Search

Helical assemblies of proteins, which consist of a two-dimensional lattice of identical subunits arranged with helical symmetry, are a common structural motif in nature. For membrane proteins, crystallization protocols can induce helical arrangements and take advantage of the symmetry found in these assemblies for the structural determination of target proteins. Modern advances in the field of electron cryo-microscopy (cryo-EM), in particular the advent of direct electron detectors, have opened the potential for structure determination of membrane proteins in such assemblies at high resolution. The nature of the symmetry in helical crystals of membrane proteins means that a single image potentially contains enough information for three-dimensional structural determination. With the current direct electron detectors, we have never been closer to making this a reality. Here, we present a protocol detailing the preparation of helical crystals, with an emphasis on further cryo-EM analysis and structural determination of the sarco(endo)plasmic reticulum Ca 2+ -ATPase in the presence of regulatory subunits such as phospholamban.

Electron crystallography of two-dimensional crystalline arrays is a powerful alternative for the structure determination of membrane proteins. The advantages offered by this technique include a native membrane environment and the ability to closely correlate function and dynamics with crystalline preparations and structural data. Herein, we provide a detailed protocol for the reconstitution and two-dimensional crystallization of the sarcoplasmic reticulum calcium pump (also known as Ca(2+)-ATPase or SERCA) and its regulatory subunits phospholamban and sarcolipin.

The various isoforms of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) are responsible for the Ca(2+) uptake from the cytosol into the endoplasmic or sarcoplasmic reticulum (ER/SR). In some tissues, the activity of SERCA can be modulated by binding partners, such as phospholamban and sarcolipin. The activity of SERCA can be characterized by its apparent affinity for Ca(2+) as well as maximal enzymatic velocity. Both parameters can be effectively determined by the protocol described here. Specifically, we describe the measurement of the rate of oxalate-facilitated (45)Ca uptake into the SR of crude mouse ventricular homogenates. This protocol can easily be adapted for different tissues and animal models as well as cultured cells.

Molecular modeling and simulation are useful tools in structural biology, allowing the formulation of functional hypotheses and interpretation of spectroscopy experiments. Here, we describe a method to construct in silico models of a fluorescent fusion protein construct, where a cyan fluorescent protein (CFP) is linked to the actuator domain of the Sarco/Endoplasmic Reticulum Ca(2+)-ATPase (SERCA). This CFP-SERCA construct is a biosensor that can report on structural dynamics in the cytosolic headpiece of SERCA. Molecular modeling and FRET experiments allow us to generate new structural and mechanistic models that better describe the conformational landscape and regulation of SERCA. The methods described here can be applied to the creation of models for any fusion protein constructs and also describe the steps needed to simulate FRET results using molecular models.

Fluorescence measurements as monitored with either extrinsic or intrinsic probes constitute important ways with which to study the molecular properties of macromolecules. With high-quality spectrofluorimeters, it is, e.g., possible kinetically to follow local conformational changes, induced by ligands and inhibitors, with a sensitivity that is unsurpassed by any other physicochemical technique. We demonstrate here with Ca(2+) and two specific inhibitors of SERCA how this can be done by measurements of the intrinsic fluorescence of the tryptophan residues of SERCA.

The photoactivation of aryl azides is commonly employed as a means to covalently attach cross-linking and labeling reagents to proteins, facilitated by the high reactivity of the resultant aryl nitrenes with amino groups present in the protein side chains. We have developed a simple and reliable assay for the determination of the ATP binding affinity of native or recombinant sarcoplasmic reticulum Ca(2+)-ATPase, taking advantage of the specific photolabeling of Lys(492) in the Ca(2+)-ATPase by [γ-(32)P]2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine 5'-triphosphate ([γ-(32)P]TNP-8N3-ATP) and the competitive inhibition by ATP of the photolabeling reaction. The method allows determination of the ATP affinity of Ca(2+)-ATPase mutants expressed in mammalian cell culture in amounts too minute for conventional equilibrium binding studies. Here, we describe the synthesis and purification of the [γ-(32)P]TNP-8N3-ATP photolabel, as well as its application in ATP affinity measurements.

The SERCA calcium ATPase, probably the most well-investigated membrane protein both from a biophysical and structural view, can be purified from native source in substantial quantities, making it a favorable target when conducting biochemical experiments and structure determination, e.g., by X-ray crystallography.

