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leftThe Membrane Protein Structural Biology Consortium (MPSBC) is one of nine centers for membrane
right protein structure determination that were established in July 2010 as part of the PSI:Biology phase of the National Institutes of Health’s Protein Structure Initiative (PSI). 

MPSBC is a consortium involving three laboratories headed by scientists with diverse and complementary expertise. The senior investigators are:

  • Dr. Mark E. Dumont, Department of Biochemistry and Biophysics at the University of Rochester School of Medicine and Dentistry,
  • Dr. Michael C. Wiener, Department of Molecular Physiology and Biological Physics at the University of Virginia, and
  • Dr. Michael G. Malkowski, Department of Structural Biology at the Hauptman-Woodward Institute in Buffalo NY.

The three different laboratories bring together a range of pre-existing facilities and personnel, ongoing structural projects, ties to collaborators working on important targets, and existing infrastructures for interacting with the broader scientific community.  In addition, Dr. Peter Nollert of Emerald BioSystems (Bainbridge Island, WA) is a subcontractor specializing in lipidic cubic phase (LCP) crystallization.  Professor Da-Neng Wang (New York University) serves as a one-person advisory board, helping the consortium to prioritize and achieve its goals.

Transmembrane proteins (TMPs) comprise a large fraction of most genomes, play critical roles in cell physiology, and are targets of diverse, widely used drugs.  However, understanding their functions and the design of agents to modulate their actions are hampered by a lack of knowledge about the three-dimensional structures of TMPs, especially TMPs from eukaryotic sources.  MPSBC’s goal is to overcome the technical barriers associated with membrane protein structural biology, use improved technologies to solve several TMP structures, and disseminate these improvements.  MPSBC will establish facilities for high-throughput screening of detergent compatibilities and for high-throughput crystallization trials that can be used by the general biological and structural biology communities.

Structure Determination.
MPSBC has established a pipeline for structure determination with the following features: 1) Target selection is designed to identify multiple related targets, such as orthologs and mutated proteins that minimize predicted disordered regions. 2) The primary focus is on expression in yeast and E. coli where high-throughput (HT) approaches allow rapid expression testing of multiple constructs in parallel. 3) Multiple related targets from initial expression testing will be subjected to intermediate level purification to provide enough protein for pre-crystallization screening using biophysical and biochemical approaches and for small scale crystallization screening to allow determination of optimal protein concentrations and solvent conditions. An important aspect of this step is the use of a HT screen for detergent compatibility developed in the Wiener laboratory. 4) Multiple crystallization and crystal optimization approaches including HT microbatch under oil at the Hauptman-Woodward Institute and lipidic cubic phase screening at Emerald BioSystems will be used to maximize the likelihood of obtaining “winning” crystals suitable for structure determination.

Biological Targets.
MPSBC focuses on structure determinations of three classes of TMPs, selected based on their intrinsic biological and medical interest as well as their suitability for establishment of the technology development aspects of this project. Wherever possible, the selection of classes of targets is also based on the existence of ongoing collaborations with non-structural laboratories maintaining active interests in specific classes of proteins. Emphasis is primarily on targets that can be expressed in the yeast and bacterial expression systems that form the core of MPSBC’s production pipeline and technology development efforts; however, insect cell expression will be used for targets that do not express in yeast or E. coli.  MPSBC’s targets are as follows:

1) Eukaryotic SLC4 family proteins related to human AE1 (Band 3), the erythrocyte Cl-/HCO3- exchanger. These proteins play critical roles in processes as diverse as circulatory oxygen and CO2 transport, ion and pH homeostasis, and nutrient uptake. However, the detailed molecular mechanisms of such transport remain poorly understood.
2) Members of the Major Facilitator Superfamily. This family, responsible for transporting a wide variety of substrates, comprises more than 25% of all known transporters.
3) Oligopeptide transporters related to the SLC15 family, which are involved in nutrient and drug uptake in mammals.

Transmembrane Enzymes Involved in Lipid Synthesis and Lipid Attachment to Proteins.
1) Protein modifications associated with the attachment of prenyl groups.
2) A recently discovered class of enzymes that catalyzes the attachment of palmitoyl groups to proteins.
3) TMPs involved in sphingolipid and endocannabinoid synthesis.

Oligomeric Complexes of Proteins containing 7 transmembrane segments and proteins containing 1 transmembrane segment (7+1TM Complexes).
1) Several classes of G Protein Coupled Receptors (GPCRs) require co-expression of a single pass transmembrane partner for effective subcellular targeting and function of the receptor. Co-expression and co-crystallization of such complexes is expected to provide a benign and less perturbative way of stabilizing GPCRs, as well as providing an important picture of the mechanism by which these interactions affect targeting and signaling function.
2) A separate 7+1TM complex that mediates bile acid transport across membranes.
3) A 14+1TM complex involved in monocarboxylate transport.

Community Outreach.
The MPSBC project will establish community-accessible facilities for HT screening of detergent solubilization and crystallization conditions for TMP targets. The Wiener Laboratory has recently established a protocol for rapid testing of a large number of detergents to determine their effects on protein solubility and compatibility with subsequent purification. Since these procedures are readily adaptable to automation, they will be incorporated into a screening facility allowing rapid analysis of TMP targets. After initial testing and validation of these procedures against standard targets, the facility will be made available to the greater biological and structural biology communities. Significantly, for several years the Hauptman-Woodward Institute has been offering high-throughput crystallization screening (HTS) for soluble proteins. The HTS Lab has already had more than 900 users. During the last 18 months, the HTS Lab has also offered its first crystallization screen tailor-made for TMP targets. This screen is based, in part, on the phase partitioning behavior of detergents.

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