High-Yield α-Synuclein Purification and Ionic Strength Modification Pivotal to Seed Amplification Assay Performance and Reproducibility.

Alpha-synuclein seed amplification assays (αSyn-SAAs) have emerged as promising diagnostic tools for Parkinson's disease (PD) by detecting misfolded αSyn and amplifying the signal through cyclic shaking and resting in vitro. Recently, our group and others have shown that multiple biospecimens, including CSF, skin, and submandibular glands (SMGs), can be used to seed the aggregation reaction and robustly distinguish between patients with PD and non-disease controls. The ultrasensitivity of the assay affords the ability to detect minute quantities of αSyn in peripheral tissues, but it also produces various technical challenges of variability. To address the problem of variability, we present a high-yield αSyn protein purification protocol for the efficient production of monomers with a low propensity for self-aggregation. We expressed wild-type αSyn in BL21 Escherichia coli, lysed the cells using osmotic shock, and isolated αSyn using acid precipitation and fast protein liquid chromatography (FPLC). Following purification, we optimized the ionic strength of the reaction buffer to distinguish the fluorescence maximum (Fmax) separation between disease and healthy control tissues for enhanced assay performance. Our protein purification protocol yielded high quantities of αSyn (average: 68.7 mg/mL per 1 L of culture) and showed highly precise and robust αSyn-SAA results using brain, skin, and SMGs with inter-lab validation.

Defective pgsA contributes to increased membrane fluidity and cell wall thickening in Staphylococcus aureus with high-level daptomycin resistance.

Daptomycin is a membrane-targeting last-resort antimicrobial therapeutic for the treatment of infections caused by methicillin- and/or vancomycin-resistant Staphylococcus aureus. In the rare event of failed daptomycin therapy, the source of resistance is often attributable to mutations directly within the membrane phospholipid biosynthetic pathway of S. aureus or in the regulatory systems that control cell envelope response and membrane homeostasis. Here we describe the structural changes to the cell envelope in a daptomycin-resistant isolate of S. aureus strain N315 that has acquired mutations in the genes most commonly reported associated with daptomycin resistance: mprF, yycG, and pgsA. In addition to the decreased phosphatidylglycerol (PG) levels that are the hallmark of daptomycin resistance, the mutant with high-level daptomycin resistance had increased branched-chain fatty acids (BCFAs) in its membrane lipids, increased membrane fluidity, and increased cell wall thickness. However, the successful utilization of isotope-labeled straight-chain fatty acids (SCFAs) in lipid synthesis suggested that the aberrant BCFA:SCFA ratio arose from upstream alteration in fatty acid synthesis rather than a structural preference in PgsA. Transcriptomics studies revealed that expression of pyruvate dehydrogenase (pdhB) was suppressed in the daptomycin-resistant isolate, which is known to increase BCFA levels. While complementation with an additional copy of pdhB had no effect, complementation of the pgsA mutation resulted in increased PG formation, reduction in cell wall thickness, restoration of normal BCFA levels, and increased daptomycin susceptibility. Collectively, these results demonstrate that pgsA contributes to daptomycin resistance through its influence on membrane fluidity and cell wall thickness, in addition to phosphatidylglycerol levels. IMPORTANCE: The cationic lipopeptide antimicrobial daptomycin has become an essential tool for combating infections with Staphylococcus aureus that display reduced susceptibility to ß-lactams or vancomycin. Since daptomycin's activity is based on interaction with the negatively charged membrane of S. aureus, routes to daptomycin-resistance occur through mutations in the lipid biosynthetic pathway surrounding phosphatidylglycerols and the regulatory systems that control cell envelope homeostasis. Therefore, there are many avenues to achieve daptomycin resistance and several different, and sometimes contradictory, phenotypes of daptomycin-resistant S. aureus, including both increased and decreased cell wall thickness and membrane fluidity. This study is significant because it demonstrates the unexpected influence of a lipid biosynthesis gene, pgsA, on membrane fluidity and cell wall thickness in S. aureus with high-level daptomycin resistance.

Defective pgsA contributes to increased membrane fluidity and cell wall thickening in S. aureus with high-level daptomycin resistance.

