Piotr G. Fajer

Myosin cleft movement and its coupling to actomyosin dissociation

It has long been known that binding of actin and binding of nucleotides to myosin are antagonistic, an observation that led to the biochemical basis for the crossbridge cycle of muscle contraction. Thus ATP binding to actomyosin causes actin dissociation, whereas actin binding to the myosin accelerates ADP and phosphate release. Structural studies have indicated that communication between the actin- and nucleotide-binding sites involves the opening and closing of the cleft between the upper and lower 50K domains of the myosin head. Here we test the proposal that the cleft responds to actin and nucleotide binding in a reciprocal manner and show that cleft movement is coupled to actin binding and dissociation. We monitored cleft movement using pyrene excimer fluorescence from probes engineered across the cleft.

Structure of the inhibitory region of troponin by site directed spin labeling electron paramagnetic resonance

Site-directed spin labeling EPR (SDSL-EPR) was used to determine the structure of the inhibitory region of TnI in the intact cardiac troponin ternary complex. Maeda and collaborators have modeled the inhibitory region of TnI (skeletal 96–112: the structural motif that communicates the Ca2+ signal to actin) as a kinked α-helix [Vassylyev, D., Takeda, S., Wakatsuki, S., Maeda, K. & Maeda, Y. (1998) Proc. Natl. Acad. Sci. USA 95, 4847–4852), whereas Trewhella and collaborators have proposed the same region to be a flexible β-hairpin [Tung, C. S., Wall, M. E., Gallagher, S. C. & Trewhella, J. (2000) Protein Sci. 9, 1312–1326]. To distinguish between the two models, residues 129–145 of cardiac TnI were mutated sequentially to cysteines and labeled with the extrinsic spin probe, MTSSL. Sequence-dependent solvent accessibility was measured as a change in power saturation of the spin probe in the presence of the relaxation agent. In the ternary complex, the 129–137 region followed a pattern characteristic of a regular 3.6 residues/turn α-helix. The following region, residues 138–145, showed no regular pattern in solvent accessibility. Measurements of 4 intradomain distances within the inhibitory sequence, using dipolar EPR, were consistent with an α-helical structure. The difference in side-chain mobility between the ternary (C⋅I⋅T) and binary (C⋅I) complexes revealed a region of interaction of TnT located at the N-terminal end of the inhibitory sequence, residues 130–135. The above findings for the troponin complex in solution do not support either of the computational models of the binary complex; however, they are in very good agreement with a preliminary report of the x-ray structure of the cardiac ternary complex [Takeda, S. Yamashita, A., Maeda, K. & Maeda, Y. (2002) Biophys. J. 82, 832].

General method for multiparameter fitting of high-resolution EPR spectra using a simplex algorithm

A general method for fitting experimental electron paramagnetic resonance spectra to numerically simulated multiparameter model spectra has been developed. The goal of this work is to provide a quantitative and reliable method for evaluating spectral simulations and extracting the maximum information possible from experimental EPR spectra. The calculation involves a minimization of gx2, in which a downhill simplex algorithm is used as the search procedure for varying the parameters that determine the simulated model spectra. This method is applied to determine (1) magnetic tensors and linewidths from spectra of randomly oriented (powder) samples, (2) complex orientational distributions from spectra of oriented assemblies, and (3) exponential recovery times in time-domain EPR. The reliability of the calculation was verified by successful applications to simulated spectra for which the correct results were known, and by showing that the same results were obtained independently of initial assumptions or the convergence path followed, for both simulated and experimental spectra. Estimates of uncertainties in the fitted parameters were obtained by determining the standard deviations from multiple independent calculations with different initial values.

