Conventional and saturation transfer EPR spectroscopy of Na+/K(+)-ATPase modified with different maleimide-nitroxide derivatives.

The membranous Na+/K(+)-ATPase from Squalus acanthias has been covalently modified on either Class I or Class II sulphydryl groups using derivatives of 3-(maleimidomethyl)-1-oxyl-2,2,5,5-tetramethylpyrrolidine with substituents of different charge and hydrophobicity attached at the remaining unsubstituted position of the pyrrolidine ring. The substituent groups used were a methyl and a hexyl ester, and di- and tri-methylammonium ethyl esters, as well as the parent underivatized compound. Additionally, another series of maleimide-nitroxides differing (by zero to seven intervening atoms) in the length of the linking group between the maleimide and the pyrrolidine moieties was used. The sites of attachment have been characterized in terms of the rotational mobility and environmental polarity by using conventional and saturation transfer EPR spectroscopy of these spin-labelled reagents. This provides a further sub-classification of the primary Class I and Class II SH-groups on the alpha-subunit of the enzyme, which differ both in their reactivity and influence on the Na+/K(+)-ATPase activity.     

Solution conformation of lactate dehydrogenase as studied by saturation transfer ESR spectroscopy.

Several binary and ternary inhibitor and 'dead end' complexes of pig heart lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) were studied by saturation transfer ESR spectroscopy by means of an active NAD analog, spin-labeled at N6. The mobility of the spin-label depends on the nature of small molecules bound at the remote catalytic end of the coenzyme. The spin-label was found to serve as a reporter group monitoring the conformation of the peptide loop that is folded down over the active cleft in crystals of ternary complexes. The data suggest a fluctuation of the loop between open and closed forms in solution. The structure of the inhibitor molecules has been correlated with their ability to stabilize a more closed conformation of the loop.     

Measurement of rotational molecular motion by time-resolved saturation transfer electron paramagnetic resonance.

We have used saturation-recovery electron paramagnetic resonance (SR-EPR), a time-resolved saturation transfer EPR technique, to measure directly the microsecond rotational diffusion of spin-labeled proteins. SR-EPR uses an intense microwave pulse to saturate a spin population having narrow distribution of orientations with respect to the magnetic field. The time evolution of the signal is then observed. The signal increases in time as saturation is relieved by spin-lattice relaxation (Tl) as well as by saturation transfer due to spectral diffusion (Tsd), which is a function of rotational diffusion (Tr) and spectral position. In the presence of both events, the recovery is biphasic, with the initial phase related to both Tr and Tl, and the second phase determined only by Tl. We have measured the saturation recoveries of spin-labeled hemoglobin tumbling in media of known viscosities as a function of rotational correlation time (Tr) and pulse duration (tp). The Tr values estimated from the initial phase of recovery were in good agreement with theory. Variation of the pulse time can also be used to determine Tr. For tp less than Tsd, the recoveries were observed to be biphasic, for tp greater than Tsd a single-exponential. T1 values were determined from the recoveries after pulses quenching spectral diffusion or from the second phase of recovery after shorter pulses. These results demonstrate that SR-EPR is applicable to the study of motion of spin-labeled proteins. Its time resolution should provide a significant advantage over steady state techniques, particularly in the case of motional anisotropy or system heterogeneity.     

Dynamics of tropomyosin in muscle fibers as monitored by saturation transfer EPR of bi-functional probe

The dynamics of four regions of tropomyosin was assessed using saturation transfer electron paramagnetic resonance in the muscle fiber. In order to fully immobilize the spin probe on the surface of tropomyosin, a bi-functional spin label was attached to i,i+4 positions via cysteine mutagenesis. The dynamics of bi-functionally labeled tropomyosin mutants decreased by three orders of magnitude when reconstituted into "ghost muscle fibers". The rates of motion varied along the length of tropomyosin with the C-terminus position 268/272 being one order of magnitude slower then N-terminal domain or the center of the molecule. Introduction of troponin decreases the dynamics of all four sites in the muscle fiber, but there was no significant effect upon addition of calcium or myosin subfragment-1.     

Deuteron field-cycling relaxation spectroscopy and translational water diffusion in protein hydration shells.

The deuterated hydration shells of bovine serum (BSA) albumin, and purple membrane sheets have been studied by the aid of deuteron field-cycling relaxation spectroscopy. The deuteron Larmor frequency range was 10(3) to 10(8) Hz. The temperature and the water content has been varied. The data distinguish translational diffusion on the protein surface from macromolecular tumbling or exchange with free water. A theory well describing all dependences has been developed on this basis. All parameters have successfully been tested concerning consistency with other sources of information. The concept is considered as a major relaxation scheme determining, apart from cross-relaxation effects, the water proton relaxation in tissue.     

