Phospholipid acyl chain rotational dynamics are independent of headgroup structure in unilamellar vesicles containing binary mixtures of dioleoyl-phosphatidylcholine and dioleoyl-phosphatidylethanolamine.

We have examined relationships between phospholipid headgroup structure and acyl chain dynamics, and their respective roles in modulating the physical properties of biological membranes. Fluorescence lifetime and anisotropy measurements were used to assess structural changes involving the lipid acyl chains in homogeneous populations of small and large unilamellar vesicles containing binary mixtures of dioleoyl-phosphatidylcholine (PC) and dioleoyl-phosphatidylethanolamine (PE) in the liquid-crystalline (Lalpha) phase. These measurements involve three different fluorescent lipid analogs containing diphenylhexatriene (DPH) linked to either a trimethylamine moiety (i.e., TMA-DPH) or the sn-1 position of monostearoyl-phospholipids containing PC or PE headgroups (i.e., DPH-PC and DPH-PE). The average lifetimes, rotational correlation times, and order parameters associated with DPH-PC and DPH-PE are virtually identical, and are not affected by alterations in the PE content of the membrane. These results suggest that the average cross-sectional areas of the phospholipid acyl chains of DOPE and DOPC relative to the membrane normal are similar in these unilamellar vesicles. Since PC headgroups are larger than those of PE, differences in the relative orientation of the phosphocholine and phosphoethanolamine moieties relative to the membrane surface probably function to maintain optimal van der Waals contact interactions between acyl chains. On the other hand, the average lifetime associated with TMA-DPH, whose chromophoric group is near the membrane surface, increases with increasing PE content. The position of TMA-DPH relative to the membrane surface does not change, since the rotational dynamics of TMA-DPH are independent of the PE concentration. Therefore, alterations in the average lifetime of TMA-DPH results from polarity differences near the membrane surface at the level of the glycerol backbone. These results are discussed in terms of how differences in the average conformation of the glycerol backbones or phospholipid headgroups of PE and PC have the potential to regulate membrane function.

Decreased conformational stability of the sarcoplasmic reticulum Ca-ATPase in aged skeletal muscle.

Sarcoplasmic reticulum (SR) membranes purified from young adult (4-6 months) and aged (26-28 months) Fischer 344 male rat skeletal muscle were compared with respect to the functional and structural properties of the Ca-ATPase and its associated lipids. While we find no age-related alterations in (1) expression levels of Ca-ATPase protein, and (2) calcium transport and ATPase activities, the Ca-ATPase isolated from aged muscle exhibits more rapid inactivation during mild (37 degrees C) heat treatment relative to that from young muscle. Saturation-transfer EPR measurements of maleimide spin-labeled Ca-ATPase and parallel measurements of fatty acyl chain dynamics demonstrate that, accompanying heat inactivation, the Ca-ATPase from aged skeletal muscle more readily undergoes self-association to form inactive oligomeric species without initial age-related differences in association state of the protein. Neither age nor heat inactivation results in differences in acyl chain dynamics of the bilayer including those lipids at the lipid-protein interface. Initial rates of tryptic digestion associated with the Ca-ATPase in SR isolated from aged muscle are 16(+/- 2)% higher relative to that from young muscle. indicating more solvent exposure of a portion of the cytoplasmic domain. During heat inactivation these structural differences are amplified as a result of immediate and rapid further unfolding of the Ca-ATPase isolated from aged muscle relative to the delayed unfolding of the Ca-ATPase isolated from young muscle. Thus age-related alterations in the solvent exposure of cytoplasmic peptides of the Ca-ATPase are likely to be critical to the loss of conformational and functional stability.

Adaptive changes in lipid composition of skeletal sarcoplasmic reticulum membranes associated with aging.