Daptomycin is a membrane-targeting last-resort antimicrobial therapeutic for the treatment of infections caused by methicillin- and/or vancomycin-resistant Staphylococcus aureus. In the rare event of failed daptomycin therapy, the source of resistance is often attributable to mutations directly within the membrane phospholipid biosynthetic pathway of S. aureus or in the regulatory systems that control cell envelope response and membrane homeostasis. Here we describe the structural changes to the cell envelope in a daptomycin-resistant isolate of S. aureus strain N315 that has acquired mutations in the genes most commonly reported associated with daptomycin-resistance: mprF, yycG, and pgsA. In addition to the decreased phosphatidylglycerol (PG) levels that are the hallmark of daptomycin-resistance, the mutant with high-level daptomycin resistance had increased branched-chain fatty acids (BCFAs) in its membrane lipids, increased membrane fluidity, and increased cell wall thickness. However, the successful utilization of isotope-labeled straight-chain fatty acids (SCFAs) in lipid synthesis suggested that the aberrant BCFA:SCFA ratio arose from upstream alteration in fatty acid synthesis rather than a structural preference in PgsA. RT-qPCR studies revealed that expression of pyruvate dehydrogenase (pdhB) was suppressed in the daptomycin-resistant isolate, which is known to increase BCFA levels. While complementation with an additional copy of pdhB had no effect, complementation of the pgsA mutation resulted in increased PG formation, reduction in cell wall thickness, restoration of normal BCFA levels, and increased daptomycin susceptibility. Collectively, these results demonstrate that pgsA contributes to daptomycin resistance through its influence on membrane fluidity and cell wall thickness, in addition to phosphatidylglycerol levels.

Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor.

Bacillus anthracis adenylyl cyclase toxin edema factor (EF) is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM) leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, thus reducing production of reactive oxygen species (ROS) used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met) residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut) with Met to leucine (Leu) substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils.

Solution structure and properties of AlgH from Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the algH gene regulates the cellular concentrations of a number of enzymes and the production of several virulence factors, and is suggested to serve a global regulatory function. The precise mechanism by which the algH gene product, the AlgH protein, functions is unknown. The same is true for AlgH family members from other bacteria. In order to lay the groundwork for understanding the physical underpinnings of AlgH function, we examined the structure and physical properties of AlgH in solution. Under reducing conditions, results of NMR, electrophoretic mobility, and sedimentation equilibrium experiments indicate AlgH is predominantly monomeric and monodisperse in solution. Under nonreducing conditions intra and intermolecular disulfide bonds form, the latter promoting AlgH oligomerization. The high-resolution solution structure of AlgH reveals alpha/beta-sandwich architecture fashioned from ten beta strands and seven alpha helices. Comparison with available structures of orthologues indicates conservation of overall structural topology. The region of the protein most strongly conserved structurally also shows the highest amino acid sequence conservation and, as revealed by hydrogen-deuterium exchange studies, is also the most stable. In this region, evolutionary trace analysis identifies two clusters of amino acid residues with the highest evolutionary importance relative to all other AlgH residues. These frame a partially solvent exposed shallow hydrophobic cleft, perhaps identifying a site for intermolecular interactions. The results establish a physical foundation for understanding the structure and function of AlgH and AlgH family proteins and should be of general importance for further investigations of these and related proteins.

Membranous adenylyl cyclase 1 activation is regulated by oxidation of N- and C-terminal methionine residues in calmodulin.

Membranous adenylyl cyclase 1 (AC1) is associated with memory and learning. AC1 is activated by the eukaryotic Ca(2+)-sensor calmodulin (CaM), which contains nine methionine residues (Met) important for CaM-target interactions. During ageing, Met residues are oxidized to (S)- and (R)-methionine sulfoxide (MetSO) by reactive oxygen species arising from an age-related oxidative stress. We examined how oxidation by H2O2 of Met in CaM regulates CaM activation of AC1. We employed a series of thirteen mutant CaM proteins never assessed before in a single study, where leucine is substituted for Met, in order to analyze the effects of oxidation of specific Met. CaM activation of AC1 is regulated by oxidation of all of the C-terminal Met in CaM, and by two N-terminal Met, M36 and M51. CaM with all Met oxidized is unable to activate AC1. Activity is fully restored by the combined catalytic activities of methionine sulfoxide reductases A and B (MsrA and B), which catalyze reduction of the (S)- and (R)-MetSO stereoisomers. A small change in secondary structure is observed in wild-type CaM upon oxidation of all nine Met, but no significant secondary structure changes occur in the mutant proteins when Met residues are oxidized by H2O2, suggesting that localized polarity, flexibility and structural changes promote the functional changes accompanying oxidation. The results signify that AC1 catalytic activity can be delicately adjusted by mediating CaM activation of AC1 by reversible Met oxidation in CaM. The results are important for memory, learning and possible therapeutic routes for regulating AC1.

