Spin-label studies of the lipid and protein components of erythrocyte membranes. A comparison of electron paramagnetic resonance and saturation transfer electron paramagnetic resonance methods.

We have used both a protein spin label and a lipid spin probe to study some of the slow motions of proteins and of lipids, respectively, in intact erythrocyte membranes. Three electron paramagnetic resonance (EPR) methods, conventional (V1) EPR, second harmonic out-of-phase absorption saturation transfer (ST) EPR (V'2), and first harmonic out-of-phase dispersion ST EPR (U'1) were used to compare the experimental methods and spectral sensitivities with different kinds of molecular motions in human erythrocyte membranes under different experimental conditions. The results show that the V'2 display is relatively more sensitive to the protein motion, while the U'1 display appears more sensitive to the lipid motions, and the V'2 display is substantially more convenient to obtain than the U'1 display.     

A spin label study on human low density lipoprotein

Selective labeling with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-maleimide of human serum LDL has been performed. The spin-labeled LDL exhibited an ESR spectrum containing signals of a strongly immobilized component only. The signals were completely reversible between 4 degrees C and 37 degrees C and fairly stable at each temperature. The spin-labeled LDL which was prepared by the usual method exhibited an ESR spectrum containing signals of both strongly immobilized and weakly immobilized components (5, 6). The latter was unstable above 25 degrees C and changed irreversibly. The strongly binding site showed higher affinity for the nitroxide radical than the weakly binding site, and two kinds of the strongly binding site were demonstrated kinetically. The rate of binding of the nitroxide radical to the two kinds of strongly binding site were estimated to be 4.7 x 10(4) M-1 . day-1 and 0.16 x 10(4) M-1 . day-1 at pH 7.4 and 4 degrees C, respectively. Both the strongly immobilized and weakly immobilized radicals were reduced with ascorbate at the same rate. It was also shown on gel filtration of the SDS-treated LDL derivatives that the strongly immobilized component was on the apoprotein B moiety, whereas either noncovalent binding to LDL or binding to some small molecular species other than protein was suggested for the weakly immobilized component.     

Saturation transfer electron paramagnetic resonance of spin-labeled muscle fibers. Dependence of myosin head rotational motion on sarcomere length

We have investigated the orientation and rotational mobility of spin-labeled myosin heads in muscle fibers as a function of the sarcomere length in the absence of ATP. An iodoacetamide spin label was used to label selectively two-thirds of the sulfhydryl-1 groups in glycerinated rabbit psoas muscle. Conventional electron paramagnetic resonance experiments were used to determine the orientation distribution of the probes relative to the fiber axis, and saturation transfer experiments were used to detect sub-millisecond rotational motion. When fibers are at sarcomere length 2.3 microns (full overlap), spin-labeled heads have a high degree of orientational order. The probes are in a single, narrow orientation distribution (full width 15 degrees), and they exhibit no detectable sub-millisecond rotational motion. When fibers are stretched (sarcomere length increased), either before or after labeling, disorder and microsecond mobility increase greatly, in proportion to the fraction of myosin heads that are no longer in the overlap zone between the thick and thin filaments. Saturation transfer difference spectra show that a fraction of myosin heads equal to the fraction outside the overlap zone have much more rotational mobility than those in fibers at full overlap, and almost as much as in synthetic myosin filaments. The most likely interpretation is that some of the probes, corresponding approximately to the fraction of heads in the overlap zone, remain oriented and immobile, while the rest are highly disordered (angular spread greater than 90 degrees) and mobile (microsecond rotational motion). Thus, it appears that myosin heads are rigidly immobilized by actin, but they rotate through large angles on the microsecond time-scale when detached from actin, even in the absence of ATP.     