We have undertaken a detailed examination of changes associated with aging in lipid composition and corresponding physical properties of hindlimb skeletal sarcoplasmic reticulum (SR) membranes isolated from young (5 months), middle-aged (16 months), and old (28 months) Fischer strain 344 rats. Silica gel HPLC chromatography was used to separate phospholipid headgroup species. Subsequent reversed-phase HPLC was used to resolve fatty acid chain compositions of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol species. For all three phospholipid pools, significant age-related variations are observed in the abundance of multiple molecular species, particularly those having polyunsaturated fatty acid chains. Using mass spectrometry (fast atom bombardment and tandem techniques) to distinguish ester- from ether-linked phosphatidylethanolamine species, we demonstrate that overall plasmenylethanolamine content is substantially increased with age, from 48 mol% to 62 mol%. A substantial increase is also observed in the single molecular species 18:0-20:4 phosphatidylinositol suggesting implications for signalling pathways. In addition, associated with senescence we find a significant increase in the rigidifying lipid, cholesterol. Despite these changes in lipid composition of different aged animals, the average bilayer fluidity examined at several bilayer depths with stearic acid spin labels, is not altered. Neither do we find differences in the rotational mobility of maleimide spin-labeled Ca(2+)-ATPase, as determined from saturation-transfer electron paramagnetic resonance, which is sensitive to both the fluidity of lipids directly associated with the Ca(2+)-ATPase and to its association with proteins.

Age-related decrease in brain synaptic membrane Ca2+-ATPase in F344/BNF1 rats.

We used Fisher 344/Brown Norway hybrid rats (F344/BNF1) to determine whether previously reported decreases in brain synaptic plasma membrane (SPM) Ca2+-ATPase activity in inbred F344 rats also occurred in the hybrids. Plasma membrane Ca2+-ATPase (PMCA) activity in SPMs from F344/BNF1 rat brains showed a progressive age-dependent decrease in Vmax from 60.9 +/- 3.7 nmol Pi/mg/min (n = 6) in 5-month rats to 32.4 +/- 3.6 nmol Pi/mg/min (n = 6) in 34-month animals, with no change in K (act) for Ca2+. Immunoreactive PMCA in SPMs also decreased by approximately 20% at 34 months, and the calmodulin (CaM) bound to membranes following extraction with EDTA also declined progressively with age. The effectiveness of CaM in stimulating PMCA activity was significantly lower when CaM was purified from the brains of old vs. young F344 rats and when CaM from 5-month rats was oxidized in vitro. These results indicate: 1) that PMCA activity in SPMs from longer lived F344/BNF1 hybrids also decreases with age; 2) that part of the reduction in PMCA activity is due to loss of PMCA from the membranes; and 3) that age-related structural changes in CaM may decrease its interaction with proteins in SPMs.

Diastereoselective reduction of protein-bound methionine sulfoxide by methionine sulfoxide reductase.

Methionine sulfoxide (MetSO) in calmodulin (CaM) was previously shown to be a substrate for bovine liver peptide methionine sulfoxide reductase (pMSR, EC 1.8.4.6), which can partially recover protein structure and function of oxidized CaM in vitro. Here, we report for the first time that pMSR selectively reduces the D-sulfoxide diastereomer of CaM-bound L-MetSO (L-Met-D-SO). After exhaustive reduction by pMSR, the ratio of L-Met-D-SO to L-Met-L-SO decreased to about 1:25 for hydrogen peroxide-oxidized CaM, and to about 1:10 for free MetSO. The accumulation of MetSO upon oxidative stress and aging in vivo may be related to incomplete, diastereoselective, repair by pMSR.

Decrease in Ca-ATPase activity in aged synaptosomal membranes is not associated with changes in fatty acyl chain dynamics.