Proton-induced reactivity of NO⁻ from a {CoNO}8 complex.

Research on the one-electron reduced analogue of NO, namely nitroxyl (HNO/NO(-)), has revealed distinguishing properties regarding its utility as a therapeutic. However, the fleeting nature of HNO requires the design of donor molecules. Metal nitrosyl (MNO) complexes could serve as potential HNO donors. The synthesis, spectroscopic/structural characterization, and HNO donor properties of a {CoNO}(8) complex in a pyrrole/imine ligand frame are reported. The {CoNO}(8) complex [Co(LN4(PhCl))(NO)] (1) does not react with established HNO targets such as Fe(III) hemes or Ph3P. However, in the presence of stoichiometric H(+) 1 behaves as an HNO donor. Complex 1 readily reacts with [Fe(TPP)Cl] or Ph3P to afford the {FeNO}(7) porphyrin or Ph3P═O/Ph3P═NH, respectively. In the absence of an HNO target, the {Co(NO)2}(10) dinitrosyl (3) is the end product. Complex 1 also reacts with O2 to yield the corresponding Co(III)-η(1)-ONO2 (2) nitrato analogue. This report is the first to suggest an HNO donor role for {CoNO}(8) with biotargets such as Fe(III)-porphyrins.

Determinants of affinity and activity of the anti-sigma factor AsiA.

The AsiA protein is a T4 bacteriophage early gene product that regulates transcription of host and viral genes. Monomeric AsiA binds tightly to the sigma(70) subunit of Escherichia coli RNA polymerase, thereby inhibiting transcription from bacterial promoters and phage early promoters and coactivating transcription from phage middle promoters. Results of structural studies have identified amino acids at the protomer-protomer interface in dimeric AsiA and at the monomeric AsiA-sigma(70) interface and demonstrated substantial overlap in the sets of residues that comprise each. Here we evaluate the contributions of individual interfacial amino acid side chains to protomer-protomer affinity in AsiA homodimers, to monomeric AsiA affinity for sigma(70), and to AsiA function in transcription. Sedimentation equilibrium, dynamic light scattering, electrophoretic mobility shift, and transcription activity measurements were used to assess affinity and function of site-specific AsiA mutants. Alanine substitutions for solvent-inaccessible residues positioned centrally in the protomer-protomer interface of the AsiA homodimer, V14, I17, and I40, resulted in the largest changes in free energy of dimer association, whereas alanine substitutions at other interfacial positions had little effect. These residues also contribute significantly to AsiA-dependent regulation of RNA polymerase activity, as do additional residues positioned at the periphery of the interface (K20 and F21). Notably, the relative contributions of a given amino acid side chain to RNA polymerase inhibition and activation (MotA-independent) by AsiA are very similar in most cases. The mainstay for intermolecular affinity and AsiA function appears to be I17. Our results define the core interfacial residues of AsiA, establish roles for many of the interfacial amino acids, are in agreement with the tenets underlying protein-protein interactions and interfaces, and will be beneficial for a general, comprehensive understanding of the mechanistic underpinnings of bacterial RNA polymerase regulation.

Mechanism of calmodulin recognition of the binding domain of isoform 1b of the plasma membrane Ca(2+)-ATPase: kinetic pathway and effects of methionine oxidation.

