Michael Wiener, Ph.D.

Michael Wiener, Ph.D.

wiener.jpg

Michael C. Wiener, Ph.D.
Associate Professor


E-mail
Address:
mwiener@virginia.edu
Telephone: 434-243-2731/2730
Address: Molecular Physiology
and Biological Physics
University of Virginia
PO Box 800736
Charlottesville, VA 22908-0736
Membrane Protein Crystallography & Molecular Biophysics

The structure/function paradigm is of central import in modern molecular biology. High-resolution structures, determined by x-ray crystallography, of proteins involved in biological processes provide insight into their function. These structures provide a basis for the design of future experiments to probe more deeply the precise molecular mechanisms that underlie biological activity. In addition to providing fundamental insight into function and mechanism, structures of proteins involved in disease and other pathophysiological states can serve as targets for structure-based drug design.

We propose a structure-based approach to the determination of membrane protein function. Our goal is to solve x-ray crystal structures of channel, transport and receptor proteins, and to use these structures in conjunction with other results to understand the molecular basis of function. While thousands of soluble protein structures have been solved (at a rate of about three per day!), very few integral membrane proteins have been solved to high resolution. Major technical, scientific and intellectual challenges involved include: expression of multimilligram quantities of recombinant protein for crystallization experiments, crystallization from detergent-solubilized protein solutions where the properties of the detergent may be as or more significant than those of the protein, and determination of a structure from crystals that may not be of very high quality. The 'payoff' of this research endeavor, however, is large; the structure of a membrane protein at atomic (or near-atomic) resolution is a significant achievement.

In addition to structure determination, there are research opportunities in the lab for the utilization or development of expression systems, and for biophysical studies of membrane protein crystallization. A range of proteins are under consideration for study, either alone or in collaboration with other investigators in or outside of the university. Many of these are of major biological and/or medical interest. Some of the current projects in my laboratory include:

1.The Molecular Basis of Protein-Mediated Water Transport

Protein-mediated water transport is a fundamental physiological process in all organisms. It is carried out by integral membrane proteins, aquaporins, that function as water channels. Aquaporins serve as passive, diffusion-limited channels to dissipate osmotic gradients that form across cell membranes. Currently, six different human aquaporins have been discovered, each with a different distribution in bodily organs, tissues and cells. This heterogeneous distribution predicts significant roles for human aquaporins in both normal physiology and disease. Mutations in one aquaporin, aquaporin-2, are responsible for nephrogenic diabetes insipidus. Others are implicated in the maintenance of water homeostasis in erythrocytes, kidney, lung, brain and salivary gland. The aquaporins are likely targets for the future development of therapeutic agents directed to prevention or control of edema and fluid balance. Crystals of human aquaporin-1 have been obtained, and structure determination is underway.

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2. Structural Dissection of Bacterial Active Transport Pathways

Bacteria possess active transport pathways for the binding and uptake of essential compounds that they do not themselves make biosynthetically. These pathways consist of an outer membrane receptor which binds substrate with high specificity and affinity, and a complex of proteins in the inner membrane. One of these inner membrane proteins spans the periplasmic space and functions to couple the transmembrane potential of the inner membrane to drive transport across the outer membrane. Overexpression, purification and crystallization of the vitamin B12 receptor BtuB is underway, in collaboration with Prof. Robert Kadner of the Microbiology Department. Other proteins in this pathway will also be examined structurally, alone or in complex with the receptor.

These transport pathways are also utilized by bacterial protein toxins (bacteriocins) and bacteriophage to gain entry into the target cell. One class of bacteriocins are the channel-forming colicins, which bind to specific outer membrane receptors, translocate across the outer membrane and form voltage-gated channels in the inner membrane. These various functions are performed by different domains of the protein. A single colicin molecule is sufficient to kill a bacterium, and thus Local Imageare both more selective and effective than conventional antibiotics. We solved the structure of the soluble form of colicin Ia.

 

A ribbon representation of the structure of colicin Ia with blue-to-red coloring in the N-to-C direction. The scale bar indicates 50Å. The molecule is approximately 210Å long and consists of three functional domains separated by a pair of helices each 160Å long. Three domains, R (receptor-binding), T (translocation) and C (channel-forming) are indicated.

Selected publications :

Conformational exchange in a membrane transport protein is altered in protein crystals. Freed DM, Horanyi PS, Wiener MC, Cafiso DS.
Biophys J. 2010 Sep 8;99(5):1604-10.PMID: 20816073

A high-throughput differential filtration assay to screen and select detergents for membrane proteins. Vergis JM, Purdy MD, Wiener MC.
Anal Biochem. 2010 Dec 1;407(1):1-11. Epub 2010 Jul 25. PMID: 20667442

Design and characterization of a constitutively open KcsA. Cuello LG, Jogini V, Cortes DM, Sompornpisut A, Purdy MD, Wiener MC, Perozo E.
FEBS Lett. 2010 Mar 19;584(6):1133-8. PMID: 20153331

Coupling of calcium and substrate binding through loop alignment in the outer-membrane transporter BtuB.
Gumbart J, Wiener MC, Tajkhorshid E.
J Mol Biol. 2009 Nov 13;393(5):1129-42. Epub 2009 Sep 9.  PMID: 19747487

Mechanics of force propagation in TonB-dependent outer membrane transport.
Gumbart J, Wiener MC, Tajkhorshid E.
Biophys J. 2007 Jul 15;93(2):496-504. Epub 2007 Apr 20. PMID: 17449669

When worlds colloid.
Wiener MC.
Protein Sci. 2006 Dec;15(12):2679-81. No abstract available. PMID: 17132858