We have examined lipid peroxidation (LPO) and fatty acid acyl chain dynamics in synaptosomal membranes isolated from aged rat (Fischer 344 x Brown Norway F1 hybrids) brains, correlating these results with measurements of enzymatic activity of the synaptic plasma membrane Ca2(+)-ATPase (PMCA). Calcium-dependent ATPase activity in these membranes exhibits progressive decreases with a maximal loss of activity with age of approximately 35%. The sensitivity of this membrane-bound ion transporter to the lipid composition of the surrounding membrane, as well as the high abundance of oxidatively sensitive polyunsaturated fatty acyl chains in synaptosomal membranes, suggests that this age-related loss in catalytic turnover may result from LPO-mediated protein modification and/or changes in the physical structure of the bilayer. However, high-performance liquid chromatography analysis of 2,4-dinitrophenylhydrazone derivatives reveals no significant age-related increases in the content of reactive aldehydes (malondialdehyde, formaldehyde, acetaldehyde or acetone) which comprise breakdown products of lipid peroxidation. Electron paramagnetic resonance measurements employing 5- and 12-stearic acid spin labels with the nitroxide reporter groups at two depths in the bilayer were used to assess the fatty acyl chain dynamics (fluidity) of synaptosomal membranes. The resulting spectra demonstrate anisotropic lipid dynamics of two populations of lipids, i.e. lipids in direct association with membrane proteins (boundary lipids) and bulk lipids that do not directly associate with proteins. The nanosecond dynamics of both lipid populations is unaltered with age indicating that any compositional changes occurring with age are insufficient to result in alterations in bilayer fluidity relevant to PMCA activity. Thus, the observed age-related decline in PMCA activity may be explained by direct modification of membrane protein.

Protein oxidation and age-dependent alterations in calcium homeostasis.

Alterations in the capacity to maintain normal calcium homeostasis have been suggested to underlie the reduced cellular function characteristic of the aging process, and to predispose the senescent organism to a host of diverse pathologies including cancer, heart disease, and a range of muscle and neurodegenerative diseases. Therefore, critical to the eventual treatment of many age-related diseases has been the identification of both post-translational modifications and the underlying structural changes that result in an age-related decline in the function of critical calcium regulatory proteins. In brain, multiple methionines within the calcium signaling protein calmodulin (CaM) are oxidized to their corresponding methionine sulfoxides during aging, resulting in an inability to activate a range of target proteins, including the plasma membrane (PM) Ca-ATPase involved in the maintenance of the low intracellular calcium levels necessary for intracellular signaling. Likewise, changes in the transport activity of the PM-Ca-ATPase occur during aging. In muscle, the function of the SERCA2a isoform of the Ca-ATPase within the sarcoplasmic reticulum (SR) declines during aging as a result of the nitration of selected tyrosines. The age-related loss-of-function of these critical calcium regulatory proteins are consistent with observed increases in intracellular calcium levels within senescent cells. A possible regulatory role for these post-translational modifications is discussed, since they have the potential to be reversed following the restoration of normal cellular redox conditions by intracellular repair enzymes that are specific for these post-translational modifications. It is suggested that the reversible oxidation of critical calcium regulatory proteins within excitable cells by reactive oxygen species functions to enhance cellular survival under conditions of oxidative stress by reducing the energy expenditure within excitable cells. Thus, a diminished ability to efficiently generate cellular ATP may ultimately underlie the loss of calcium homeostasis and cellular function during aging.

Oxidative stress and protein aggregation during biological aging.