Exchange rates at the lipid-protein interface of the myelin proteolipid protein determined by saturation transfer electron spin resonance and continuous wave saturation studies.

The microwave saturation properties of various spin-labeled lipids in reconstituted complexes of the myelin proteolipid protein with dimyristoyl phosphatidylcholine have been studied both by conventional and saturation transfer electron spin resonance (ESR) spectroscopy. In the fluid phase, the conventional ESR spectra consist of a fluid and a motionally restricted (i.e., protein-associated) component, whose relative proportions can be determined by spectral subtractions and depend on the selectivity of the particular spin-labeled lipid for the protein. At 4 degrees C when the bulk lipid is in the gel phase, the integrated intensity of the saturation transfer ESR spectra displays a linear dependence on the fraction of motionally restricted lipid that is deduced from the conventional ESR spectra in the fluid phase, indicating the presence of distinct populations of free and protein-interacting lipid with no exchange between them on the saturation transfer ESR time scale in the gel phase. At 30 degrees C when the bulk lipid is in the fluid phase, the saturation transfer integral displays a nonlinear dependence on the fraction of motionally restricted lipid, consistent with exchange between the two lipid populations on the saturation transfer ESR time scale in the fluid phase. For lipid spin labels with different selectivities for the protein in complexes of fixed lipid/protein ratio, the data in the fluid phase are consistent with a constant (diffusion-controlled) on-rate for exchange at the lipid-protein interface. Values ranging between 1 and 9 x 10(6) s-1 are estimated for the intrinsic off-rates for exchange of spin-labeled stearic acid and phosphatidylcholine, respectively, at 30 degrees C. Conventional continuous wave saturation experiments lead to similar conclusions regarding the lipid exchange rates in the fluid and gel phases of the lipid/protein recombinants. The ESR saturation studies therefore demonstrate exchange on the time scale of the nitroxide spin-lattice relaxation at the lipid-protein interface of myelin proteolipid/dimyristoyl phosphatidylcholine complexes in the fluid phase but not in the gel phase.     

Translational diffusion of fluorescent proteins by molecular fourier imaging correlation spectroscopy

The ability to noninvasively observe translational diffusion of proteins and protein complexes is important to many biophysical problems. We report high signal/noise (>or=250) measurements of the translational diffusion in viscous solution of the fluorescent protein, DsRed. This is carried out using a new technique: molecular Fourier imaging correlation spectroscopy (M-FICS). M-FICS is an interferometric method that detects a collective Fourier component of the fluctuating density of a small population of fluorescent molecules, and provides information about the distribution of molecular diffusivities. A theoretical analysis is presented that expresses the detected signal fluctuations in terms of the relevant time-correlation functions for molecular translational diffusion. Furthermore, the role played by optical orientational degrees of freedom is established. We report Fickian self-diffusion of the DsRed tetramer at short timescales. The long-time deviation of our data from Fickian behavior is used to determine the variance of the distribution of the protein self-diffusion coefficient. We compare our results to the expected outcomes for 1), a bi-disperse distribution of protein species, and 2), dynamic disorder of the host solvent.     

Accessibility of nitroxide side chains: absolute Heisenberg exchange rates from power saturation EPR

In site-directed spin labeling, the relative solvent accessibility of spin-labeled side chains is taken to be proportional to the Heisenberg exchange rate (W(ex)) of the nitroxide with a paramagnetic reagent in solution. In turn, relative values of W(ex) are determined by continuous wave power saturation methods and expressed as a proportional and dimensionless parameter Pi. In the experiments presented here, NiEDDA is characterized as a paramagnetic reagent for solvent accessibility studies, and it is shown that absolute values of W(ex) can be determined from Pi, and that the proportionality constant relating them is independent of the paramagnetic reagent and mobility of the nitroxide. Based on absolute exchange rates, an accessibility factor is defined (0 < rho < 1) that serves as a quantitative measure of side-chain solvent accessibility. The accessibility factors for a nitroxide side chain at 14 different sites in T4 lysozyme are shown to correlate with a structure-based accessibility parameter derived from the crystal structure of the protein. These results provide a useful means for relating crystallographic and site-directed spin labeling data, and hence comparing crystal and solution structures.     

Saturation transfer EPR spectroscopy on spin-labeled muscle fibers using a loop-gap resonator.

