Rapid, high-yield expression and purification of Ca2+-ATPase regulatory proteins for high-resolution structural studies

Phospholamban (PLB) and sarcolipin (SLN) are small integral membrane proteins that regulate the Ca(2+)-ATPases of cardiac and skeletal muscle, respectively, and directly alter their calcium transport properties. PLB interacts with and regulates the cardiac Ca(2+)-ATPase at submaximal calcium concentrations, thereby slowing relaxation rates and reducing contractility in the heart. SLN interacts with and regulates the skeletal muscle Ca(2+)-ATPase in a mechanism analogous to that used by PLB. While these regulatory interactions are biochemically and physiologically well characterized, structural details are lacking. To pursue structural studies, such as electron cryo-microscopy and X-ray crystallography, large quantities of over-expressed and purified protein are required. Herein, we report a modified method for producing large quantities of PLB and SLN in a rapid and efficient manner. Briefly, recombinant wild-type PLB and SLN were over-produced in Escherichia coli as maltose binding protein fusion proteins. A tobacco etch virus protease site allowed specific cleavage of the fusion protein and release of recombinant PLB or SLN. Selective solubilization with guanidine-hydrochloride followed by reverse-phase HPLC permitted the rapid, large-scale production of highly pure protein. Reconstitution and measurement of ATPase activity confirmed the functional interaction between our recombinant regulatory proteins and Ca(2+)-ATPase. The inhibitory properties of the over-produced proteins were consistent with previous studies, where the inhibition was relieved by elevated calcium concentrations. In addition, we show that our recombinant PLB and SLN are suitable for high-resolution structural studies.     

Sarcolipin, the shorter homologue of phospholamban, forms oligomeric structures in detergent micelles and in liposomes.

The human 31-amino acid integral membrane protein sarcolipin (SLN), which regulates the sarcoplasmic reticulum Ca-ATPase in fast-twitch skeletal muscle, was chemically synthesized. Appropriate synthesis and purification strategies were used to achieve high purity and satisfactory yields of this hydrophobic and poorly soluble protein. Structural and functional properties of SLN were analyzed and compared with the homologous region of human phospholamban (PLB) comprising residues Ala(24)-Leu(52) (PLB-(24-52)), the regulatory protein of the cardiac sarcoplasmic reticulum Ca-ATPase. Circular dichroism spectroscopy showed that SLN is a predominantly alpha-helical protein and that the secondary structure is highly resistant to SDS and thermal denaturation. In this respect SLN is remarkably similar to PLB-(24-52). However, SLN is monomeric in SDS gels, whereas PLB-(24-52) shows a monomer-pentamer equilibrium typical for native PLB. Analytical ultracentrifugation experiments revealed that SLN oligomerizes in the presence of the nonionic detergents octylpolyoxyethylene and octyl glucoside in a concentration-dependent manner. No plateau was observed, and a pentameric state was only reached at much higher protein concentrations compared with PLB-(24-52). Chemical cross-linking showed that also in liposomes SLN has the ability to self-associate to oligomers. PLB-(24-52) specifically oligomerized to pentamers in the presence of octylpolyoxyethylene as well as in liposomes at low protein concentrations. In the presence of octylpolyoxyethylene pentamers were the main oligomeric species, whereas in liposomes monomers and dimers were predominant. Increasing the protein concentration led to self-association of PLB-(24-52) pentamers in the presence of octylpolyoxyethylene. Functional reconstitution of Ca-ATPase with PLB-(24-52) and SLN in liposomes showed that both proteins regulate the Ca-ATPase in a similar manner.     

Structure and orientation of sarcolipin in lipid environments.

Sarcolipin (SLN) is a 31 amino acid integral membrane protein that regulates Ca-ATPase activity in skeletal muscle. Here, we report the three-dimensional structure and topology of synthetic SLN in lipid environments, as determined by solution and solid-state NMR spectroscopy. 2D solution NMR experiments were performed on SLN solubilized in sodium dodecyl sulfate (SDS) micelles. We found that SLN adopts a highly defined alpha-helical conformation from F9 through R27, with a backbone RMSD of 0.65 A and a side chain RMSD of 1.66 A. The N-terminus (M1 through L8) and the C-terminus (S28 through Y31) are mostly unstructured. The orientation of the SLN was determined using one-dimensional (15)N NMR solid-state spectroscopy. The protein was incorporated into phospholipid bilayers prepared from a mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine. The (15)N chemical shift solid-state spectra from selectively labeled SLN samples indicate that SLN orients perpendicularly to the plane of the membrane bilayers. These results support the proposed mechanism of Ca-ATPase regulation of SLN via protein-protein intramembranous interactions between the highly conserved transmembrane domains of the Ca-ATPase and the conserved transmembrane domain of SLN.     