A spin-label study on human high density lipoprotein

Human plasma high density lipoproteins (HDL) have been labeled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (NEM-TEMPO). The spin-labeled HDL exhibited an ESR spectrum containing signals of both strongly immobilized and weakly immobilized components by the reaction with a high concentration of NEM-TEMPO, while an ESR spectrum containing only signals of a strongly immobilized component range between 4 degrees C and 37 degrees C, the signal height of the strongly immobilized component exhibited reversible temperature-dependent changes, whereas that of the weakly immobilized component changed irreversibly at temperatures above 25 degrees C. The activation energy of the irreversible change was estimated to be 26 kcal per mol. The strongly immobilized component was derived from NEM-TEMPO which modified apolipoprotein A-I covalently, while the weakly immobilized component was derived from NEM-TEMPO noncovalently bound to HDL. The rate of binding of NEM-TEMPO to either the strongly binding or weakly binding sites and the number of the strongly binding sites in apolipoprotein A-I were estimated to be 125 M-1.day-1 and 1.78, respectively. The binding of NEM-TEMPO to the strongly binding sites was suppressed greatly by pretreatment of HDL with 2,4,6-trinitrobenzene sulfonic acid (TNBS). The slow reaction and suppression with TNBS suggest that NEM-TEMPO binds to some amino acid residue, probably a lysine residue, in apoprotein A-I. The strongly immobilized and weakly immobilized components were reduced almost completely by ascorbate at the same rate, 0.048 min-1 at pH 7.4 and at 4 degrees C.     

Absence of nonlinear responses in cells and tissues exposed to RF energy at mobile phone frequencies using a doubly resonant cavity

A doubly resonant cavity was used to search for nonlinear radiofrequency (RF) energy conversion in a range of biological preparations, thereby testing the hypothesis that living tissue can demodulate RF carriers and generate baseband signals. The samples comprised high‐density cell suspensions (human lymphocytes and mouse bone marrow cells); adherent cells (IMR‐32 human neuroblastoma, G361 human melanoma, HF‐19 human fibroblasts, N2a murine neuroblastoma (differentiated and non‐differentiated) and Chinese hamster ovary (CHO) cells) and thin sections or slices of mouse tissues (brain, kidney, muscle, liver, spleen, testis, heart and diaphragm). Viable and non‐viable (heat killed or metabolically impaired) samples were tested. Over 500 cell and tissue samples were placed within the cavity, exposed to continuous wave (CW) fields at the resonant frequency (f) of the loaded cavity (near 883 MHz) using input powers of 0.1 or 1 mW, and monitored for second harmonic generation by inspection of the output at 2f. Unwanted signals were minimised using low pass filters (≤1 GHz) at the input to, and high pass filters (≥1 GHz) at the output from, the cavity. A tuned low noise amplifier allowed detection of second harmonic signals above a noise floor as low as −169 dBm. No consistent second harmonic of the incident CW signals was detected. Therefore, these results do not support the hypothesis that living cells can demodulate RF energy, since second harmonic generation is the necessary and sufficient condition for demodulation. Bioelectromagnetics 31:556–565, 2010. © 2010 Wiley‐Liss, Inc.     

Insect crossbridges, relaxed by spin-labeled nucleotide, show well-ordered 90 degrees state by X-ray diffraction and electron microscopy, but spectra of electron paramagnetic resonance probes report disorder.

The structure of glycerinated Lethocerus insect flight muscle fibers, relaxed by spin-labeled ATP and vanadate (Vi), was examined using X-ray diffraction, electron microscopy and electron paramagnetic resonance (e.p.r.) spectra. We obtained excellent relaxation of MgATP quality as determined by mechanical criteria, using vanadate trapping of 2' spin-labeled 3' deoxyATP at 3 degree C. In rigor fibers, when the diphosphate analog is bound in the absence of Vi, the probes on myosin heads are well-ordered, in agreement with electron microscopic and X-ray patterns showing that myosin heads are ordered when attached strongly to actin. In relaxed muscle, however, e.p.r. spectra report orientational disorder of bound (Vi-trapped) spin-labeled nucleotide, while electron microscopic and X-ray patterns both show well-ordered bridges at a uniform 90 degrees angle to the filament axis. The spin-labeled nucleotide orientation is highly disordered, but not completely isotropic; the slight anisotropy observed in probe spectra is consistent with a shift of approximately 10% of probes from angles close to 0 degrees to angles close to 90 degrees. Measurements of probe mobility suggest that the interaction between probe and protein remains as tight in relaxed fibers as in rigor, and thus that the disorder in relaxed fibers arises from disorders of (or within) the protein and not from disorder of the probe relative to the protein. Fixation of the relaxed fibers with glutaraldehyde did not alter any aspect of the spectrum of the Vi-trapped analog, including the slight order observed, showing that the extensive inter- and intra-molecular cross-linking of the first step of sample preparation for electron microscopy had not altered relaxed crossbridge orientations. Two models that may reconcile the apparently disparate results obtained on relaxed fibers are presented: (1) a rigid myosin head could possess considerable disorder in the regular array about the thick filament; or (2) the nucleotide site could be on a disordered, probably distal, domain of myosin, while a more proximal region is well ordered on the thick filament backbone. Our findings suggest that when e.p.r. probes signal disorder of a local site or domain, this is complementary, not contradictory, to signals of general order. The e.p.r. spectra show that a portion of the myosin molecule can be disordered at the same time as the X-ray diffraction and electron microscopy show the bulk of myosin head mass to be uniformly oriented and regularly arrayed.     