Practical Pulsed Dipolar ESR (DEER)

The recent resurgence of ESR (electron spin resonance) in structural biology is in large part due to the development of distance measurements. This application was made possible by targeting of specific sites/domains by cysteine mutagenesis. Four techniques were developed to cover various distance ranges: exchange ESR for the very short distances (4–8 A) (Miick et al. 1992); static and dynamic dipolar cw ESR (continuous wave ESR) for the 8–25 A distance range (McHaourab et al. 1997; Rabenstein and Shin 1995); and two pulsed methods: DEER (double electron electron resonance) (Milov et al. 1981) and DQC (double quantum coherence) (Borbat et al. 2002) for distances between 17 and 80 A. The first two methods rely on line shape broadening and are thus limited to strong interactions (short distances). The pulsed methods extract weaker dipolar interactions from spin coherence and are thus sensitive to longer interspin distances. In addition, distances can be measured between the nitroxides and paramagnetic metals that enhance the relaxation of nitroxides (Budker et al. 1995; Voss et al. 1995). All these techniques have been extensively tested and verified across the full distance range of sensitivity on model systems including organic biradicals (Jeschke et al. 2000), proteins (Sale et al. 2005) and DNA (Borbat et al. 2004; Schiemann et al. 2004). The use of extrinsic spin labels for distance measurements allows targeting of specific sites by side-directed spin labeling, which is of great advantage.

Sensitivity of saturation transfer ESR spectra to anisotropic rotation. Application to membrane systems

Abstract The sensitivity to axial rotation of the various diagnostic regions in the 9 GHz saturation transfer ESR spectra of nitroxide spin labels has been investigated. The rate of change of resonant field position with molecular rotation was calculated for individual molecular rotations, and the values summed over all molecular rotations which contribute to a particular field position. Both off axial and axial rotations have been considered, with the rotation axis parallel to the nitroxide z axis or parallel to the y axis. The effect of off-axial rotation restricted within a cone is to increase sensitivity to axial rotation, but otherwise the results are qualitatively similar to unrestricted off-axial rotation. The partial averaging of the nitroxide tensors by rapid, restricted oscillation is considered, and can be allowed for by defining the diagnostic regions at isosbestic points which are insensitive to the averaging. Plots of the relative sensitivities to anisotropic rotation and of the axial/off-axial discrimination of the various diagnostic regions are presented, and the results have been applied to data from spin-labeled phosphatidylcholine and spin-labeled cholestane i in el phase bilayers of dipalmitoyl phosphatidylcholine.

Site directed spin labelling and pulsed dipolar electron paramagnetic resonance (double electron–electron resonance) of force activation in muscle

The recent development of site specific spin labelling and advances in pulsed electron paramagnetic resonance (EPR) have established spin labelling as a viable structural biology technique. Specific protein sites or whole domains can be selectively targeted for spin labelling by cysteine mutagenesis. The secondary structure of the proteins is determined from the trends in EPR signals of labels attached to consecutive residues. Solvent accessibility or label mobility display periodicities along the labelled polypeptide chain that are characteristic of β-strands (periodicity of 2 residues) or α-helices (3.6 residues). Low-resolution 3D structure of proteins is determined from the distance restraints. Two spin labels placed within 60-70 A of each other create a local dipolar field experienced by the other spin labels. The strength of this field is related to the interspin distance, αr -3 . The dipolar field can be measured by the broadening of the EPR lines for the short distances (8-20 A) or for the longer distances (17-70 A) by the pulsed EPR methods, double electron-electron resonance (DEER) and double quantum coherence (DQC). A brief review of the methodology and its applications to the multisubunit muscle protein troponin is presented below.

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.

Orientational disorder and motion of weakly attached cross-bridges

In a relaxed muscle fiber at low ionic strength, the cross-bridges may well be in states comparable to the one that precedes the cross-bridge power stroke (Schoenberg, M. 1988. Adv. Exp. Med. Biol. 226:189-202). Using electron paramagnetic resonance (EPR) and (saturation transfer) electron paramagnetic resonance (ST-EPR) techniques on fibers labeled with maleimide spin label, under low ionic strength conditions designed to produce a majority of weakly-attached heads, we have established that (a) relaxed labeled fibers show a speed dependence of chord stiffness identical to that of unlabeled, relaxed fibers, suggesting similar rapid dissociation and reassociation of cross-bridges; (b) the attached relaxed heads at low ionic strength are nearly as disordered as in relaxation at physiological ionic strength where most of the heads are detached from actin; and (c) the microsecond rotational mobility of the relaxed heads was only slightly restricted compared to normal ionic strength, implying great motional freedom despite attachment. The differences in head mobility between low and normal ionic strength scale with filament overlap and are thus due to acto-myosin interactions. The spectra can be modeled in terms of two populations: one identical to relaxed heads at normal ionic strength (83%), the other representing a more oriented population of heads (17%). The spectrum of the latter is centered at approximately the same angle as the spectrum in rigor but exhibits larger (40 degrees) axial probe disorder with respect to the fiber axis. Alternatively, assuming that the chord stiffness is proportional to the fraction of attached crossbridges, the attached fraction must be even more disordered than 400, with rotational mobility nearly as great as for detached cross-bridges.