Comparable levels of Ca-ATPase inhibition by phospholamban in slow-twitch skeletal and cardiac sarcoplasmic reticulum

Alterations in expression levels of phospholamban (PLB) relative to the sarcoplasmic reticulum (SR) Ca-ATPase have been suggested to underlie defects of calcium regulation in the failing heart and other cardiac pathologies. To understand how variation in PLB expression relative to that of the Ca-ATPase can modulate calcium transport, we have investigated the inhibition of the Ca-ATPase by PLB in native SR membranes from slow-twitch skeletal and cardiac muscle and in reconstituted proteoliposomes. Quantitative immunoblotting in combination with affinity-purified protein standards was used to measure protein concentrations of PLB and of the Ca-ATPase. Functional inhibition of the Ca-ATPase was determined from both the calcium concentrations for half-maximal activation (Ca(1/2)) and the shift in the calcium concentrations following release of PLB inhibition (i.e., (Delta)Ca(1/2)) by incubation with monoclonal antibodies against PLB, which are equivalent to phosphorylation of PLB by cAMP-dependent protein kinase. We report that equivalent levels of PLB inhibition and antibody-induced activation ((Delta)Ca(1/2) = 0.25 +/- 0.02 microM) are observed in SR membranes from slow-twitch skeletal and cardiac muscle, where molar stoichiometries of PLB expressed per Ca-ATPase vary, respectively, from 0.9 +/- 0.1 to 4.1 +/- 0.8. Similar levels of inhibition to those observed in isolated SR vesicles were observed using reconstituted proteoliposomes following co-reconstitution of affinity-purified Ca-ATPase with PLB. These results indicate that total expression levels of one PLB per Ca-ATPase result in full inhibition of the Ca-ATPase and, based on the measured K(D) (140 +/- 30 microM), suggests one PLB complexed with two Ca-ATPase molecules is sufficient for full inhibition of activity. Therefore, the excess PLB expressed in the heart over that required for inhibition suggests a capability for graded responses of the Ca-ATPase activity to endogenous kinases and phosphatases that modulate the level of phosphorylation necessary to relieve inhibition of the Ca-ATPase by PLB.     

Novel strategies to overcome expression problems encountered with toxic proteins: Application to the production of Lac repressor proteins for NMR studies

NMR studies of structural aspects of allosteric regulation by the Lac repressor requires overexpression and isotope labeling of the protein. The size of the repressor makes it a challenging target, putting constraints on both expression conditions and sample preparation methods to overcome problems associated with studies of larger proteins by NMR. We optimized protocols for the production of deuterated functionally active thermostable dimeric Lac repressor and its core domain mutants. The Lac repressor core domain has never been obtained as a recombinant protein, possibly due to the observed toxicity to the host cells. We overcame the core domain induced toxicity by co-expression of this domain with the full length Lac repressor, combined with a stringent control of culture conditions. Significant overexpression was only obtained if during all stages of pre-culturing the bacteria were kept in their exponential growth phase at low density. The sensitivity of NMR measurements is dramatically affected by buffer conditions; we therefore used a thermofluor buffer optimization screen to determine the optimal buffer conditions. The combined thermofluor and NMR screening method yielded thermostable fully functional Lac repressor domain samples suitable for high-resolution NMR studies. The optimized procedures to adapt Escherichia coli to growth in D₂O, to overcome toxicity, and to optimize protein sample conditions provides a broad range of universally applicable techniques for production of larger proteins for NMR spectroscopy.     