Biological aging is a fundamental process that represents the major risk factor with respect to the development of cancer, neurodegenerative, and cardiovascular diseases in vertebrates. It is, therefore, evident that the molecular mechanisms of aging are fundamental to understand many disease processes. In this regard, the oxidation and nitration of intracellular proteins and the formation of protein aggregates have been suggested to underlie the loss of cellular function and the reduced ability of senescent animals to withstand physiological stresses. Since oxidatively modified proteins are thermodynamically unstable and assume partially unfolded tertiary structures that readily form aggregates, it is likely that oxidized proteins are intermediates in the formation of amyloid fibrils. It is, therefore, of interest to identify oxidatively sensitive protein targets that may play a protective role through their ability to down-regulate energy metabolism and the consequent generation of reactive oxygen species (ROS). In this respect, the maintenance of cellular calcium gradients represents a major energetic expense, which links alterations in intracellular calcium levels to ATP utilization and the associated generation of ROS through respiratory control mechanisms. The selective oxidation or nitration of the calcium regulatory proteins calmodulin and Ca-ATPase that occurs in vivo during aging and under conditions of oxidative stress may represent an adaptive response to oxidative stress that functions to down-regulate energy metabolism and the associated generation of ROS. Since these calcium regulatory proteins are also preferentially oxidized or nitrated under in vitro conditions, these results suggest an enhanced sensitivity of these critical calcium regulatory proteins, which modulate signal transduction processes and intracellular energy metabolism, to conditions of oxidative stress. Thus, the selective oxidation of critical signal transduction proteins probably represents a regulatory mechanism that functions to minimize the generation of ROS through respiratory control mechanisms. The reduction of the rate of ROS generation, in turn, will promote cellular survival under conditions of oxidative stress, when reactive oxygen and nitrogen species overwhelm cellular antioxidant defense systems, by minimizing the non-selective oxidation of a range of biomolecules. Since protein aggregation occurs if protein repair and degradative systems are unable to act upon oxidized proteins and restore cellular function, the reduction of the oxidative load on the cell by the down-regulation of the electron transport chain functions to minimize protein aggregation. Thus, ROS function as signaling molecules that fine-tune cellular metabolism through the selective oxidation or nitration of calcium regulatory proteins in order to minimize wide-spread oxidative damage and protein aggregation. Oxidative damage to cellular proteins, the loss of calcium homeostasis and protein aggregation contribute to the formation of amyloid deposits that accumulate during biological aging. Critical to understand the relationship between these processes and biological aging is the identification of oxidatively sensitive proteins that modulate energy utilization and the associated generation of ROS. In this latter respect, oxidative modifications to the calcium regulatory proteins calmodulin (CaM) and the sarco/endoplasmic reticulum Ca-ATPase (SERCA) function to down-regulate ATP utilization and the associated generation of ROS associated with replenishing intracellular ATP through oxidative phosphorylation. Reductions in the rate of ROS generation, in turn, will minimize protein oxidation and facilitate intracellular repair and degradative systems that function to eliminate damaged and partially unfolded proteins. Since the rates of protein repair or degradation compete with the rate of protein aggregation, the modulation of intracellular calcium concentrations and energy metabolism through the selective oxidation or nitration of critical signal transduction proteins (i.e. CaM or SERCA) is thought to maintain cellular function by minimizing protein aggregation and amyloid formation. Age-dependent increases in the rate of ROS generation or declines in cellular repair or degradation mechanisms will increase the oxidative load on the cell, resulting in corresponding increases in the concentrations of oxidized proteins and the associated formation of amyloid.

Decreased plasma membrane calcium transport activity in aging brain.

We have assessed the functional properties of both calmodulin (CaM) and the plasma membrane Ca(2+)-ATPase in brains of young, middle aged, and old Fisher 344 rats. Under optimal conditions of saturating Ca2+ and ATP, the CaM-activated Ca(2+)-ATPase activity was decreased with increasing age, particularly when CaM isolated from the brains of aged rats was used to stimulate the enzyme. In the case of CaM, structural modifications within the primary sequence of the protein from aged brains were identified. We found that during normal biological aging approximately 6 methionine residues were modified to their corresonding sulfoxide per CaM, and no other amino acids were modified. Some aspects of the age-related decline in the effectiveness of CaM as an activator of Ca(2+)-ATPase could be simulated using a range of reactive oxygen species (including hydrogen peroxide and oxoperoxynitrite) and, in the latter case, the extent of oxidative modification of specific methionine residues was directly related to their surface accessibility. The pattern of oxidative modification of the methionines in the aged CaM was less straightforward, though both in vitro oxidation of CaM and aging within the brain markedly decreased the functional properties of this important Ca(2+)-regulating protein.

Ordered and cooperative binding of opposing globular domains of calmodulin to the plasma membrane Ca-ATPase.