Outer membrane active transport: structure of the BtuB:TonB complex.
Shultis DD, Purdy MD, Banchs CN, Wiener MC.
Science. 2006 Jun 2;312(5778):1396-9. PMID: 16741124

TonB-dependent outer membrane transport: going for Baroque?
Wiener MC.
Curr Opin Struct Biol. 2005 Aug;15(4):394-400. Review. PMID: 16039843

Comparative structural analysis of TonB-dependent outer membrane transporters: implications for the transport cycle.
Chimento DP, Kadner RJ, Wiener MC.
Proteins. 2005 May 1;59(2):240-51. PMID: 15739205

A pedestrian guide to membrane protein crystallization.
Wiener MC.
Methods. 2004 Nov;34(3):364-72. Review. PMID: 15325654

Membrane protein expression and production: effects of polyhistidine tag length and position.
Mohanty AK, Wiener MC.
Protein Expr Purif. 2004 Feb;33(2):311-25. PMID: 14711520

Enzymatic E-colicins bind to their target receptor BtuB by presentation of a small binding epitope on a coiled-coil scaffold.
Mohanty AK, Bishop CM, Bishop TC, Wimley WC, Wiener MC.
J Biol Chem. 2003 Oct 17;278(42):40953-8. Epub 2003 Aug 4. PMID: 12902336

Substrate-induced transmembrane signaling in the cobalamin transporter BtuB.
Chimento DP, Mohanty AK, Kadner RJ, Wiener MC.
Nat Struct Biol. 2003 May;10(5):394-401. PMID: 12652322

Inhibition of tobacco etch virus protease activity by detergents.
Mohanty AK, Simmons CR, Wiener MC.
Protein Expr Purif. 2003 Jan;27(1):109-14. PMID: 12509992

Thiol-reactive lanthanide chelates for phasing protein X-ray diffraction data.
Purdy MD, Ge P, Chen J, Selvin PR, Wiener MC.
Acta Crystallogr D Biol Crystallogr. 2002 Jul;58(Pt 7):1111-7. Epub 2002 Jun 20. PMID: 12077430

Bacterial export takes its Tol.
Wiener MC.
Structure. 2000 Sep 15;8(9):R171-5. Review. PMID: 10986468

Mesoscopic surfactant organization and membrane protein crystallization.
Wiener MC, Verkman AS, Stroud RM, van Hoek AN.
Protein Sci. 2000 Jul;9(7):1407-9. PMID: 10933509

Expression, purification, and initial structural characterization of YadQ, a bacterial homolog of mammalian ClC chloride channel proteins.
Purdy MD, Wiener MC.
FEBS Lett. 2000 Jan 21;466(1):26-8. PMID: 10648805

Crystal structure of colicin Ia.
Wiener M, Freymann D, Ghosh P, Stroud RM.
Nature. 1997 Jan 30;385(6615):461-4. PMID: 9009197

A lectin screening method for membrane glycoproteins: application to the human CHIP28 water channel (AQP-1).
Wiener MC, van Hoek AN.
Anal Biochem. 1996 Oct 15;241(2):267-8. No abstract available. PMID: 8921199

Structure of a fluid dioleoylphosphatidylcholine bilayer determined by joint refinement of x-ray and neutron diffraction data. III. Complete structure.
Wiener MC, White SH.
Biophys J. 1992 Feb;61(2):434-47. PMID: 1547331

Structure of a fluid dioleoylphosphatidylcholine bilayer determined by joint refinement of x-ray and neutron diffraction data. II. Distribution and packing of terminal methyl groups.
Wiener MC, White SH.
Biophys J. 1992 Feb;61(2):428-33. PMID: 1547330

Structure of a fluid dioleoylphosphatidylcholine bilayer determined by joint refinement of x-ray and neutron diffraction data. I. Scaling of neutron data and the distributions of double bonds and water.
Wiener MC, King GI, White SH.
Biophys J. 1991 Sep;60(3):568-76. PMID: 1932548

Transbilayer distribution of bromine in fluid bilayers containing a specifically brominated analogue of dioleoylphosphatidylcholine.
Wiener MC, White SH.
Biochemistry. 1991 Jul 16;30(28):6997-7008. PMID: 2069956

Fluid bilayer structure determination by the combined use of x-ray and neutron diffraction. II. "Composition-space" refinement method.
Wiener MC, White SH.
Biophys J. 1991 Jan;59(1):174-85. PMID: 2015382

Fluid bilayer structure determination by the combined use of x-ray and neutron diffraction. I. Fluid bilayer models and the limits of resolution.
Wiener MC, White SH.
Biophys J. 1991 Jan;59(1):162-73. PMID: 2015381

Structure of the fully hydrated gel phase of dipalmitoylphosphatidylcholine.
Wiener MC, Suter RM, Nagle JF.
Biophys J. 1989 Feb;55(2):315-25. PMID: 2713445

Relations for lipid bilayers. Connection of electron density profiles to other structural quantities.
Nagle JF, Wiener MC.
Biophys J. 1989 Feb;55(2):309-13. PMID: 2713444

Structure of fully hydrated bilayer dispersions.
Nagle JF, Wiener MC.
Biochim Biophys Acta. 1988 Jul 7;942(1):1-10. PMID: 3382651

Specific volumes of lipids in fully hydrated bilayer dispersions.
Wiener MC, Tristram-Nagle S, Wilkinson DA, Campbell LE, Nagle JF.
Biochim Biophys Acta. 1988 Feb 18;938(2):135-42. PMID: 2829963