The N Terminus of Sarcolipin Plays an Important Role in Uncoupling Sarco-endoplasmic Reticulum Ca2+-ATPase (SERCA) ATP Hydrolysis from Ca2+ Transport

The sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA) is responsible for intracellular Ca²⁺ homeostasis. SERCA activity in muscle can be regulated by phospholamban (PLB), an affinity modulator, and sarcolipin (SLN), an uncoupler. Although PLB gets dislodged from Ca²⁺-bound SERCA, SLN continues to bind SERCA throughout its kinetic cycle and promotes uncoupling of Ca²⁺ transport from ATP hydrolysis. To determine the structural regions of SLN that mediate uncoupling of SERCA, we employed mutagenesis and generated chimeras of PLB and SLN. In this study we demonstrate that deletion of SLN N-terminal residues ²ERSTQ leads to loss of the uncoupling function even though the truncated peptide can target and constitutively bind SERCA. Furthermore, molecular dynamics simulations of SLN and SERCA interaction showed a rearrangement of SERCA residues that is altered when the SLN N terminus is deleted. Interestingly, transfer of the PLB cytosolic domain to the SLN transmembrane (TM) and luminal tail causes the chimeric protein to lose SLN-like function. Further introduction of the PLB TM region into this chimera resulted in conversion to full PLB-like function. We also found that swapping PLB N and C termini with those from SLN caused the resulting chimera to acquire SLN-like function. Swapping the C terminus alone was not sufficient for this conversion. These results suggest that domains can be switched between SLN and PLB without losing the ability to regulate SERCA activity; however, the resulting chimeras acquire functions different from the parent molecules. Importantly, our studies highlight that the N termini of SLN and PLB influence their respective unique functions.     

Oligomeric interactions of sarcolipin and the Ca-ATPase

We have detected directly the interactions of sarcolipin (SLN) and the sarcoplasmic reticulum Ca-ATPase (SERCA) by measuring fluorescence resonance energy transfer (FRET) between fusion proteins labeled with cyan fluorescent protein (donor) and yellow fluorescent protein (acceptor). SLN is a membrane protein that helps control contractility by regulating SERCA activity in fast-twitch and atrial muscle. Here we used FRET microscopy and spectroscopy with baculovirus expression in insect cells to provide direct evidence for: 1) oligomerization of SLN and 2) regulatory complex formation between SLN and the fast-twitch muscle Ca-ATPase (SERCA1a isoform). FRET experiments demonstrated that SLN monomers self-associate into dimers and higher order oligomers in the absence of SERCA, and that SLN monomers also bind to SERCA monomers in a 1:1 binary complex when the two proteins are coexpressed. FRET experiments further demonstrated that the binding affinity of SLN for itself is similar to that for SERCA. Mutating SLN residue isoleucine-17 to alanine (I17A) decreased the binding affinity of SLN self-association and converted higher order oligomers into monomers and dimers. The I17A mutation also decreased SLN binding affinity for SERCA but maintained 1:1 stoichiometry in the regulatory complex. Thus, isoleucine-17 plays dual roles in determining the distribution of SLN homo-oligomers and stabilizing the formation of SERCA-SLN heterodimers. FRET results for SLN self-association were supported by the effects of SLN expression in bacterial cells. We propose that SLN exists as multiple molecular species in muscle, including SERCA-free (monomer, dimer, oligomer) and SERCA-bound (heterodimer), with transmembrane zipper residues of SLN serving to stabilize oligomeric interactions.     

Protein-Protein Interactions in Calcium Transport Regulation Probed by Saturation Transfer Electron Paramagnetic Resonance

We have used electron paramagnetic resonance (EPR) to probe the homo- and heterooligomeric interactions of reconstituted sarcoplasmic reticulum Ca-ATPase (SERCA) and its regulator phospholamban (PLB). SERCA is responsible for restoring calcium to the sarcoplasmic reticulum to allow muscle relaxation, whereas PLB inhibits cardiac SERCA unless phosphorylated at Ser¹⁶. To determine whether changes in protein association play essential roles in regulation, we detected the microsecond rotational diffusion of both proteins using saturation transfer EPR. Peptide synthesis was used to create a fully functional and monomeric PLB mutant with a spin label rigidly coupled to the backbone of the transmembrane helix, while SERCA was reacted with a Cys-specific spin label. Saturation transfer EPR revealed that sufficiently high lipid/protein ratios minimized self-association for both proteins. Under these dilute conditions, labeled PLB was substantially immobilized after co-reconstitution with unlabeled SERCA, reflecting their association to form the regulatory complex. Ser¹⁶ phosphorylation slightly increased this immobilization. Complementary measurements with labeled SERCA showed no change in mobility after co-reconstitution with unlabeled PLB, regardless of its phosphorylation state. We conclude that phosphorylating monomeric PLB can relieve SERCA inhibition without changes in the oligomeric states of these proteins, indicating a structural rearrangement within the heterodimeric regulatory complex.     

