We have used site-directed spin labeling and electron paramagnetic resonance (EPR) to map interactions between the transmembrane (TM) domains of the sarcoplasmic reticulum Ca-ATPase (SERCA) and phospholamban (PLB) as affected by PLB phosphorylation. In the cardiac sarcoplasmic reticulum, PLB binding to SERCA results in Ca-dependent enzyme inhibition, which is reversed by PLB phosphorylation at Ser16. Previous spectroscopic studies on SERCA-PLB have largely focused on the cytoplasmic domain of PLB, showing that phosphorylation induces a structural shift in this domain relative to SERCA. However, SERCA inhibition is due entirely to TM domain interactions. Therefore, we focus here on PLB's TM domain, attaching Cys-reactive spin labels at five different positions. In each case, continuous-wave EPR indicated moderate spin-label mobility, with the addition of SERCA revealing two populations, one indistinguishable from PLB alone and another with more restricted rotational mobility, presumably due to SERCA-binding. Phosphorylation had no effect on the rotational mobility of either component but significantly decreased the mole fraction of the restricted component. Solvent-accessibility experiments using power-saturation EPR and saturation-recovery EPR confirmed that these two spectral components were SERCA-bound and unbound PLB and showed that phosphorylation increased the overall lipid accessibility of the TM domain by increasing the fraction of unbound PLB. However-based on these results-at physiological levels of SERCA and PLB, most SERCA would have bound PLB even after phosphorylation. Additionally, no structural shift in the TM domain of SERCA-bound PLB was detected, as there were no significant changes in membrane insertion depth or its accessibility. Therefore, we conclude that under physiological conditions, the phosphorylation of PLB induces little or no change in the interaction of the TM domain with SERCA, so relief of inhibition is predominantly due to the previously observed structural shift in the cytoplasmic domain.

Direct time-domain simulation of continuous-wave (CW) EPR spectra from molecular dynamics (MD) trajectories has become increasingly popular, especially for proteins labeled with nitroxide spin labels. Due to the time-consuming nature of simulating adequately long MD trajectories, two approximate methods have been developed to reduce the MD trajectory length required for modeling EPR spectra: hindered Brownian diffusion (HBD) and hidden Markov models (HMMs). Here, we assess the accuracy of these two approximate methods relative to direct simulations from MD trajectories for three spin-labeled protein systems (a simple helical peptide, a soluble protein, and a membrane protein) and two nitroxide spin labels with differing mobilities (R1 and TOAC). We find that the HMMs generally outperform HBD. Although R1 dynamics partially resembles hindered Brownian diffusion (HBD), HMMs accommodate the multiple dynamic timescales for the transitions between rotameric states of R1 that cannot be captured accurately by a HBD model, even when a three-angle orientational potential and recently developed estimation methods for the rotational diffusion tensor are utilized. We show that TOAC dynamics closely resembles slow multi-site exchange between twist-boat and chair ring puckering states, and we establish for the first time that this motion is modeled well by a HMM with up to four states, but not by HBD. All MD trajectory data processing, stochastic trajectory simulations, and CW EPR spectral simulations are implemented in EasySpin, a free software package for MATLAB.

JULY 15, I960 Table I. The room temperature values of k for the d 1 andd 3 metals in percent. a ' nnn-= 3 = 4 = 5 d> 21 Sc: 0. 57 La: 0 24 .63 & i 2 3 V: 4 lNb: 7J Ta: d 3 0.58 ; 0.87 : 1.1 a W. D. Knight, reference 1. b L. H. Bennett and J. I. Budnick, Bull. Am. Phys. Soc. j>, 242 (1960).ever, the large increase as one proceeds from 3d 1 to 3d 3 or M 1 to 5d 3 is not expected. 5 We suggest that this results from the enhanced s -electron polarization produced by the s-d exchange interaction.It is noteworthy that the linewidths of Sc, V, Nb, and La are all considerably larger than the direct nuclear dipole-dipole interaction would allow. Since all these elements have single isotopes of overwhelming abundance any indirectThe properties of the nuclear magnetic resonance (NMR) of Mn 55 in a and /3 Mn have been investigated as a function of field and temperature. 1 A very broad asymmetric NMR (67/" 2 50 oe) was observed in aMn at 295°K. The center of gravity of the resonance corresponds to a negative Knight shift (k) (see Table I). Since there are four magnetically inequivalent sites, 2 it was first thought that the line shape was the result of superposition of several resonances. However, a negligible field dependence was observed for the shape of the line. The a phase is known to have an antiferromagnetic transition at 95°K. 3 Correspondingly, no resonance was visible at 77°K and 20.2°K in the randomly oriented powdered sample.In /3Mn only a single resonance line was found, although there occur two physically inequivalent sites. The value of k observed as a function of exchange interaction would not produce line broadening. 6 The field independence of the linewidth and the line shapes exclude both quadrupolar and anisotropic magnetic contributions. These facts, combined with the Gaussian shape and the temperature independence of the linewidth, indicate a pseudodipolar origin for the observed line shapes. . This author reports a Curie-Weiss behavior with the Curie constant indicating an effective magnetic moment of one-half of a Bohr magneton. 3 R. M, Bozorth (private communication). 4 K. Yosida, Phys. Rev. 106, 893 (1957). 5 One must of course allow for the fact that all of the 4/ elements occur between La and Ta. 6 M. A. Ruderman and C" Kittel, PhySo Rev. 96, 99 (1954). Table Io The temperature dependence of the Knight shift in/3Mn as observed at 14.245 Mc/sec. An error of ± 0. 02 % exists in each of the values given, k was determined with respect to the Mn 55 NMR in a saturated solution of LiMn0 4 and in KMn0 4 . The field for resonance in the two solutions is identical to one part in 10 5 . No chemical shifts have been reported for Mn 55 in the literature. T(°K) k(%) 1.8 -0.11 4o2 -0.11 20o2 -0.12 77 -0.13 190 -0.13 295 -0.13 NUCLEAR MAGNETIC RESONANCE IN a AND 0 MANGANESE