Preparation and characterization of spin-labeled oligonucleotides for DNA hybridization.

Sequence-specific spin-labeled oligodeoxynucleotides with conformation-sensitive electron paramagnetic resonance (EPR) signals are synthesized and examined as solution-phase nucleic acid hybridization probes. Either a proxyl or tempo ring linked to the C(5) position of deoxyuridine (dU) by a nonrigid two-atom methylamino tether is incorporated within 15-mers by phosphotriester chemistry yielding stable spin-labeled probes with distinctive EPR specific activity (AEPR) values. The AEPR is greater for a proxyl-labeled than for a tempo-labeled probe and is consistent with EPR data of enzymatically labeled 26-mers [Bobst, A. M., Pauly, G. T., Keyes, R. S., and Bobst, E. V. (1988) FEBS Lett. 228, 33-36], after normalizing for percent labeling. The spectral characteristics of the free probes and the probe/target complexes are similar to those of enzymatically spin-labeled nucleic acids containing a different nonrigid two-atom-tethered spin label [Bobst, A. M., Kao, S.-C., Toppin, R. C., Ireland, J. C., and Thomas, I. E. (1984) J. Mol. Biol. 173, 63-70]. The presence of target DNA is detected in solution by EPR spectroscopy and the assay is based on the characteristic line-shape change associated with hybridization. The EPR spectra of free and bound probe reflect little interference from changes in global dynamics of the probe, and the line-shape change upon complexation results primarily from a change in local base dynamics. The presence or absence of hybridization can be detected in a loop-gap resonator with about 1 pmol of spin-labeled 15-mer within minutes.     

Conformation of spin-labeled tropomyosin in reconstituted muscle thin filaments in response to calcium ion and heavy meromyosin

Tropomyosin (TM) exists in thermal equilibrium between a highly structured N state, a partially unfolded X state, and a completely unfolded D state, i.e., N in equilibrium X in equilibrium D. The strongly immobilized electron spin resonance (ESR) spectral component of spin-labeled TM corresponds to TM in the N state and the weakly immobilized component to TM in the X state below the main unfolding transition and to TM in the D state above this transition [Graceffa, P., & Lehrer, S. S. (1984) Biochemistry 23, 2606-2612]. The addition of actin, troponin (TN), and heavy meromyosin (HMM) to spin-labeled TM reduces the ratio of weakly to strongly immobilized labels, indicating a shift in the N in equilibrium X in equilibrium D equilibrium toward the N state. At 37 degrees C, for spin-labeled TM alone K (=X/N) greater than 1.0 with some TM in the D state, K = 0.8 for spin-labeled TM bound to actin, and K less than 0.05 for spin-labeled TM bound to actin + TN +/- Ca2+, actin + HMM + TN +/- Ca2+, and actin + HMM. Thus, actin + TN dramatically shifts the TM structure to the N conformation with little further effect upon addition of Ca2+ or HMM. The temperature at which spin-labeled TM begins to dissociate from a protein complex was determined from the temperature dependence of the ESR spectra.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effect of proteolysis on the electron spin resonance spectra of maleimide spin labeled erythrocyte membrane

The ratio of low-field amplitudes of weakly and strongly immobilized signals of ESR spectra of a maleimide spin label bound to erythrocyte membranes (hw/hs) increases progressively during incubation at 37 degrees C. This increase is due to the 'self-digestion' of membrane proteins by endogenous proteinases and is attenuated by proteinase inhibitors. Digestion of membranes with chymotrypsin also increases the hw/hs ratio. These results suggest a need for a careful interpretation of data from spin-labeled membrane proteins, especially in experiments involving prolonged incubations of membrane preparations when the proteolytic effects may be significant.     