Tuning different expression parameters to achieve soluble recombinant proteins in E. coli: advantages of high-throughput screening

Proteins are the main reagents for structural, biomedical, and biotechnological studies; however, some important challenges remain concerning protein solubility and stability. Numerous strategies have been developed, with some success, to mitigate these challenges, but a universal strategy is still elusive. Currently, researchers face a plethora of alternatives for the expression of the target protein, which generates a great diversity of conditions to be evaluated. Among these, different promoter strength, diverse expression host and constructs, or special culture conditions have an important role in protein solubility. With the arrival of automated high-throughput screening (HTS) systems, the evaluation of hundreds of different conditions within reasonable cost and time limits is possible. This technology increases the chances to obtain the target protein in a pure, soluble, and stable state. This review focuses on some of the most commonly used strategies for the expression of recombinant proteins in the enterobacterium Escherichia coli, including the use of HTS for the production of soluble proteins.     

Structure and function of integral membrane protein domains resolved by peptide-amphiphiles: application to phospholamban

We have used synthetic lipidated peptides ("peptide-amphiphiles") to study the structure and function of isolated domains of integral transmembrane proteins. We used 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis to prepare full-length phospholamban (PLB(1-52)) and its cytoplasmic (PLB(1-25)K: phospholamban residues 1-25 plus a C-terminal lysine), and transmembrane (PLB(26-52)) domains, and a 38-residue model alpha-helical sequence as a control. We created peptide-amphiphiles by linking the C-terminus of either the isolated cytoplasmic domain or the model peptide to a membrane-anchoring, lipid-like hydrocarbon tail. Circular dichroism measurements showed that the model peptide-amphiphile, either in aqueous suspension or in lipid bilayers, had a higher degree of alpha-helical secondary structure than the unlipidated model peptide. We hypothesized that the peptide-amphiphile system would allow us to study the function and structure of the PLB(1-25)K cytoplasmic domain in a native-like configuration. We compared the function (inhibition of the Ca-ATPase in reconstituted membranes) and structure (via CD) of the PLB(1-25) amphiphile to that of PLB and its isolated transmembrane and cytoplasmic domains. Our results indicate that the cytoplasmic domain PLB(1-25)K has no effect on Ca-ATPase (calcium pump) activity, even when tethered to the membrane in a manner mimicking its native configuration, and that the transmembrane domain of PLB is sufficient for inhibition of the Ca-ATPase.     

A simple protocol for the production of highly deuterated proteins for biophysical studies

Highly deuterated protein samples expand the biophysics and biological tool kit by providing, among other qualities, contrast matching in neutron diffraction experiments and reduction of dipolar spin interactions from normally protonated proteins in magnetic resonance studies, impacting both electron paramagnetic resonance and NMR spectroscopy. In NMR applications, deuteration is often combined with other isotopic labeling patterns to expand the range of conventional NMR spectroscopy research in both solution and solid-state conditions. However, preparation of deuterated proteins is challenging. We present here a simple, effective, and user-friendly protocol to produce highly deuterated proteins in Escherichia coli cells. The protocol utilizes the common shaker flask growth method and the well-known pET system (which provides expression control via the T7 promotor) for large-scale recombinant protein expression. One liter expression typically yields 5 to 50 mg of highly deuterated protein. Our data demonstrate that the optimized procedure produces a comparable quantity of protein in deuterium (²H₂O) oxide M9 medium compared with that in ¹H₂O M9 medium. The protocol will enable a broader utilization of deuterated proteins in a number of biophysical techniques.     