Calmodulin (CaM) binds to a domain near the C-terminus of the plasma membrane Ca2+-ATPase (PMCA), causing the release of this domain and relief of its autoinhibitory function. We investigated the kinetics of dissociation and binding of Ca2+-CaM with a 28-residue peptide [C28W(1b)] corresponding to the CaM-binding domain of isoform 1b of PMCA. CaM was labeled with a fluorescent probe on either the N-terminal domain at residue 34 or the C-terminal domain at residue 110. Formation of complexes of CaM with C28W(1b) results in a decrease in the fluorescence yield of the fluorophore, allowing the kinetics of dissociation or binding to be detected. Using a maximum entropy method, we determined the minimum number and magnitudes of rate constants required to fit the data. Comparison of the fluorescence changes for CaM labeled on the C-terminal or N-terminal domain suggests sequential and ordered binding of the C-terminal and N-terminal domains of CaM with C28W(1b). For dissociation of C28W(1b) from CaM labeled on the N-terminal domain, we observed three time constants, indicating the presence of two intermediate states in the dissociation pathway. However, for CaM labeled on the C-terminal domain, we observed only two time constants, suggesting that the fluorescence label on the C-terminal domain was not sensitive to one of the kinetic steps. The results were modeled by a kinetic mechanism in which an initial complex forms upon binding of the C-terminal domain of CaM to C28W(1b), followed by binding of the N-terminal domain, and then formation of a tight binding complex. Oxidation of methionine residues in CaM resulted in significant perturbations to the binding kinetics. The rate of formation of a tight binding complex was reduced, consistent with the poorer effectiveness of oxidized CaM in activating the Ca2+ pump.

High-affinity and cooperative binding of oxidized calmodulin by methionine sulfoxide reductase.

Methionines can play an important role in modulating protein-protein interactions associated with intracellular signaling, and their reversible oxidation to form methionine sulfoxides [Met(O)] in calmodulin (CaM) and other signaling proteins has been suggested to couple cellular redox changes to protein functional changes through the action of methionine sulfoxide reductases (Msr). Prior measurements indicate the full recovery of target protein activation upon the stereospecific reduction of oxidized CaM by MsrA, where the formation of the S-stereoisomer of Met(O) selectively inhibits the CaM-dependent activation of the Ca-ATPase. However, the physiological substrates of MsrA remain unclear, as neither the binding specificities nor affinities of protein targets have been measured. To assess the specificity of binding and its possible importance in the maintenance of CaM function, we have measured the kinetics of repair and the binding affinity between oxidized CaM and MsrA. Reduction of Met(O) in fully oxidized CaM by MsrA is sensitive to the protein fold, as repair of the intact protein is incomplete, with >6 Met(O) remaining in each CaM following MsrA reduction. In contrast, following proteolytic digestion, MsrA is able to fully reduce one-half of the oxidized methionines, indicating that surface-accessible Met(O) within folded proteins need not be substrates for MsrA repair. Mutation of the active site (i.e., C72S) in MsrA permitted equilibrium-binding measurements using both ensemble and single-molecule fluorescence correlation spectroscopy measurements. We observe cooperative binding of two MsrA to each CaMox with an apparent affinity (K = 70 +/- 10 nM) that is 3 orders of magnitude greater than the Michaelis constant (KM = 68 +/- 4 microM). The high-affinity and cooperative interaction between MsrA and CaMox suggests an important regulatory role of MsrA in the binding and reduction of Met(O) in functionally sensitive proteins, such that multiple MsrA proteins are recruited to simultaneously bind and reduce Met(O) in highly oxidized proteins. Given the suggested role of Met(O) in modulating reversible binding interactions between proteins associated with cellular signaling, these results indicate an ability of MsrA to selectively reduce Met(O) within highly surface-accessible sequences to maintain cellular function as part of an adaptive response to oxidative stress.

Structural and functional effects of tryptophans inserted into the membrane-binding and substrate-binding sites of human group IIA phospholipase A2.

Phospholipase A(2) (PLA(2)) enzymes become activated by binding to biological membranes and hydrolyze phospholipids to free fatty acids and lyso-phospholipids, the precursors of inflammatory mediators. To understand the functional significance of amino acid residues at key positions, we have studied the effects of the substitution of Val(3) (membrane binding surface) and Phe(5) (substrate binding pocket) of human group IIA PLA(2) by tryptophan on the structure and function of the enzyme. Despite the close proximity of the sites of mutations, the V3W mutation results in substantial enhancement of the enzyme activity, whereas the F5W mutant demonstrates significantly suppressed activity. A structural analysis of all three proteins free in buffer and bound to membranes indicates that large differences in activities result from distinct conformational changes in PLA(2)s upon membrane binding. Although PLA(2) and the V3W mutant demonstrate a decrease in helical content and an increase in helix flexibility, the F5W mutant experiences partial distortion of the alpha-helical structure presumably resulting from the tendency of Trp(5) to insert into the membrane. Furthermore, whereas the PLA(2) and the V3W mutant bind to the membrane at similar and apparently productive-mode orientation, the F5W mutant binds to membranes with a distinctly different orientation. It is suggested that both the stimulatory effect of the V3W mutation and the inhibitory effect of the F5W mutation result from the high affinity of Trp for the membrane-water interface. Although Trp(3) at the membrane binding face of PLA(2) facilitates the proper membrane binding of the enzyme, Trp(5) in the internal substrate binding site causes partial unwinding of the N-terminal helix in order to interact with the membrane.