The tryptic digestion of spin-labelled heavy meromyosin

The S₁ thiol groups of heavy meromyosin (HMM) have been selectively spin labeled with a paramagnetic analog of iodoacetamide (10) and the effects of tryptic digestion on the ESR spectrum and ATPase activity have been studied. The loss of ATPase activity on tryptic digestion occurs at the same rate with spin-labeled or unlabeled HMM suggesting that spin labeling produces no major change in the conformation of HMM. ESR spectra indicate that spin labels bound to S₁ groups of HMM are strongly immobilized; spectra of subfragment-1 isolated from tryptic digests of spin-labeled HMM are the same as those of labeled HMM. ESR spectra of S₁-spin-labeled peptides produced by tryptic digestion of HMM indicate essentially no immobilization of labels, the spectra being similar to that of a solution of free labels. The ESR spectrum of an unfractionated digest of HMM exhibits a peak attributable to strongly immobilized labels on HMM and subfragment-1 and a peak attributable to weakly immobilized labels bound to peptides. The rate at which spin-labeled peptides are released on tryptic digestion can be measured on the unfractionated digest by the decrease in the ESR peak corresponding to HMM and subfragment-1. The appearance of peptides containing spin-labeled S₁ groups parallels the loss of ATPase activity. No evidence has been found for the existence of an enzymatically active subfragment-1 lacking S₁ thiol groups.     

Spin-labeling studies of rat liver NADPH-cytochrome p450 reductase: conformation and function relationship.

ESR spin-labeling studies designed to yield information regarding the relationship between function and conformation of rat liver NADPH-cytochrome P450 reductase (EC 1.6.4.2) were carried out. The purified enzyme was spin labeled by a nitroxide derivative of p-chloromercuribenzoate. Two conditions for spin labeling were employed: (i) the presence of NADP+, yielding an active site-protected spin-labeled reductase, and (ii) the absence of NADP+, yielding completely spin-labeled reductase. Reductase in which the active site was protected by binding NADP+ and then spin-labeled retains most of its enzymatic activity; on the other hand, completely spin-labeled reductase is devoid of any enzymatic activity. Completely spin-labeled reductase yields a two-component resolved ESR spectrum that reflects two classes of spin-labeled binding sites, a strongly immobilized (S) and a weakly immobilized (W) site. The ratio of W/S provides a valuable parameter for studying the relationship between function and conformation. Structural perturbants, such as urea, KCl, and pH, were employed to determine their effects on the activity of the enzyme and their relationship to changes in the conformational state of the reductase. It was further observed that the enzymatically active spin-labeled derivative generated superoxide radical in the presence of NADPH and cytochrome c, which in turn reduced completely the attached spin-label.     

A doubly resonant cavity for detection of RF demodulation by living cells

We describe the design, construction, and operating characteristics of a doubly resonant cylindrical microwave cavity. This cavity has been developed to allow a search for nonlinear RF energy conversion in biological cells. Cells with a diode-like nonlinearity could demodulate a modulated RF carrier wave and generate low frequency signals in an exposed biological preparation. The cavity is designed to be resonant on the TE(111) mode at about 890 MHz and on the TE(113) mode at about 1780 MHz. The cavity performs exactly as designed and has proved capable of detecting the nonlinearity in a microscopic Schottky diode test structure. The sensitivity is sufficient to detect any nonlinearity in a collection of biological cells that could have any potential biological significance.     