Previously, saturation transfer (ST-EPR) studies of biomolecular dynamics have involved the use of a resonant cavity and the V'2 display (absorption, second harmonic, out of phase). In the present study, we replaced the resonant cavity with a loop-gap resonator and used the U'1 display (dispersion, first harmonic, out of phase) to study spin-labeled muscle fibers. The new resonator and display showed several advantages over those previously used. It produced virtually noiseless U'1 spectra on a 0.4 microliter sample using a 4 min scan; previous U'1 experiments on spin-labeled muscle, using a conventional rectangular cavity, resulted in an unacceptably low signal-to-noise ratio. The high filling factor of the resonator facilitated the study of these extremely small fiber bundles and permitted high microwave field intensities to be achieved at much lower incident microwave power levels, thus greatly enhancing the signal-to-noise ratio in U'1 experiments. This reduction in the noise level made it possible to benefit from the other advantages of U'1 over V'2, such as stronger signals, simpler line shapes, and simpler data analysis. For these muscle fiber samples, the resulting sensitivity (signal/noise/sample volume) of the U'1 signals was greater than 100 times that of V'2 signals obtained in a conventional cavity. Another advantage of the U'1 display is that signals from weakly immobilized probes, i.e., probes that have nanosecond rotational mobility relative to the labeled protein (myosin), are greatly suppressed relative to strongly immobilized probes. This reduces the ambiguity of spectral analysis, and eliminates the need for chemical treatments [e.g., using K3Fe(CN)6] that were previously required in muscle fibers and other systems. Further suppression of this weakly immobilized component was achieved in U'1 spectra by increasing the microwave power and decreasing the field modulation frequency.     

Measurement of proton relaxation rates in proteins

Summary Five different types of experiment are described which make it possible to measure various relaxation rates of selected protons in crowded spectra of macromolecules such as proteins: longitudinal spin-lattice relaxation rates =1/T₁, transverse relaxation rates =1/T₂ measured under conditions of free precession, transverse relaxation rates ¹ _LOCK=1/T₁ measured under conditions of spin-locking, and transverse relaxation rates ^DQC=1/T₂ ^DQC and ^ZQC=1/T₂ ^ZQC of double- and zero-quantum coherences. The surprisingly large discrepancy between the transverse rates ^t and ^t is discussed in detail. To separate overlapping proton signals, the experimental schemes involve one or several magnetization transfer steps, using a doubly selective homonuclear Hartmann-Hahn method. Numerous variants of the basic ideas can be conceived, depending on the extent of signal overlap and on the topology of the networks of scalar couplings. Applications are shown to H and H of Tyr²³, to H, H and H of Cys³⁰, and to H and H of Ala²⁴ in bovine pancreatic trypsin inhibitor (BPTI).     

Lecithin translational diffusion studied by pulsed nuclear magnetic resonance.

The translational diffusion coefficient of egg yolk and dilauroyl lecithin in optically isotropic phases containing sodium cholate has been measured using the pulsed NMR magnetic field gradient method. After a correction for geometrical factors the measured diffusion coefficient is found to agree well with previous determinations in phospholipid systems. The experimental data imply that the cubic mesophase of the lecithin-sodium cholate-water system contains continuous lipid aggregates. A possible model of the arrangement of the different amphiphile molecules in the cubic phase is discussed.     

Measurement of local diffusion coefficients in biofilms by microinjection and confocal microscopy

A new technique for the determination of local diffusion coefficients in biofilms is described. It is based on the microinjection of fluorescent dyes and quantitative analysis of the subsequent plume formation using confocal laser microscopy. The diffusion coefficients of fluorescein (MW 332), TRITC-IgG (MW 150000) and phycoerythrin (MW 240000) were measured in the cell clusters and interstitial voids of a heterogeneous biofilm. The diffusivities measured in the voids were close to the theoretical values in water. Fluorescein had the same diffusivity in cell clusters, voids, and sterile medium. TRITC-IgG did not diffuse in cell clusters, presumably due to binding to the cell cluster matrix. After treatment of the biofilm with bovine serum albumin, binding capacity decreased and the diffusion coefficient could be measured. The diffusivity of phycoerythrin in cell clusters was impeded by 41%, compared to interstitial voids. From the diffusion data of phycoerythrin it was further calculated that the cell cluster matrix had the characteristics of a gel with 0.6 nm thick fibers and pore diameters of 80 nm. (c) 1997 John Wiley & Sons, Inc.     