Single-molecule characterization of the dynamics of calmodulin bound to oxidatively modified plasma-membrane Ca2+-ATPase.

We used single-molecule fluorescence spectroscopy to probe the conformation of calmodulin (CaM) bound to oxidatively modified plasma-membrane Ca(2+)-ATPase (PMCAox). We found that oxidative modification altered the coupling between the ATP binding domain and the autoinhibitory domain. Oxidative modification of PMCA is known to result in a loss of activity for the enzyme. Conformations of PMCAox-CaM complexes were probed by single-molecule polarization modulation spectroscopy, which measured the orientational mobility of fluorescently labeled CaM bound to PMCAox. We detected an enhanced population of PMCAox-CaM complexes with a low orientational mobility in the presence of ATP, whereas nonoxidized PMCA-CaM complexes existed almost exclusively in a high-mobility state in the presence of ATP. We have previously attributed such high-mobility states to PMCA-CaM complexes with a dissociated autoinhibitory/CaM binding domain, whereas the lower-mobility state was attributed to autoinhibited PMCA-CaM complexes with a nondissociated autoinhibitory domain [Osborn, K. D., et al. (2004) Biophys. J. 87, 1892-1899]. In the absence of ATP, the orientational mobility distributions are similar for CaM complexed with oxidized PMCA or nonoxidized PMCA. These results suggest that oxidative modification of PMCA reduced the propensity of the autoinhibitory domain to dissociate from binding sites near the catalytic core of the enzyme with bound nucleotide upon CaM stimulation in the presence of Ca(2+). This interpretation was further supported by chymotrypsin proteolysis, which probes the tightness of binding of the autoinhibitory domain to sites near the catalytic core of the enzyme. Enhanced proteolysis was observed for PMCA upon binding CaM or ATP. In contrast, proteolysis was partially blocked for oxidatively modified PMCA, even in the presence of ATP.

Cloning, high yield overexpression, purification, and characterization of AlgH, a regulator of alginate biosynthesis in Pseudomonas aeruginosa.

The most common cause of mortality among cystic fibrosis sufferers is infection by antibiotic resistant strains of Pseudomonas aeruginosa. Means to control these strains continue to be an important goal. An integral component of the ability of many of these strains to defy antibiotic therapies is the protection afforded by the mucoexopolysaccharide alginate. Production of alginate by P. aeruginosa is tightly regulated at the transcriptional level. AlgH, a putative transcriptional regulator, is involved in regulating alginate biosynthesis as well as nucleoside diphosphate kinase activity and succinyl coenzyme A synthetase activity in P. aeruginosa. Sequence homologues are found in many bacterial species. Here, we describe a method for high level overexpression and high yield/high purity production of AlgH for biophysical and functional studies. The algH gene was cloned and AlgH was overexpressed in Escherichia coli using a commercially available vector with an inducible T7 promoter. We purified the recombinantly produced protein using a rapid classical purification scheme. The yield of purified protein, either isotopically labeled for NMR studies or unlabeled, is excellent (30-37 mg of purified protein per liter of minimal media culture), as is the purity (>95% pure). Analysis of the secondary structure using circular dichroism and NMR indicates that the protein is comprised of both beta-sheet and alpha-helical secondary structural elements. Heteronuclear NMR spectra indicate that AlgH is a monodisperse, folded globular protein. This rapid, high yield, and high purity method for AlgH production will permit further biophysical characterization of this protein including high resolution structural studies.

Sampling unfolding intermediates in calmodulin by single-molecule spectroscopy.