The P-type ATPases form an autophosphorylated intermediate with ATP, and its isomeric transition and hydrolysis are obligatory events in the ATP-driven pump and thus for the energy coupling. The analyses of these reactions are therefore crucial for understanding the mechanism of the pump function and diseases caused by its defects. Here we describe the methods to analyze these processes in the transport cycle with a representative member of P-type ATPase family, SERCA1a, sarco(endo)plasmic reticulum Ca(2+)-ATPase.

Lipids play an important role in maintaining P-type ATPase structure and function, and often they are crucial for ATPase activity. When the P-type ATPases are in the membrane, they are surrounded by a mix of different lipid species with varying aliphatic chain lengths and saturation, and the complex interplay between the lipids and the P-type ATPases are still not well understood. We here describe a robust method to exchange the majority of the lipids surrounding the ATPase after solubilisation and/or purification with a target lipid of interest. The method is based on an ultracentrifugation step, where the protein sample is spun through a dense buffer containing large excess of the target lipid, which results in an approximately 80-85 % lipid exchange. The method is a very gently technique that maintains protein folding during the process, hence allowing further characterization of the protein in the presence of a target lipid of interest.

The SERCA isoform 1a is constructed to transport 2 Ca(2+) ions across the sarcoplasmic reticulum membrane coupled to the hydrolysis of one molecule of MgATP. However, observed coupling ratios for Ca(2+) transported/ATP hydrolzyed are usually less than 2:1, since part of the Ca(2+) accumulated at high intravesicular concentrations by the active transport of Ca(2+) leaks out of the vesicles because of Ca(2+)-induced Ca(2+) exchange. However, in the presence of a high concentration of oxalate (5 mM) Ca(2+) will precipitate as Ca-oxalate inside the vesicles and thereby be prevented from leaking out and, in addition, this treatment will reduce the intravesicular free concentration of Ca(2+) to a level where optimal coupling ratios of 2:1 can be achieved.

Electron microscopy (EM) has made possible to solve the structure of many proteins. However, the resolution of some of the EM maps is too low for interpretation at the atomic level, which is particularly important to describe function. We describe methods that combine low-resolution EM data with atomic structures for different conformations of the same protein in order to produce atomic models compatible with the EM map.We illustrate these methods with EM data from decavanadate-induced tubular crystals of a pseudo-phosphorylated intermediate of Ca-ATPase and the various atomic structures of other intermediates available in the Protein Data Bank (PDB). Determination of atomic structure permits not only to analyse protein-protein interactions in the crystals, but also to localize residues in the proximity of the crystallizing agent both within Ca-ATPase and between Ca-ATPase molecules.

Heterologous SERCA1a Ca(2+)-ATPase (sarco-endoplasmic reticulum Ca(2+)-adenosine triphosphatase isoform 1a) from rabbit was expressed in yeast Saccharomyces cerevisiae as a fusion protein, with a biotin acceptor domain (BAD) linked to the SERCA C-terminus by a thrombin cleavage site. Thanks to the pYeDP60 vector, the recombinant protein was expressed under the control of a galactose-inducible promoter. Biotinylation of the protein occurred directly in yeast. Optimizing the number of galactose induction steps and increasing the amount of Gal4p transcription factor both improved expression. Lowering the temperature from 28 to 18 degrees C during expression enhanced the recovery of detergent-extractible active protein. In the "light membrane fraction," thought to mainly contain internal membranes, we are able to recover about 14-18 mg Ca(2+)-ATPase per liter of yeast culture in a bioreactor. Solubilization of this membrane fraction by n-dodecyl beta-D: -maltopyranoside (DDM) allowed us to recover the largest amount of active protein. The in vivo biotinylated recombinant protein was then bound to a streptavidin-Sepharose resin. Selective elution of the biotinylated SERCA1a was carried out after thrombin action on the resin-bound protein. We were able to obtain 200-500 microg/L of yeast culture of a 50% pure SERCA1a that displays an ATPase activity similar to that of the native rabbit Ca(2+)-ATPase. To succeed in crystallization, an additional size exclusion chromatography step was necessary. This step increases purity to 70%, removes aggregated protein and exchanges DDM for C(12)E(8).

This chapter reviews the molecular biology, biochemical, and NMR methods that we used to study the structural dynamics, membrane topology, and interaction of phospholamban (PLN), a small regulatory membrane protein involved in the regulation of the sarcoplasmic reticulum Ca-ATPase (SERCA). In particular, we show the progression of our research from the initial hypotheses toward understanding the molecular mechanisms of SERCA's regulation, including the effects of PLN oligomerization and posttranslational phosphorylation. Finally, we show how the knowledge of the molecular mechanism of the structural dynamics and topology of free and bound proteins can lead to the rational design of PLN analogs for possible use in gene therapy.