We have investigated the mechanisms of activation of the plasma membrane (PM) Ca-ATPase by calmodulin (CaM), which result in enhanced calcium transport rates and the maintenance of low intracellular calcium levels. We have isolated the amino- or carboxyl-terminal domains of CaM (i.e. CaMN or CaMC), permitting an identification of their relative specificity for binding to sites on either the PM Ca-ATPase or a peptide (C28W) corresponding to the CaM-binding sequence. We find that either CaMN or CaMC alone is capable of productive interactions with the PM Ca-ATPase that induces enzyme activation. There are, however, large differences in the affinity and specificity of binding between CaMN and CaMC and either C28W or the PM Ca-ATPase. The initial binding interaction between CaMC and the PM Ca-ATPase is highly specific, having approximately 10,000-fold greater affinity in comparison with CaMN. However, following the initial association of either CaMC or CaMN, there is a 300-fold enhancement in the affinity of CaMN for the secondary binding site. Thus, while CaMC binds with a high affinity to the two CaM-binding sites within the PM Ca-ATPase in a sequential manner, CaMN binds cooperatively with a lower affinity to both binding sites. These large differences in the binding affinities and specificities of the amino- and carboxyl-terminal domains ensure that CaM binding to the PM Ca-ATPase normally involves the formation of a specific complex in which the initial high affinity association of the carboxyl-terminal domain promotes the association of the amino-terminal domain necessary for enzyme activation.

Methodology for increased precision in saturation transfer electron paramagnetic resonance studies of rotational dynamics.

Microsecond rotational motions of nitroxide spin labels are measured primarily with saturation transfer electron paramagnetic resonance (ST-EPR). In the present study we have used model system experiments to quantitatively evaluate different ST-EPR spectral parameters, both in-phase and out-of-phase, with an emphasis on techniques for suppressing the interference from weakly immobilized probes. Analyses of both systematic and random errors show that maximum sensitivity to small changes in correlation time and minimum ambiguity of interpretation are best achieved by combining measurements of both spectral line-shape, i.e., the ratio of line-heights, and spectral intensity, i.e., the absolute amplitude of either a position within a spectrum or a spectral integral. Errors in the measurement of correlation times for the two types of parameters tend to be complementary. Integrated intensity parameters are particularly useful in measuring microsecond probe motions in the presence of weakly immobilized components. We confirm that integrated intensity parameters are sometimes effective in rejecting signals from weakly immobilized probes, but the effectiveness of this rejection is more limited than previously supposed and depends on the type of parameter being measured. We describe procedures for evaluating and minimizing errors due to weakly immobilized probes, emphasizing the advantages of a new kind of intensity parameter obtained from integrated in-phase spectra. We provide detailed descriptions of experimental procedures, along with calibration plots of the most useful spectral parameters vs. rotational correlation time, which should make it possible for workers in other laboratories, using different instruments and sample geometries, to reproduce spectra quantitatively and to make accurate correlation time measurements.

Applications of new saturation transfer electron paramagnetic resonance methodology to the rotational dynamics of the Ca-ATPase in sarcoplasmic reticulum membranes.

The presence of small amounts of weakly immobilized probes can result in large systematic errors in the measurement of correlation times (tau r) from saturation transfer EPR spectra. However, we have recently developed experimental methodology to minimize these errors (Squier and Thomas, Biophys. J., 49:921-935). In the present study we have applied this methodology to the measurement of the rotational motion of the Ca-ATPase in sarcoplasmic reticulum. This analysis involves the estimate of tau r from line-shape parameters (spectral line-height ratios) and intensity parameters (spectral integral), coupled with digital subtractions to remove spectral components corresponding to weakly immobilized probes. We have analyzed the ST-EPR spectra of the Ca-ATPase over a range of temperatures and find that, unlike line-shape parameters, intensity parameters are little affected by the subtraction of the weakly immobilized spectral component (W). Thus, tau r values from intensity parameters are a more reliable measurement of rotational motion. As reported previously, an analysis with line-shape parameters yields a nonlinear Arrhenius plot of protein mobility. However, the plot is linear when intensity parameters or corrected spectra are used, consistent with the theory for the hydrodynamic properties of a membrane protein of unchanging size and shape in a fluid bilayer. An analysis with line-shape parameters yields different effective tau r values in different spectral regions, and these tau r values are temperature-dependent. However, correction of spectra for W yields temperature-independent tau r ratios, indicating that the motional anisotropy is temperature-independent. Obtaining a good match for the weakly immobilized spectral component remains a major difficulty in the quantitative analysis of ST-EPR spectra using line-shape parameters. This study shows that intensity parameters can be used to avoid this problem, making the ST-EPR technique applicable in cases that were previously resistant to analysis.

