondary structural elements is very different from those originally reported. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The interaction of water molecules with purple membrane suspension using 2H double-quantum filter, 1H and 2H diffusion nuclear magnetic resonance

Bacteriorhodopsin is a membrane protein of the purple membrane (PM) of Halobacterium salinarum, which is isolated as sheets of highly organized two-dimensional hexagonal microcrystals and for which water molecules play a crucial role that affects its function as a proton pump. In this paper we used single- and double-quantum (2)H NMR as well as (1)H and (2)H diffusion NMR to characterize the interaction of water molecules with the PM in D(2)O suspensions. We found that, under the influence of a strong magnetic field on a concentrated PM sample (0.61 mM), the PM sheets affect the entire water population and a residual quadrupolar splitting (upsilon(q) approximately 5.5 Hz, 298 K, at 11.7 T) is observed for the D(2)O molecules. We found that the residual quadrupolar coupling, the creation time in which a maximal DQF signal was obtained (tau(max)), and the relative intensity of the (2)H DQF spectrum of the water molecules in the PM samples (referred to herein as NMR order parameters) are very sensitive to temperature, dilution, and chemical modifications of the PM. In concentrated PM samples in D(2)O, these NMR parameters seem to reflect the relative organization of the PM. Interestingly, we have observed that some of these parameters are sensitive to the efficiency of the trimer packing, as concluded from the apo-membrane behavior. The data for dionized blue membrane, partially delipidated sample, and detergent-treated PM show that these D(2)O NMR order parameters, which are magnetic field dependent, are sensitive to the structural integrity of the PM. In addition, we revealed that heating the PM sample inside or outside the NMR magnet has, after cooling, a different effect on the NMR characteristics of the water molecules in the concentrated PM suspensions. The difference in the D(2)O NMR order parameters for the PM samples, which were heated and cooled in the presence and in the absence of a strong magnetic field, corroborates the conclusions that the above D(2)O order parameters are indirect reflections of both microscopic and macroscopic order of the PM samples. In addition, (1)H NMR diffusion measurements showed that at least three distinct water populations could be identified, based on their diffusion coefficients. These water populations seem to correlate with different water populations previously reported for the PM system.     

Solution structure determination of the two DNA-binding domains in the Schizosaccharomyces pombe Abp1 protein by a combination of dipolar coupling and diffusion anisotropy restraints

We have solved the solution structure of the N-terminal region of the fission yeast centromere protein, Abp1, bound to a 21-base pair DNA fragment bearing its recognition site (Mw = 30 kDa). Although the two DNA-binding domains in the Abp1 protein were defined well by a conventional NOE-based NMR methodology, the overall structure of the Abp1 protein was poorly defined, due to the lack of interdomain distance restraints. Therefore, we additionally used residual dipolar couplings measured in a weakly aligned state, and rotational diffusion anisotropies. Neither the NH residual dipolar couplings nor the backbone ¹⁵N T ₁/T ₂ data were sufficient to determine the overall structure of the Abp1 protein, due to spectral overlap. We used a combination of these two orientational restraints (residual dipolar coupling and rotational diffusion anisotropy), which significantly improved the convergence of the overall structures. The range of the observed T ₁/T ₂ ratios was wider (20–50 for the secondary structure regions of Abp1) than the previously reported data for several globular proteins, indicating that the overall shape of the Abp1DNA complex is ellipsoid. This extended form would facilitate the recognition of the two separate sites in the relatively long DNA sequence by the DNA-binding domains of Apb1.     

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.     

Estimation of the shape and size of fribrinogen in solution from its hydrodynamic properties using theories for bead models and cylinders

The translational and rotational diffusion coefficients and the intrinsic viscosity of fibrinogen in solution are used to estimate its size, shape and hydration. Experimental data of the three hydrodynamic properties taken from the literature are compared with theoretical predictions for several molecular geometries that have been observed by electron microscopy. Modern theories for the hydrodynamics of bead models and cylindrical particles are employed in the calculations. The discrepancy between experimental results and theoretical predictions for spherical particles rules out the dodecahedral model and indicates that fibrinogen is elongated. The Hall-Slayter nodular model and its refinements perform better but still underestimate the size of the hydrated molecule. The best agreement between theoretical and experimental values is found for a cylindrical particle with length and diameter of about 48 and 6.8 nm, respectively. The hydration is calculated to be 3 g water/g protein. We speculate that, to accommodate such a large amount of water, fibrinogen in solution should be appreciably hydrated.     

The influence of melittin on the rotation of band 3 protein in the human erythrocyte membrane

The rotational mobility of band 3, a protein constituent of the human erythrocyte membrane, was measured by observing the flash-induced transient dichroism of the triplet probe eosin maleimide. In the presence of melittin, a pharmacologically active polypeptide from honey bee (Apis mellifera) venom, a dose-dependent loss of rotational mobility was detected. With acetylated melittin, the ability to immobilise is reduced. Succinylated melittin, however, is devoid of immobilising activity.The possible relevance of these findings to the normal mode of action of melittin was examined by comparing the relative abilities of the native, acetylated and succinylated melittins to lyse erythrocytes and synergise with phospholipase A₂, another constituent of bee venom. For both these properties, the order of effectiveness is native melittin > acetyl melittin > succinyl melittin = 0, the same as their order of effectiveness in immobilising band 3.A mechanism is proposed in which melittin is anchored in the membrane by its hydrophobic N-terminus, while its cationic C-terminal moiety binds to negatively charged residues on membrane proteins. This leads either directly or indirectly to protein aggregation and hence loss of mobility. From a detailed comparison of the different effects of the melittin derivatives, it is concluded that melittin may function in vivo by aggregating membrane proteins in order to allow phospholipase A₂ to gain access to the membrane bilayer and commence catalysis.