Membrane Protein Structural Biology Consortium (MPSBC) is one of
nine centers for membrane
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
- 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.
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.
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
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
2) A separate 7+1TM complex that mediates bile acid transport across
3) A 14+1TM complex involved in monocarboxylate transport.
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.
back to top