Selective detection of the rotational dynamics of the protein-associated lipid hydrocarbon chains in sarcoplasmic reticulum membranes.

We have developed a saturation transfer EPR (ST-EPR) method to measure selectively the rotational dynamics of those lipids that are motionally restricted by integral membrane proteins and have applied this methodology to measure lipid-protein interactions in native sarcoplasmic reticulum (SR) membranes. This analysis involves the measurement of spectral saturation using a series of six stearic acid spin labels that are labeled with a nitroxide at different carbon atom positions. A large amount of spectral saturation is observed for spin labels in native SR membranes, but not for spin labels in dispersions of extracted SR lipids, implying that the motional properties of those lipids interacting with the Ca-ATPase, i.e., the boundary or annular lipid, can be directly measured without the need for spectral subtraction procedures. A comparison of the motional properties of the restricted lipid, measured by ST-EPR, with those measured by digital subtraction of conventional EPR spectra qualitatively agree, for in both cases the Ca-ATPase restricts the rotational mobility of a population of lipids, whose rotational mobility increases as the nitroxide is positioned toward the center of the bilayer. However, the ability of ST-EPR to directly measure the motionally restricted lipid in a model-independent means provides the greater precision necessary to measure small changes in the rotational dynamics of the lipid at the protein-lipid interface, providing a valuable tool in clarifying the relationship between the physical nature of the protein-lipid interface and membrane function.

Relationship between protein rotational dynamics and phosphoenzyme decomposition in the sarcoplasmic reticulum Ca-ATPase.

We have investigated the role of large-scale protein rotational mobility in the reaction mechanism of the Ca-ATPase in sarcoplasmic reticulum using conditions that have previously been found to inhibit selectively phosphoenzyme decomposition, i.e. 1) partial delipidation (by detergent extraction or phospholipase treatment) and 2) the addition of nonaqueous solvents (dimethyl sulfoxide, glycerol, and sucrose). Using saturation-transfer electron paramagnetic resonance to probe the microsecond rotational motion of the spin-labeled Ca-ATPase, we find that both calcium-dependent ATPase activity and protein rotational mobility decrease in parallel, suggesting that protein mobility is important to the enzymatic step(s) involving phosphoenzyme decomposition. Using conventional EPR to measure the nanosecond rotational dynamics of spin-labeled lipid hydrocarbon chains, we find that neither the removal of lipid nor the addition of nonaqueous solvents significantly affects the lipid dynamics. We propose that the physical mode of inactivation under these conditions is the reduction in protein mobility through enforced protein-protein interactions, the result of which is a reduction in a motion essential for Ca-ATPase activity.

Calcium-dependent stabilization of the central sequence between Met(76) and Ser(81) in vertebrate calmodulin.

Spin-label electron paramagnetic resonance (EPR) provides optimal resolution of dynamic and conformational heterogeneity on the nanosecond time-scale and was used to assess the structure of the sequence between Met(76) and Ser(81) in vertebrate calmodulin (CaM). Previous fluorescence resonance energy transfer and anisotropy measurements indicate that the opposing domains of CaM are structurally coupled and the interconnecting central sequence adopts conformationally distinct structures in the apo-form and following calcium activation. In contrast, NMR data suggest that the opposing domains of CaM undergo independent rotational dynamics and that the sequence between Met(76) and Ser(81) in the central sequence functions as a flexible linker that connects two structurally independent domains. However, these latter measurements also resolve weak internuclear interactions that suggest the formation of transient helical structures that are stable on the nanosecond time-scale within the sequence between Met(76) and Asp(80) in apo-CaM (H. Kuboniwa, N. Tjandra, S. Grzekiek, H. Ren, C. B. Klee, and A. Bax, 1995, Nat. Struct. Biol. 2:768-776). This reported conformational heterogeneity was resolved using site-directed mutagenesis and spin-label EPR, which detects two component spectra for 1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)-methanethiosulfonate spin labels (MTSSL) bound to CaM mutants T79C and S81C that include a motionally restricted component. In comparison to MTSSL bound within stable helical regions, the fractional contribution of the immobilized component at these positions is enhanced upon the addition of small amounts of the helicogenic solvent trifluoroethanol (TFE). These results suggest that the immobilized component reflects the formation of stable secondary structures. Similar spectral changes are observed upon calcium activation, suggesting a calcium-dependent stabilization of the secondary structure. No corresponding changes are observed in either the solvent accessibility to molecular oxygen or the maximal hyperfine splitting. In contrast, more complex spectral changes in the line-shape and maximal hyperfine splitting are observed for spin labels bound to sites that undergo tertiary contact interactions. These results suggest that spin labels at solvent-exposed positions within the central sequence are primarily sensitive to backbone fluctuations and that either TFE or calcium binding stabilizes the secondary structure of the sequence between Met(76) and Ser(81) and modulates the structural coupling between the opposing domains of CaM.