No abstract

The millimeter wave paramagnetic resonance spectra of Ni ++ , V ++ , and Cr +++ have been measured in ZnF 2 . Evidence was found that the extra charge of Cr +++ is compensated by an F~" ion, sitting on one of two available nonequivalent neighboring sites. The antiferromagnetic resonance line in N1F2 which has been postulated by Moriya, is now expected to occur at 320 kMc/sec. T HE paramagnetic spectra of the ions Ni 4-4 ", V+ 4 ", and Cr 44 " 4 " have been measured in the diamagnetic ZnF 2 lattice. They are described by the spin HamiltonianThe parameters for these ions are summarized in Table I. In Ni 44-, V 44 ", and Cr 4 " 4-1 ", spectrum A, the % axis is the [001] axis of ZnF 2 and the x axis follows [110]. In these spectra we do not know the sign of E. In Cr 444-, spectrum B, the z axis is in a (110) plane, 9° off the [110] axis, and the y axis follows the [110] axis.The Cr +++ spectra have previously been measured by Tinkham, 1 but we find 20% smaller zero field splittings than he predicted. We agree, however, with Tinkham's measurement of the directions of the main axes of the spin Hamiltonian, and also with his conclusion that they are due to the presence of an anion which compensates the excess charge on Cr" H_f , and which can occupy either one of two nonequivalent positions. The fact that Cr 44 " 4-can be introduced in considerable amounts, together with recent measurements in CaF 2 2 suggests F~ as the compensating ion.The D term in Ni 4 " 4 " has been predicted by Stout 3 from susceptibility measurements. Moriya 4 has shown, that the Ni 44 " spins are aligned along the [110] axes of 1 M. Tinkham, Proc. Roy. Soc. (London) A236, 535 (1956).

cations. A discrete set of high probability binding sites were identified at the interface between catalytic and association domains of neighboring subunits, far from the Mg 2þ binding sites required to coordinate ATP in the catalytic cleft. Next, we experimentally characterized the effects of divalent cations on CaMKII structure using fluorescence, light scattering and electron microscopy. We found that some, but not all, divalent ions including Zn 2þ promote the reversible formation of self-assembled CaMKII fibers that can extend for several mm. When associated in fibers, CaMKII activity is compromised; however, Ca 2þ /CaM activation of the enzyme disassembles fibers and the loss of activity is reversed. Interestingly, fiber assembly does not interfere with the autophosphorylation of CaMKII at T286, an autoregulatory modification, which confers autonomous activity in monodisperse CaMKII. We compile our results with structural and mechanistic data from literature to propose a model of Zn 2þ -mediated CaMKII fiber formation, where Ca 2þ /CaM regulates fiber formation through allosteric modification of a divalent ion site identified in our electrostatic map.

Antiferromagnetic and paramagnetic resonance absorption has been measured in CuF2·2H2O. At 1.4°K, the antiferromagnetic resonances in zero field occur at 95.84±0.05 kMc and 95.91±0.05 kMc. The easy direction of magnetization occurs at an angle of 3.5° with the c axis. This orientation is in agreement with neutron diffraction and single-crystal susceptibility measurements. Due to strong exchange narrowing, narrow paramagnetic resonance lines were observed. The g values are gξ=2.07, gη=2.08, and gζ=2.42. The principal axes are nearly coinciding with the ligand directions. From the g values, the energy levels and wave functions of the Cu++ ions are determined. A discussion of the anisotropy is given. The dipolar part is calculated numerically, and the anisotropic exchange of pseudodipolar type is determined, using the fact that ω1∼ω2 and the known easy magnetization direction. From the total anisotropy determined in this way, and the perpendicular susceptibility measured by Williams, the antiferromagnetic resonance frequency is predicted to occur at 130 kMc. The agreement with experiment is not unreasonable in view of the reduction of this frequency by the zero-point oscillations of the spin waves. A brief discussion of the superexchange interaction in this crystal is given.

Adenosine triphosphate (ATP) binding cassette (ABC) transporters are ubiquitous molecular motor proteins that translocate various substrates across cell membranes at the expense of ATP hydrolysis. Although it is known that