Isolation of a highly active PSII-LHCII supercomplex from thylakoid membranes by a direct method

Abstract We have developed a simple and novel method to isolate a highly pure and active photosystem (PS) II complex, directly from thylakoid membranes. This complex is a discrete particle and contains all the proteins of the oxygen evolving complex and a set of chlorophyll a/b binding proteins. The intactness of both the donor side and the acceptor side has resulted in a very high oxygen evolution activity and therefore offers a superior experimental system to that of PSII enriched membrane fragments in which there is heterogeneity in activities and biochemical composition. z 1999 Federation of European Biochemical Societies

Chapter fourteen – the mechanisms of Mg2+ and Co2+ transport by the CorA family of divalent cation transporters

The metal ions Mg2+ and Co2+ are essential for life, although to different degree. They have similar chemical and physical properties, but their slight differences result in Mg2+ to be the most abundant metal ion in living cells and the trace element Co2+ being toxic at relatively low concentrations. Specialized transporters have evolved in living cells to supply and balance the Mg2+ and Co2+ need of the cells. The current knowledge of the molecular mechanisms of Mg2+ and Co2+ -specific transporters is very limited at this point. Recently, there has been remarkable advances to understand the CorA family, a family of transporters that are able to transport both ions. These new data have increased our insights in how Mg2+ and Co2+ are translocated across membranes. Presently, CorA is probably the best system to study the mechanisms of Mg2+ and Co2+ transport. This chapter discusses the mechanisms through which CorA selects, transports, and regulates the translocation of its substrate. In addition, we highlight the physical and chemical properties of the substrates, which are important parameters required for better understanding of the transporter action

An efficient strategy for high-throughput expression screening of recombinant integral membrane proteins

The recombinant expression of integral membrane proteins is considered a major challenge, and together with the crystallization step, the major hurdle toward routine structure determination of membrane proteins. Basic methodologies for high-throughput (HTP) expression optimization of soluble proteins have recently emerged, providing statistically significant success rates for producing such proteins. Experimental procedures for handling integral membrane proteins are generally more challenging, and there have been no previous comprehensive reports of HTP technology for membrane protein production

Structural insights into the mechanisms of Mg2+ uptake, transport, and gating by CorA

Despite the importance of Mg2+ for numerous cellular activities, the mechanisms underlying its import and homeostasis are poorly understood. The CorA family is ubiquitous and is primarily responsible for Mg2+ transport. However, the key questions—such as, the ion selectivity, the transport pathway, and the gating mechanism—have remained unanswered for this protein family. We present a 3.2 Å resolution structure of the archaeal CorA from Methanocaldococcus jannaschii, which is a unique complete structure of a CorA protein and reveals the organization of the selectivity filter, which is composed of the signature motif of this family. The structure reveals that polar residues facing the channel coordinate a partially hydrated Mg2+ during the transport. Based on these findings, we propose a unique gating mechanism involving a helical turn upon the binding of Mg2+ to the regulatory intracellular binding sites, and thus converting a polar ion passage into a narrow hydrophobic pore. Because the amino acids involved in the uptake, transport, and gating are all conserved within the entire CorA family, we believe this mechanism is general for the whole family including the eukaryotic homologs.Published Versio

Engineering membrane protein overproduction in Escherichia coli

Membrane proteins play a fundamental role in human disease and therapy, but suffer from a lack of structural and functional information compared to their soluble counterparts. The paucity of membrane protein structures is primarily due to the unparalleled difficulties in obtaining detergent-solubilized membrane proteins at sufficient levels and quality. We have developed an in vitro evolution strategy for optimizing the levels of detergent-solubilized membrane protein that can be overexpressed and purified from recombinant Escherichia coli. Libraries of random mutants for nine membrane proteins were screened for expression using a novel implementation of the colony filtration blot. In only one cycle of directed evolution were significant improvements of membrane protein yield obtained for five out of nine proteins. In one case, the yield of detergent-solubilized membrane protein was increased 40-fold

Comprehensive analysis and identification of the human STIM1 domains for structural and functional studies

STIM1 is a Ca2+ sensor within the ER membrane known to activate the plasma membrane store-operated Ca2+ channel upon depletion of its target ion in the ER lumen. This activation is a crucial step to initiate the Ca2+ signaling cascades within various cell types. Human STIM1 is a 77.4 kDa protein consisting of various domains that are involved in Ca2+ sensing, oligomerization, and channel activation and deactivation. In this study, we identify the domains and boundaries in which functional and stable recombinant human STIM1 can be produced in large quantities. To achieve this goal, we cloned nearly 200 constructs that vary in their initial and terminal residues, length and presence of the transmembrane domain, and we conducted expression and purification analyses using these constructs. The results revealed that nearly half of the constructs could be expressed and purified with high quality, out of which 25% contained the integral membrane domain. Further analyses using surface plasmon resonance, nuclear magnetic resonance and a thermostability assay verified the functionality and integrity of these constructs. Thus, we have been able to identify the most stable and well-behaved domains of the hSTIM1 protein, which can be used for future in vitro biochemical and biophysical studies.Published versio

Co2+ Selectivity of Thermotoga maritima CorA and Its Inability to Regulate Mg2+ Homeostasis Present a New Class of CorA Proteins*

CorA is a family of divalent cation transporters ubiquitously present in bacteria and archaea. Although CorA can transport both Mg2+ and Co2+ almost equally well, its main role has been suggested to be that of primary Mg2+ transporter of prokaryotes and hence the regulator of Mg2+ homeostasis. The reason is that the affinity of CorA for Co2+ is relatively low and thus considered non-physiological. Here, we show that Thermotoga maritima CorA (TmCorA) is incapable of regulating the Mg2+ homeostasis and therefore cannot be the primary Mg2+ transporter of T. maritima. Further, our in vivo experiments confirm that TmCorA is a highly selective Co2+ transporter, as it selects Co2+ over Mg2+ at >100 times lower concentrations. In addition, we present data that show TmCorA to be extremely thermostable in the presence of Co2+. Mg2+ could not stabilize the protein to the same extent, even at high concentrations. We also show that addition of Co2+, but not Mg2+, specifically induces structural changes to the protein. Altogether, these data show that TmCorA has the role of being the transporter of Co2+ but not Mg2+. The physiological relevance and requirements of Co2+ in T. maritima is discussed and highlighted. We suggest that CorA may have different roles in different organisms. Such functional diversity is presumably a reflection of minor, but important structural differences within the CorA family that regulate the gating, substrate selection, and transport