Beamline characterization of a dielectric-filled reentrant cavity resonator as beam current monitor for a medical cyclotron facility

At PSI (Paul Scherrer Institute), Switzerland, a superconducting cyclotron called “COMET” delivers proton beam of 250 MeV pulsed at 72.85 MHz for proton radiation therapy. Measuring proton beam currents (0.1–10nA) is of crucial importance for the treatment safety and is usually performed with invasive monitors such as ionisation chambers (ICs) which degrade the beam quality. A new non-invasive beam current monitor working on the principle of electromagnetic resonance is built to replace ICs in order to preserve the beam quality delivered. The fundamental resonance frequency of the resonator is tuned to 145.7 MHz, which is the second harmonic of the pulse rate, so it provides signals proportional to beam current. The cavity resonator installed in the beamline of the COMET is designed to measure beam currents for the energy range 238–70 MeV. Good agreement is reached between expected and measured resonator response over the energy range of interest. The resonator can deliver beam current information down to 0.15 nA for a measurement integration time of 1 s. The cavity resonator might be applied serving as a safety monitor to trigger interlocks within the existing domain of proton radiation therapy. Low beam currents limit the abilities to detect sufficiently, however, with the potential implementation of FLASH proton therapy, the application of cavity resonator as an online beam-monitoring device is feasible.     

Fluorescence polarization of skeletal muscle fibers labeled with rhodamine isomers on the myosin heavy chain.

Fluorescence polarization was used to examine orientational changes of Rhodamine probes in single, skinned muscle fibers from rabbit psoas muscle following either photolysis of caged nucleotides or rapid length changes. Fibers were extensively and predominantly labeled at SH1 (Cys-707) of the myosin heavy chain with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine. Results from spectroscopic experiments utilizing the two Rhodamine isomers were quite similar. Following photolysis of either caged ATP or caged ADP, probes promptly reoriented toward the muscle fiber axis. Changes in the fluorescence polarization signals with transients elicited by the photolysis of caged ATP in the presence of saturating Ca2+ greatly preceded active force generation. Photolysis of caged ADP caused only a small, rapid decrease in force but elicited changes in the fluorescence polarization signals with time course and amplitude similar to those following photolysis of caged ATP. Fluorescence polarization signals were virtually unchanged by rapid length steps in both rigor and active muscle fibers. These results indicate that structural changes monitored by Rhodamine probes at SH1 are not associated directly with the force-generating event of muscle contraction. However, the fluorescence polarization transients were slightly faster than the estimated rate of cross-bridge detachment following photolysis of caged ATP, suggesting that the observed structural changes at SH1 may be involved in the communication pathway between the nucleotide- and actin-binding sites of myosin.     

Probing of sulfhydryl groups in the adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier by maleimide spin-labels

Binding of spin-labeled maleimides to the mitochondrial ADP/ATP carrier was investigated both in mitochondria and in the detergent-solubilized carrier protein. In mitochondria, spin-label binding to the carrier was evaluated by preincubation with the inhibitor carboxyatractyloside. The membrane sidedness of SH groups in the carrier molecule was determined by chemical reduction of nitroxides on the cytosolic membrane surface by Fe2+ or by pretreatment of the mitochondria with impermeant SH reagents. These experiments suggest that each subunit of the dimeric carrier incorporates one spin-labeled maleimide. Roughly half of the carrier-bound spin-labels were found on either side of the mitochondrial membrane. The detergent-solubilized carrier protein was labeled with a series of maleimide derivatives containing a spacer of increasing length between the maleimide and nitroxide moieties. A total spin-label binding of 2-3 mol/mol of protein dimer, depending on the spin-label length, was found. The electron spin resonance spectra of the spin-labeled protein invariably showed strongly and weakly immobilized components. Increasing the distance of the nitroxide from the maleimide ring resulted in a strong increase of the contribution of the weakly immobilized component. These observations led to the conclusions that the geometrical constraint of spin-label mobility changes at a distance of about 10 A from the maleimide binding site.     

Electron spin resonance spin label studies of plasma fibronectin: effect of temperature

Plasma fibronectin was chemically modified by 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl (maleimide spin label). Only the free sulfhydryl groups of plasma fibronectin were modified by the label under the experimental conditions. The ESR spectrum of spin-labeled fibronectin showed that the sites of labeling were highly immobilized, suggesting that the sulfhydryl groups of the protein are in small, confined environments. The conversion of the strongly immobilized ESR spectrum into a weakly immobilized one was observed when the spin-labeled protein was heated from 30 to 60 degrees C, indicating the thermal unfolding of the protein molecules. The midpoint temperature for the thermal unfolding of plasma fibronectin is about 50 degrees C. The results suggest that plasma fibronectin is stable to about 40 degrees C and starts unfolding above this temperature. The rotational correlation time estimated from the ESR spectrum of spin-labeled fibronectin at 21 degrees C was about 2.0 X 10(-8) s. The rotational correlation time calculated from the Stokes-Einstein equation, assuming a rigid globular configuration for fibronectin with a Stokes radius of 10 nm, was about 7.8 X 10(-7) s. The differences in rotational correlation time by a factor of 39 between experimental and calculated values do not support a globular configuration for plasma fibronectin.     