Enhanced rotational dynamics of the phosphorylation domain of the Ca-ATPase upon calcium activation.

We have used labeling conditions that permit the specific and covalent attachment of erythrosin isothiocyanate (Er-ITC) to Lys464 within the phosphorylation domain of the Ca-ATPase in skeletal sarcoplasmic reticulum membranes. These labeling conditions do not interfere with high-affinity ATP binding, phosphoenzyme formation, or phosphoenzyme hydrolysis [Huang, S., Negash, S., and Squier, T. C. (1998) Biochemistry 37, 6949-6957]. Thus, we can use frequency-domain phosphorescence spectroscopy to measure the rotational dynamics of the Ca-ATPase stabilized in different enzymatic states corresponding to the absence of bound ligands (E), calcium activation (E x Ca2), the presence of bound nucleotide (E x ATP), and formation of phosphoenzyme (E-P). We resolve three rotational correlation times corresponding to (i) a large-amplitude domain motion of the phosphorylation domain (phi1 approximately 5 +/- 1 micros), (ii) overall protein rotational motion with respect to the membrane normal (phi2 approximately 50 +/- 10 micros), and (iii) the rotational motion of the SR vesicles (phi3 approximately 1.1 +/- 0.4 ms). No differences are observed in the rotational dynamics of E, E x ATP, or E-P, indicating that phosphoenzyme formation or nucleotide binding result in no global structural changes involving the phosphorylation domain. In contrast, calcium activation enhances the amplitude of motion of the phosphorylation domain. These observed calcium-dependent changes in rotational dynamics result from structural changes within a single Ca-ATPase polypeptide chain, since protein-protein interactions do not change upon calcium binding. Thus, calcium binding induces concerted domain motions within a single Ca-ATPase polypeptide chain that may play a critical role in facilitating substrate binding and utilization.

Variable conformation and dynamics of calmodulin complexed with peptides derived from the autoinhibitory domains of target proteins.

Calcium-saturated calmodulin (CaM) can bind and activate many target proteins through the direct association with the respective autoinhibitory domains. The CaM binding sequences within the autoinhibitory domains of these proteins have little sequence homology, and the mechanisms associated with CaM's ability to recognize and productively bind with these variable sequences is unclear. Common structural features of CaM bound to five peptides that are homologous to the autoinhibitory domains of smooth muscle myosin light chain kinase, CaM-dependent protein kinase II alpha, the plasma membrane Ca-ATPase, a MARCKS homolog, and glycogen phosphorylase kinase were assessed using frequency-domain fluorescence spectroscopy. In addition, the structural features of CaM complexed with the peptide melittin was also considered. We observe similar decreases in the average fluorescence lifetime and similar increases in the solvent accessibility of N-(1-pyrenyl)maleimide (PM) bound at Cys27 in calcium binding loop I in the amino terminal domain of CaM upon association with all six target peptides. Likewise, using fluorescence resonance energy transfer to measure the spatial separation between the opposing globular domains in CaM, we observe a similar spatial separation between the opposing globular domains of CaM bound to all six peptides. This indicates that CaM undergoes comparable structural changes upon association with all six target peptides. However, there are significant differences in the observed lifetime, solvent accessibility, correlation time associated with the segmented rotational motion of PM-CaM, and in the spatial separation between the opposing globular domains in CaM upon association with the individual target peptides, which indicates that CaM adopts a different tertiary structure that is dependent on the structural features of the bound target peptide. The correlation times associated with the overall hydrodynamic properties of CaM complexed with all six peptides are nearly identical (phi 2 approximately 10.6 +/- 0.4 ns) and are consistent with the known dimensions of CaM complexed to a peptide homologous to the CaM binding sequence of CaM-dependent protein kinase II alpha. Therefore, while these results are consistent with a common binding mechanism between CaM and all six target peptides, they indicate that the binding domains of CaM adopt different tertiary structures that allow them to bind with the variable sequences found in the autoinhibitory domains of target proteins with high affinity.