We used single-pair fluorescence resonance energy transfer (spFRET) measurements to characterize denatured and partially denatured states of the multidomain calcium signaling protein calmodulin (CaM) in both its apo and Ca(2+)-bound forms. The results demonstrate the existence of an unfolding intermediate. A CaM mutant (CaM-T34C-T110C) was doubly labeled with fluorescent probes AlexaFlour 488 and Texas Red at opposing globular domains. Single-molecule distributions of the distance between fluorophores were obtained by spFRET at varying levels of the denaturant urea. Multiple conformational states of CaM were observed, and the amplitude of each conformation was dependent on urea concentration, with the amplitude of an extended conformation increasing upon denaturation. The distributions at intermediate urea concentrations could not be adequately described as a combination of native and denatured conformations, showing that CaM does not denature via a two-state process and demonstrating that at least one intermediate is present. The intermediate conformations formed upon addition of urea were different for Ca(2+)-CaM and apoCaM. An increase in the amplitude of a compact conformation in CaM was observed for apoCaM but not for Ca(2+)-CAM upon the addition of urea. The changes in the single-molecule distributions of CaM upon denaturation can be described by either a range of intermediate structures or by the presence of a single unfolding intermediate that grows in amplitude upon denaturation. A model for stepwise unfolding of CaM is suggested in which the domains of CaM unfold sequentially.

Mediating molecular recognition by methionine oxidation: conformational switching by oxidation of methionine in the carboxyl-terminal domain of calmodulin.

The C-terminus of calmodulin (CaM) functions as a sensor of oxidative stress, with oxidation of methionine 144 and 145 inducing a nonproductive association of the oxidized CaM with the plasma membrane Ca(2+)-ATPase (PMCA) and other target proteins to downregulate cellular metabolism. To better understand the structural underpinnings and mechanism of this switch, we have engineered a CaM mutant (CaM-L7) that permits the site-specific oxidation of M144 and M145, and we have used NMR spectroscopy to identify structural changes in CaM and CaM-L7 and changes in the interactions between CaM-L7 and the CaM-binding sequence of the PMCA (C28W) due to methionine oxidation. In CaM and CaM-L7, methionine oxidation results in nominal secondary structural changes, but chemical shift changes and line broadening in NMR spectra indicate significant tertiary structural changes. For CaM-L7 bound to C28W, main chain and side chain chemical shift perturbations indicate that oxidation of M144 and M145 leads to large tertiary structural changes in the C-terminal hydrophobic pocket involving residues that comprise the interface with C28W. Smaller changes in the N-terminal domain also involving residues that interact with C28W are observed, as are changes in the central linker region. At the C-terminal helix, (1)H(alpha), (13)C(alpha), and (13)CO chemical shift changes indicate decreased helical character, with a complete loss of helicity for M144 and M145. Using (13)C-filtered, (13)C-edited NMR experiments, dramatic changes in intermolecular contacts between residues in the C-terminal domain of CaM-L7 and C28W accompany oxidation of M144 and M145, with an essentially complete loss of contacts between C28W and M144 and M145. We propose that the inability of CaM to fully activate the PMCA after methionine oxidation originates in a reduced helical propensity for M144 and M145, and results primarily from a global rearrangement of the tertiary structure of the C-terminal globular domain that substantially alters the interaction of this domain with the PMCA.

Conformational substates of calmodulin revealed by single-pair fluorescence resonance energy transfer: influence of solution conditions and oxidative modification.

A calmodulin (CaM) mutant (T34,110C-CaM) doubly labeled with fluorescence probes AlexaFluor 488 and Texas Red in opposing domains (CaM-DA) has been used to examine conformational heterogeneity in CaM by single-pair fluorescence resonance energy transfer (spFRET). Burst-integrated FRET efficiencies of freely diffusing CaM-DA single molecules yielded distributions of distance between domains of CaM-DA. We recently reported distinct conformational substates of Ca(2+)-CaM-DA and apoCaM-DA, with peaks in the distance distributions centered at approximately 28 A, 34-38 A, and 55 A [Slaughter et al. (2004) J. Phys. Chem. B 108, 10388-10397]. In the present study, shifts in the amplitudes and center distances of the conformational substates were detected with variation in solution conditions. The amplitude of an extended conformation was observed to change as a function of Ca(2+) over a free Ca(2+) range that is consistent with binding to the high affinity, C-terminal Ca(2+) binding sites, suggesting the existence of communication between lobes of CaM. Lowering pH shifted the relative amplitudes of the conformations, with a marked increase in the presence of the compact conformations and an almost complete absence of the extended conformation. In addition, the single-molecule distance distribution of apoCaM-DA at reduced ionic strength was shifted to longer distance and showed evidence of an increase in conformational heterogeneity relative to apoCaM-DA at physiological ionic strength. Oxidation of methionine residues in CaM-DA produced a substantial increase in the amplitude of the extended conformation relative to the more compact conformation. The results are considered in light of a hypothesis that suggests that electrostatic interactions between charged amino acid side chains play an important role in determining the most stable CaM conformation under varying solution conditions.