Orientation of spin-labeled light chain 2 of myosin heads in muscle fibers

Electron paramagnetic resonance (e.p.r.) spectroscopy has been used to monitor the orientation of spin labels attached rigidly to a reactive SH residue on the light chain 2 (LC2) of myosin heads in muscle fibers. e.p.r. spectra from spin-labeled myosin subfragment-1 (S1), allowed to diffuse into unlabeled rigor (ATP-free) fibers, were roughly approximated by a narrow angular distribution of spin labels centered at 66 degrees relative to the fiber axis, indicating a uniform orientation of S1 bound to actin. On the other hand, spectra from spin-labeled heavy meromyosin (HMM) were roughly approximated by two narrow angular distributions centered at 42 degrees and 66 degrees, suggesting that the LC2 domains of the two HMM heads have different orientations. In contrast to S1 or HMM, the spectra from rigor fibers, in which LC2 of endogenous myosin heads was labeled, showed a random orientation which may be due to distortion imposed by the structure of the filament lattice and the mismatch of the helical periodicities of the thick and thin filaments. However, spectra from the fibers in the presence of ATP analog 5'-adenylyl imidodiphosphate (AMPPNP) were approximated by two narrow angular distributions similar to those obtained with HMM. Thus, AMPPNP may cause the LC2 domain to be less flexible and/or the S2 portion to be more flexible, so as to release the distortion of the LC2 domain and make it return to its natural position. At high ionic strength, AMPPNP disoriented the spin labels as ATP did under relaxing conditions, suggesting that the myosin head is detached from and/or weakly (flexibly) attached to a thin filament.     

Orientation of spin-labeled nucleotides bound to myosin in glycerinated muscle fibers.

Electron paramagnetic resonance (EPR) spectroscopy of paramagnetic derivatives of ATP has been used to probe the angular distribution of myosin in glycerinated muscle fibers. Three nucleotide spin labels have been prepared with the nitroxide free radical moiety attached, via an ester linkage to either: the 2' or 3' positions of the ribose unit of ATP (SL-ATP), the 2' position of 3' deoxy ATP (2'SL-dATP), or the 3' position of 2' deoxy ATP (3'SL-dATP). In muscle fibers, these nucleotides are quickly hydrolyzed to their diphosphate forms. All three diphosphate analogues bind to the nucleotide site of myosin with similar affinities: rabbit psoas fibers, 7 X 10(3)/M; insect flight muscle, 5 X 10(3)/M; and rabbit soleus muscle, 2 X 10(4)/M. Analysis of the spectra showed that the principal z-axis of the nitroxide attached to bound nucleotides was oriented with respect to the filament axis. The principal axes of 3'SL-dADP and 2'SL-dADP appeared to be preferentially aligned at mean angles of 67 degrees +/- 4 degrees and 55 degrees +/- 5 degrees, respectively. The distribution of probes about these angles can be described by Gaussians with widths of 16 degrees +/- 4 degrees and 13 degrees +/- 5 degrees, respectively. The spectrum of bound SL-ADP was a linear combination of the spectra of the two deoxy analogues. These orientations were the same in the three muscle types examined, indicating a high degree of homology in the nucleotide binding site. Applying static strains as high as 0.2 N/mm2 to muscle fibers caused no change in the orientation of myosin-bound, spin-labeled nucleotides. When muscle fibers were stretched to decrease actin and myosin filament overlap, bound SL-ADP produced EPR spectra indicative of probes with a highly disordered angular distribution. Sodium vanadate and SL-ATP caused fiber stiffness to decrease, and the EPR spectrum of the bound analogue indicated an increase in the fraction of disoriented probes with a concomitant decrease in the fraction of oriented probes. These findings indicate that when myosin is bound to actin its nucleotide site is highly oriented relative to the fiber axis, and when this interaction is removed the orientation of the nucleotide site becomes highly disordered.