Resolved Conformational States of Spin-Labeled Ca-ATPase during the Enzymatic Cycle

Scott M. Lewis, David D Thomas

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14 Scopus citations

Abstract

We have used frequency-and time-resolved electron paramagnetic resonance (EPR) to study the effects of substrate on the nanosecond conformational dynamics of the Ca-ATPase of sarcoplasmic reticulum, as detected by an iodoacetamide spin label (IASL) attached covalently to the enzyme. We confirm previous results [Coan, C. (1983) Biochemistry 22, 5826] showing that this probe is less rotationally mobile following the addition of nucleotides (ADP, AMPPNP, ATP) and that the shape of the spectrum suggests the presence of two components. We used two approaches to enhance EPR resolution in order to resolve the spectral components and their corresponding conformational states. First, to improve resolution in the frequency (spectral) domain, we used perdeuterated IASL, which results in narrower line widths. Digital spectral analysis resolves the EPR spectrum into two components, one that is indistinguishable from the spectrum observed in the absence of ligands and another that indicates more restricted probe motions, suggesting a distinct conformation of the labeled protein. Additions of substrate ligands appear to change only the mole fractions of the two components. The mole fraction of the restricted component (fR) was 0 in the absence of ligands, but increased to about 0.5 in the presence of saturating concentrations of AMPPNP and Ca2+. In general, ATP and its analogs increase fR, with larger effects observed in the presence of Ca. However, calcium has no effect by itself (fr = 0). Both monovanadate and decavanadate increase fR, but the formation of a covalent phosphoenzyme from inorganic phosphate (E2-P) had no effect (fr = 0). Thus, although these EPR spectra are sensitive to conformational transitions in the Ca-ATPase, these transitions are not consistent with a simple E1-E2 conformational model, which predicts that the addition of calcium (to form Ca2-E1) should induce a conformation different from that induced by inorganic phosphate (to form E2-P). Our second means of resolution enhancement was in the time domain. In order to detect transients in the restricted fraction during Ca-ATPase activity, we used a laser pulse to photolyze caged ATP during EPR data acquisition. We observed a rapid increase in fR, followed by partial recovery to an intermediate steady-state level, indicating pre-steady-state conformational changes in the transport cycle. We conclude that the Ca-ATPase can exist in at least two conformations, which are distinguishable on the basis of internal protein dynamics, and whose relative populations are dependent on ligands during the active transport cycle.

Original languageEnglish (US)
Pages (from-to)7381-7389
Number of pages9
JournalBiochemistry
Volume31
Issue number32
DOIs
StatePublished - Feb 1 1992

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