A novel and simple method of production and biophysical characterization of a mini-membrane protein, Ost4p: a subunit of yeast oligosaccharyl transferase

Asparagine-linked glycosylation is an essential and highly conserved protein modification reaction. In eukaryotes, oligosaccharyl transferase (OT), a multi-subunit membrane-associated enzyme complex, catalyzes this reaction in newly synthesized proteins. In Saccharomyces cerevisiae, OT consists of nine nonidentical membrane proteins. Ost4p, the smallest subunit, bridges the catalytic subunit Stt3p with Ost3p. Mutation of transmembrane residues 18-24 in Ost4p has negative effect on OT activity, disrupts the Stt3p-Ost4p-Ost3p complex, results in temperature-sensitive phenotype, and hypoglycosylation. Heterologous expression and purification of integral membrane proteins are the bottleneck in membrane protein research. The authors report the cloning, successful overexpression and purification of recombinant Ost4p with a novel but simple method producing milligram quantities of pure protein. GB1 protein was found to be the most suitable tag for the large scale production of Ost4p. The cleavage of Ost4p conveniently leaves GB1 protein in solution eliminating further purification. The precipitated pure Ost4p is reconstituted in appropriate membrane mimetic. The recombinant protein is highly helical as indicated by the far-UV CD spectrum. The well-dispersed heteronuclear single quantum coherence spectrum indicates that this minimembrane protein is well-folded. The successful production of pure recombinant Ost4p with a novel yet simple method may have important ramification for the production of other membrane proteins.     

Recombinant expression, isotope labeling, refolding, and purification of an antimicrobial peptide, piscidin

An antimicrobial peptide, piscidin, was overexpressed as a fused form with the ubiquitin molecule in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography (IMAC). The peptide was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The expression and purification process of piscidin encountered several problems such as the lysis of the bacterial cell upon induction of the peptide production, the unwanted cleavage of the fusion protein inside the bacterial cell, and high tendency to aggregate in the aqueous environment. Such problems were alleviated by employing ubiquitin as a fusion partner for piscidin, growing the cells at a lower temperature, and changing the order of the purification steps. The yields of the fusion protein and the peptide were around 15 and 1.5 mg per liter of LB or minimal medium, respectively. The recombinant expression and purification of piscidin will enable its structural and dynamic studies using multidimensional NMR spectroscopy.     

Sarcolipin Protein Interaction with Sarco(endo)plasmic Reticulum Ca2+ATPase (SERCA) Is Distinct from Phospholamban Protein,and Only Sarcolipin Can Promote Uncoupling of the SERCA Pump

Sarco(endo)plasmic reticulum Ca²⁺ATPase (SERCA) pump activity is modulated by phospholamban (PLB) and sarcolipin (SLN) in cardiac and skeletal muscle. Recent data suggest that SLN could play a role in muscle thermogenesis by promoting uncoupling of the SERCA pump (Lee, A.G. (2002) Curr. Opin. Struct. Biol. 12, 547–554 and Bal, N. C., Maurya, S. K., Sopariwala, D. H., Sahoo, S. K., Gupta, S. C., Shaikh, S. A., Pant, M., Rowland, L. A., Bombardier, E., Goonasekera, S. A., Tupling, A. R., Molkentin, J. D., and Periasamy, M. (2012) Nat. Med. 18, 1575–1579), but the mechanistic details are unknown. To better define how binding of SLN to SERCA promotes uncoupling of SERCA, we compared SLN and SERCA1 interaction with that of PLB in detail. The homo-bifunctional cross-linker (1,6-bismaleimidohexane) was employed to detect dynamic protein interaction during the SERCA cycle. Our studies reveal that SLN differs significantly from PLB: 1) SLN primarily affects the V_max of SERCA-mediated Ca²⁺ uptake but not the pump affinity for Ca²⁺; 2) SLN can bind to SERCA in the presence of high Ca²⁺, but PLB can only interact to the ATP-bound Ca²⁺-free E2 state; and 3) unlike PLB, SLN interacts with SERCA throughout the kinetic cycle and promotes uncoupling of the SERCA pump. Using SERCA transmembrane mutants, we additionally show that PLB and SLN can bind to the same groove but interact with a different set of residues on SERCA. These data collectively suggest that SLN is functionally distinct from PLB; its ability to interact with SERCA in the presence of Ca²⁺ causes uncoupling of the SERCA pump and increased heat production.     