Effects of calcium binding and the hypertrophic cardiomyopathy A8V mutation on the dynamic equilibrium between closed and open conformations of the regulatory N-domain of isolated cardiac troponin C.

Troponin C (TnC) is the calcium-binding subunit of the troponin complex responsible for initiating striated muscle contraction in response to calcium influx. In the skeletal TnC isoform, calcium binding induces a structural change in the regulatory N-domain of TnC that involves a transition from a closed to open structural state and accompanying exposure of a large hydrophobic patch for troponin I (TnI) to subsequently bind. However, little is understood about how calcium primes the N-domain of the cardiac isoform (cTnC) for interaction with the TnI subunit as the open conformation of the regulatory domain of cTnC has been observed only in the presence of bound TnI. Here we use paramagnetic relaxation enhancement (PRE) to characterize the closed to open transition of isolated cTnC in solution, a process that cannot be observed by traditional nuclear magnetic resonance methods. Our PRE data from four spin-labeled monocysteine constructs of isolated cTnC reveal that calcium binding triggers movement of the N-domain helices toward an open state. Fitting of the PRE data to a closed to open transition model reveals the presence of a small population of cTnC molecules in the absence of calcium that possess an open conformation, the level of which increases substantially upon Ca(2+) binding. These data support a model in which calcium binding creates a dynamic equilibrium between the closed and open structural states to prime cTnC for interaction with its target peptide. We also used PRE data to assess the structural effects of a familial hypertrophic cardiomyopathy point mutation located within the N-domain of cTnC (A8V). The PRE data show that the Ca(2+) switch mechanism is perturbed by the A8V mutation, resulting in a more open N-domain conformation in both the apo and holo states.

Saturation transfer, continuous wave saturation, and saturation recovery electron spin resonance studies of chain-spin labeled phosphatidylcholines in the low temperature phases of dipalmitoyl phosphatidylcholine bilayers. Effects of rotational dynamics and spin-spin interactions

The saturation transfer electron spin resonance (STESR) spectra of 10 different positional isomers of phosphatidylcholine spin-labeled in the sn-2 chain have been investigated in the low temperature phases of dipalmitoyl phosphatidylcholine (DPPC) bilayers. The results of continuous wave saturation and of saturation recovery measurements on the conventional ESR spectra were used to define the saturation properties necessary for interpreting the STESR results in terms of the chain dynamics. Spin labels with the nitroxide group located in the center of the chain tended to segregate preferentially from the DPPC host lipids in the more ordered phases, causing spin-spin interactions which produced spectral broadening and had a very pronounced effect on the saturation characteristics of the labels. This was accompanied by a large decrease in the STESR spectral intensities and diagnostic line height ratios relative to those of spin labels that exhibited a higher degree of saturation at the same microwave power. The temperature dependence of the STESR spectra of the different spin label isomers revealed a sharp increase in the rate of rotation about the long axis of the lipid chains at approximately 25 degrees C, correlating with the pretransition of gel phase DPPC bilayers, and a progressive increase in the segmental motion towards the terminal methyl end of the chains in all phases. Prolonged incubation at low temperatures led to an increase in the diagnostic STESR line height ratios in all regions of the spectrum, reflecting the decrease in chain mobility accompanying formation of the subgel phase. Continuous recording of the central diagnostic peak height of the STESR spectra while scanning the temperature revealed a discontinuity at approximately 14-17 degrees C, corresponding to the DPPC subtransition which occurred only on the initial upward temperature scan, in addition to the discontinuity at 29-31 degrees C corresponding to the pretransition which displayed hysteresis on the downward temperature scan.