The sensitivity of carboxyl-terminal methionines in calmodulin isoforms to oxidation by H(2)O(2) modulates the ability to activate the plasma membrane Ca-ATPase.

The oxidative modification of methionines within the primary sequence of calmodulin (CaM) results in an inability to activate the PM-Ca-ATPase fully, and may contribute to alterations in calcium homeostasis under conditions of oxidative stress. To identify differences in the sensitivities of CaM isoforms to oxidative modification, we have compared the function and patterns of oxidative modification resulting from the exposure of CaM isolated from bovine testes and wheat germ to H(2)O(2). In comparison to CaM isolated from wheat germ, vertebrate CaM is functionally resistant to oxidant-induced loss of function. The decreased functional sensitivity of vertebrate CaM correlates with a 75 +/- 3% reduction in the rate of oxidative modification of a methionine near the carboxyl terminus (i.e., Met(144) or Met(145)). The extent of oxidative modification to other methionines in these CaM isoforms is similar. These results suggest that the sensitivity of Met(144) or Met(145) to oxidation modulates the ability of CaM to activate the PM-Ca-ATPase. Consistent with this interpretation, a CaM mutant in which glutamines were substituted for Met(144) and Met(145) fully activates the PM-Ca-ATPase irrespective of the oxidative modification of the other seven methionines to their corresponding methionine sulfoxides. The extent of oxidative modification to individual methionines in vertebrate CaM by H(2)O(2) correlates with the time-averaged surface accessibility of individual sulfurs calculated from molecular dynamics simulations. Thus, the sensitivity of individual methionines to oxidative modification is directly related to the solvent accessibility. These results indicate that sequence differences between vertebrate and plant CaM alter the sensitivity of methionines near the carboxyl terminus to oxidative modification because of alterations in their solvent accessibility. We suggest that these sequence differences between CaM isoforms have a regulatory role in modulating the functional sensitivity of CaM to conditions of oxidative stress.

Temperature dependence of rotational dynamics of protein and lipid in sarcoplasmic reticulum membranes.

We have investigated the relationship between function and molecular dynamics of both the lipid and the Ca-ATPase protein in sarcoplasmic reticulum (SR), using temperature as a means of altering both activity and rotational dynamics. Conventional and saturation-transfer electron paramagnetic resonance (EPR) was used to probe rotational motions of spin-labels attached either to fatty acid hydrocarbon chains or to the Ca-ATPase sulfhydryl groups in SR. EPR studies were also performed on aqueous dispersions of extracted SR lipids, in order to study intrinsic lipid properties independent of the protein. While an Arrhenius plot of the Ca-ATPase activity exhibits a clear change in slope at 20 degrees C, Arrhenius plots of lipid hydrocarbon chain mobility are linear, indicating that an abrupt thermotropic change in the lipid hydrocarbon phase is not responsible for the Arrhenius break in enzymatic activity. The presence of protein was found to decrease the average hydrocarbon chain mobility, but linear Arrhenius plots were observed both in the intact SR and in extracted lipids. Lipid EPR spectra were analyzed by procedures that prevent the production of artifactual breaks in the Arrhenius plots. Similarly, using sample preparations and spectral analysis methods that minimize the temperature-dependent contribution of local probe mobility to the spectra of spin-labeled Ca-ATPase, we find that Arrhenius plots of overall protein rotational mobility also exhibit no change in slope. The activation energy for protein mobility is the same as that of ATPase activity above 20 degrees C; we discuss the possibility that overall protein mobility may be essential to the rate-limiting step above 20 degrees C.