Phospholamban inhibits Ca-ATPase conformational changes involving the E2 intermediate

We have used steady-state fluorescence spectroscopy in combination with enzyme kinetic assays to test the hypothesis that phospholamban (PLB) stabilizes the Ca-ATPase in the E2 intermediate state. The cardiac muscle Ca-ATPase (SERCA2a) isoform was expressed either alone or coexpressed with PLB in High-Five insect cells and was isolated as insect cell microsomes. Fluorescence studies of the Ca-ATPase covalently labeled with the probe 5-(2-((iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid showed that PLB decreased the amplitude of the Ca-ATPase E2 --> E1 conformational transition by 45 +/- 3% and shifted the [Ca2+] dependence of the transition to higher Ca2+ levels (DeltaKCa = 230 nM), similar to the effect of PLB on Ca-ATPase activity. Similarly, PLB decreased the amplitude of Ca-ATPase phosphorylation by inorganic phosphate (Pi) by 55 +/- 2% and decreased slightly the affinity for Pi (DeltaK0.5 = 70 microM). However, PLB did not affect the Ca2+-dependent inhibition of Ca-ATPase phosphorylation by Pi. Finally, PLB decreased Ca-ATPase sensitivity to vanadate, increasing the IC50 value by 300 nM. The results suggest that PLB binding to Ca-ATPase stabilizes the enzyme in a conformation distinct from E2, decreasing the number of enzymes in the E2 state capable of undergoing ligand-dependent conformational changes involving the Ca-free E2 intermediate. The inability of conformation-specific ligands to fully convert this E2-like state into E1 or E2 implies that these states are not in a simple equilibrium relationship.     

Refolding out of guanidine hydrochloride is an effective approach for high-throughput structural studies of small proteins

Low in vivo solubility of recombinant proteins expressed in Escherichia coli can seriously hinder the purification of structural samples for large-scale proteomic NMR and X-ray crystallography studies. Previous results from our laboratory have shown that up to one half of all bacterial and archaeal proteins are insoluble when overexpressed in E. coli. Although a number of strategies may be used to increase in vivo protein solubility, there are no generally applicable methods, and the expression of each insoluble recombinant protein must be individually optimized. For this reason, we have tested a generic denaturation/refolding protein purification procedure to assess the number of structural samples that could be generated by using this methodology. Our results show that a denaturation/refolding protocol is appropriate for many small proteins (相似文献   

Targeted overexpression of sarcolipin in the mouse heart decreases sarcoplasmic reticulum calcium transport and cardiac contractility

The role of sarcolipin (SLN) in cardiac physiology was critically evaluated by generating a transgenic (TG) mouse model in which the SLN to sarco(endoplasmic)reticulum (SR) Ca(2+) ATPase (SERCA) ratio was increased in the ventricle. Overexpression of SLN decreases SR calcium transport function and results in decreased calcium transient amplitude and rate of relaxation. SLN TG hearts exhibit a significant decrease in rates of contraction and relaxation when assessed by ex vivo work-performing heart preparations. Similar results were also observed with muscle preparations and myocytes from SLN TG ventricles. Interestingly, the inhibitory effect of SLN was partially relieved upon high dose of isoproterenol treatment and stimulation at high frequency. Biochemical analyses show that an increase in SLN level does not affect PLB levels, monomer to pentamer ratio, or its phosphorylation status. No compensatory changes were seen in the expression of other calcium-handling proteins. These studies suggest that the SLN effect on SERCA pump is direct and is not mediated through increased monomerization of PLB or by a change in PLB phosphorylation status. We conclude that SLN is a novel regulator of SERCA pump activity, and its inhibitory effect can be reversed by beta-adrenergic agonists.     