Two dimensional diffusion of small molecules on protein surfaces: an EPR study of the restricted translational diffusion of protein-bound spin labels

Heisenberg spin exchange rates and dipole-dipole spin lattice relaxation rates for deuterated ¹⁴N- and ¹⁵N-spin labels bound selectively to the histidine His15 and to the lysines Lys13, 96, 97 of the lysozyme molecule have been determined with the aid of electron spin resonance spectroscopy. The results can be interpreted in terms of a two dimensional translational diffusion of the nitroxide tips of the spin labels along the protein surface within restricted surface areas. The spin labels are regarded as models for long amino acid side chains and as probes for the dynamics of protein and water in the vicinity of the protein surface. The translational diffusion coefficient DP_II is reduced by a factor of between six and thirty compared to the value of D found for the spin labels in bulk water, its value for T = 295 K is given by (1.3±0.6)·10^–10m²s^–1 D (2.4±0.3) 10^–11 m²s^–1. Offprint requests to: H.-J. Steinhoff     

Determination of the electron self-exchange rates of blue copper proteins by super-WEFT NMR spectroscopy

An NMR approach for determining the electron self-exchange (ESE) rate constants in blue copper proteins is presented. The approach uses the paramagnetic relaxation enhancement of resonances in 1D ¹H super-WEFT spectra of partly oxidized (paramagnetic) proteins. These spectra allow a more precise determination of the relevant paramagnetic linebroadenings than conventional 1D ¹H spectra and, thus, permit a more detailed investigation of the applicability of the linebroadenings for determining the electron exchange rates. The approach was used to estimate the ESE rate constant of plastocyanin from Anabaena variabilis. It was found that, although the rate constant can be determined accurately from a series of resonances, precise but erroneous constants are obtained from the resonances of the copper-bound residues, unless a narrow splitting of these resonances caused by the presence of two conformations is taken into account. As demonstrated here, this complication can be overcome by a correct analysis of the paramagnetic broadening of the combined double signals. Because of the high resolution and specific sensitivity of the approach it should be generally applicable to estimate electron transfer rates, k, if the paramagnetic relaxation enhancement R _2p of the resonances can be determined, and the conditions kR _2p or _pkR _2p are fulfilled, _p being the frequency separation between corresponding diamagnetic and paramagnetic sites.     

Measurement of local mass transfer coefficient in biofilms

Local mass transfer rates for an electrochemically formed microsink in an aerobic biofilm was measured by a mobile microelectrode using limiting current technique. Mass transfer coefficients varied both horizontally and vertically in the biofilm. The results implied the existence of an irregular biofilm structure consisting of microbial cell clusters surrounded by tortuous water channels. An unexpected increase of the local mass transfer coefficient just above the biofilm surface suggested the existence, of local flow instability in this region. As expected, the influence of bulk flow velocity on the local mass transfer rate decreased with increasing depth into the biofilm. Mass transfer coefficients fluctuated significantly inside microbial cell clusters, suggesting the existence of internal channels through which liquid could flow. A new conceptual model of biofilm microbial cluster structure is proposed to account for such biofilm microstructure irregularities. (c) 1995 John Wiley & Sons, Inc.     

The sensitivity of saturation transfer electron paramagnetic resonance spectra to restricted amplitude uniaxial rotational diffusion.

Computational methods have been developed to model the effects of constrained or restricted amplitude uniaxial rotational diffusion (URD) on saturation transfer electron paramagnetic resonance (ST-EPR) signals observed from nitroxide spin labels. These methods, which have been developed to model the global rotational motion of intrinsic membrane proteins that can interact with the cytoskeleton or other peripheral proteins, are an extension of previous work that described computationally efficient algorithms for calculating ST-EPR spectra for unconstrained URD (Hustedt and Beth, 1995, Biophys. J. 69:1409-1423). Calculations are presented that demonstrate the dependence of the ST-EPR signal (V'(2)) on the width (Delta) of a square-well potential as a function of the microwave frequency, the correlation time for URD, and the orientation of the spin-label with respect to the URD axis. At a correlation time of 10 micros, the V'(2) signal is very sensitive to Delta in the range from 0 to 60 degrees, marginally sensitive from 60 degrees to 90 degrees, and insensitive beyond 90 degrees. Sensitivity to Delta depends on the correlation time for URD with higher sensitivity to large values of Delta at the shorter correlation times, on the microwave frequency, and on the orientation of the spin-label relative to the URD axis. The computational algorithm has been incorporated into a global nonlinear least-squares analysis approach, based upon the Marquardt-Levenberg method (Blackman et al., 2001, Biophys. J. 81:3363-3376). This has permitted determination of the correlation time for URD and the width of the square-well potential by automated fitting of experimental ST-EPR data sets obtained from a spin-labeled membrane protein and provided a new automated method for analysis of data obtained from any system that exhibits restricted amplitude URD.