Single-molecule tracking of sub-millisecond domain motion in calmodulin.

We used single-pair fluorescence resonance energy transfer (spFRET) to track distance changes between domains of fluorescently labeled calmodulin (CaM) on the sub-millisecond time scale. In most cases, CaM remained in the same conformational substate over time periods of up to 1 ms, showing that conformational interchange occurs on a longer time scale. However, in some instances, apparent transitions between conformational substates could be detected. The magnitude of sub-millisecond motion within the dominant conformational substate also revealed fluctuations in distance between domains that were dependent on pH and ionic strength.

Single-molecule dynamics reveal an altered conformation for the autoinhibitory domain of plasma membrane Ca(2+)-ATPase bound to oxidatively modified calmodulin.

We used single-molecule polarization modulation methods to investigate the activation of the plasma membrane Ca(2+)-ATPase (PMCA) by oxidized calmodulin (CaM). Oxidative modification of methionine residues of CaM to their corresponding sulfoxides is known to inhibit the ability of CaM to activate PMCA. Single-molecule polarization methods were used to measure the orientational mobility of fluorescently labeled oxidized CaM bound to PMCA. We previously identified two distinct populations of PMCA-CaM complexes characterized by high and low orientational mobilities, with the low-mobility population appearing at a subsaturating Ca(2+) concentration [Osborn, K. D., et al. (2004) Biophys. J. 87, 1892-1899]. We proposed that the high-mobility population corresponds to PMCA-CaM complexes with a dissociated (and mobile) autoinhibitory domain, whereas the low-mobility population corresponds to PMCA-CaM complexes where the autoinhibitory domain is not dissociated and therefore the enzyme is not active. In the present experiments, performed with PMCA complexed with oxidatively modified CaM at a saturating Ca(2+) concentration, we found a large population of molecules with an orientationally immobile autoinhibitory domain. In contrast, native CaM bound to PMCA was characterized almost entirely by the more orientationally mobile population at a similar Ca(2+) concentration. The addition of 1 mM ATP to complexes of oxidized CaM with PMCA reduced but did not abolish the low-mobility population. These results indicate that the decline in the ability of oxidized CaM to activate PMCA results at least in part from its reduced ability to induce conformational changes in PMCA that result in dissociation of the autoinhibitory domain after CaM binding.

Single-molecule dynamics of the calcium-dependent activation of plasma-membrane Ca2+-ATPase by calmodulin.

The plasma membrane calcium-ATPase (PMCA) helps to control cytosolic calcium levels by pumping out excess Ca2+. PMCA is regulated by the Ca2+ signaling protein calmodulin (CaM), which stimulates PMCA activity by binding to an autoinhibitory domain of PMCA. We used single-molecule polarization methods to investigate the mechanism of regulation of the PMCA by CaM fluorescently labeled with tetramethylrhodamine. The orientational mobility of PMCA-CaM complexes was determined from the extent of modulation of single-molecule fluorescence upon excitation with a rotating polarization. At a high Ca2+ concentration, the distribution of modulation depths reveals that CaM bound to PMCA is orientationally mobile, as expected for a dissociated autoinhibitory domain of PMCA. In contrast, at a reduced Ca2+ concentration a population of PMCA-CaM complexes appears with significantly reduced orientational mobility. This population can be attributed to PMCA-CaM complexes in which the autoinhibitory domain is not dissociated, and thus the PMCA is inactive. The presence of these complexes demonstrates the inadequacy of a two-state model of Ca2+ pump activation and suggests a regulatory role for the low-mobility state of the complex. When ATP is present, only the high-mobility state is detected, revealing an altered interaction between the autoinhibitory and nucleotide-binding domains.