High-yield expression and purification of isotopically labeled norcoclaurine synthase, a Bet v 1-homologous enzyme, from Thalictrum flavum for NMR studies

The enzyme norcoclaurine synthase (NCS) found in the common meadow rue, Thalictrum flavum, and other plants shows sequence homology to members of the class 10 of pathogenesis related (PR 10) proteins that contains allergens such as the major birch pollen allergen Bet v 1, the major cherry allergen Pru av 1, and the major apple allergen Mal d 1. The enzyme is involved in the plant's secondary metabolism and is required for the production of bioactive secondary metabolites like morphine. Whereas the physiological function of PR 10 class allergens is still unknown, NCS activity has been studied in detail. Investigation of the structural properties of NCS by NMR spectroscopy can thus not only provide new information concerning the reaction mechanism of the enzyme, but is also expected to help clarify the long standing and heavily debated question on the physiological function as well as the reasons for the allergenic potential of members of this protein family. As the first important step towards the three-dimensional solution structure, we optimized expression of recombinant NCS in Escherichia coli and established an efficient purification protocol yielding high amounts of pure isotopically labeled active enzyme. The identity of NCS was confirmed by electrospray ionization mass spectrometry, and activity of the purified enzyme was determined by an assay detecting the radiolabeled reaction product. Spectroscopic analysis by NMR spectroscopy showed that the protein was properly folded with well defined tertiary structure.     

Phospholamban binds in a compact and ordered conformation to the Ca-ATPase

Mutagenesis and cross-linking measurements have identified specific contact interactions between the cytosolic and the transmembrane sequences of phospholamban (PLB) and the Ca-ATPase, and in conjunction with the high-resolution structures of PLB and the Ca-ATPase, have been used to construct models of the PLB-ATPase complex, which suggest that PLB adopts a more extended structure within this complex. To directly test these predictions, we have used fluorescence resonance energy transfer to measure the average conformation and heterogeneity between chromophores covalently bound to the transmembrane and cytosolic domains of PLB reconstituted in proteoliposomes. In the absence of the Ca-ATPase, the cytosolic domain of PLB assumes a wide range of structures relative to the transmembrane sequence, which can be described using a model involving a Gaussian distribution of distances with an average distance (Rav) of less than 21 A and a half-width (HW) of 36 A. This conformational heterogeneity of PLB is consistent with the 10 structures resolved by NMR for the C41F mutant of PLB in organic cosolvents. In contrast, PLB bound to the Ca-ATPase assumes a unique and highly ordered conformation, where Rav = 14.0 +/- 0.3 A and HW = 3.7 +/- 0.6 A. The small spatial separation between the bound chromophores on PLB is inconsistent with an extended conformation of bound PLB in current models. Thus, to satisfy known interaction sites of PLB and the Ca-ATPase, these findings suggest a reorientation of the nucleotide binding domain of the Ca-ATPase toward the bilayer surface to bring known PLB binding sites into close juxtaposition with residues near the amino-terminus of PLB. Induction of an altered conformation of the nucleotide binding domain of the Ca-ATPase by PLB binding is suggested to underlie the reduced calcium sensitivity associated with PLB inhibition of the pump.     

Two-dimensional solid-state NMR reveals two topologies of sarcolipin in oriented lipid bilayers

Sarcolipin (SLN), a 31 amino acid integral membrane protein, regulates SERCA1a and SERCA2a, two isoforms of the sarco(endo)plasmic Ca-ATPase, by lowering their apparent Ca(2+) affinity and thereby enabling muscle relaxation. SLN is expressed in both fast-twitch and slow-twitch muscle fibers with significant expression levels also found in the cardiac muscle. SLN shares approximately 30% identity with the transmembrane domain of phospholamban (PLN), and recent solution NMR studies carried out in detergent micelles indicate that the two polypeptides bind to SERCA in a similar manner. Previous 1D solid-state NMR experiments on selectively (15)N-labeled sites showed that SLN crosses the lipid bilayer with an orientation nearly parallel to the bilayer normal. With a view toward the characterization of SLN structure and its interactions with both lipids and SERCA, herein we report our initial structural and topological assignments of SLN in mechanically oriented DOPC/DOPE lipid bilayers as mapped by 2D (15)N PISEMA experiments. The PISEMA spectra obtained on uniformly (15)N-labeled protein as well as (15)N-Leu, (15)N-Ile and (15)N-Val map the secondary structure of SLN and, simultaneously, reveal that SLN exists in two distinct topologies. Both the major and the minor populations assume an orientation with the helix axis tilted by approximately 23 degrees with respect to the lipid bilayer normal, but vary in the rotation angle about the helix axis by approximately 5 degrees . The existence of the multiple populations in model membranes may be a significant requirement for SLN interaction with SERCA.