PROJECTS:
Membrane proteins are responsible for transmembrane signaling, energy
transduction, and ion and metabolite transport. Despite their importance,
and the need for structure to understand their function, few structures are
available. Membrane proteins have resisted routine crystallographic
analysis, and have been thought difficult candidates for NMR. The overall
goals of research in this laboratory are to continue to develop and apply
new solution conditions and new NMR methods to tease structural details out
of these secretive proteins.
Present work focuses on subunit c of the F1FO ATP synthase, both as a model
system and as an interesting protein in its own right. In the ATP synthase,
H+ translocation across the membrane through FO provides the driving force
for ATP synthesis on F1. Subunit c is responsible for both H+ translocation
through FO (via ionization of the buried Asp61 residue), and transmission of
long range (~100 =C5) conformational changes to the ATP binding sites on F1.
We have shown that subunit c retains native folding in organic/aqueous
solvent mixtures, permitting structural study by NMR. Introduction of a
nitroxide at a unique site allowed us to measure distances over a 10-20 =C5
range, and, from 2D difference NMR, to rapidly determine the detailed
structure of the region of the protein within 16 =C5 of the probe.
We are presently determining the complete structure of subunit c via
1H13C15N 3D and 4D NMR. Structures will be determined above and below the
pKa of the essential Asp61, to understand the nature of the conformational
change linked to energy transduction. We are developing a convenient system
to assay native folding of membrane proteins in mixed solvents and detergent
micelles, allowing us to optimize for proper folding and for favorable NMR
properties before undertaking lengthy structural studies.
We will analyze the structural features which stabilize the folding of
membrane proteins. Membrane proteins stand some of the normal folding rules
on their heads - e.g. they don=92t bury hydrophobic surface the way that=
water
soluble proteins do. We expect that clusters of aromatic residues,
intricate van der Waals packing, and connecting loops will contribute to
folding stability. Each of these elements will be varied by mutagenesis,
and folding stability will be assayed by NMR distance and dynamics
measurements. All these methods will be extended to other important membrane
proteins in the future.
SELECTED REFERENCES:
M.E. Girvin & R.H. Fillingame (1995) Determination of Local Protein
Structure by Spin Label Difference 2D NMR, Biochemistry 34, 1635.
M.E. Girvin (1994) Increased Sensitivity of COSY Spectra by Use of Constant
Time t1 Periods (CT-COSY), J. Magnetic Resonance Ser. A. 108, 99.
M.E. Girvin & R.H. Fillingame (1994) Hairpin Folding of Subunit c of F1Fo
ATP Synthase: 1H Distance Measurements to a Nitroxide-Derivatized Asp61
Biochemistry 33, 665.
INSTRUMENTATION:
The new Structural NMR Resource at Albert Einstein College of Medicine
(AECOM) occupies a space constructed to house the new Bruker DRX-600
4-channel and DRX-300 2-channel systems, along with substantial
computational resources (hardware & software). The new Resource complements
the existing 500, 400, and 200 MHz instruments which are used for
physiological NMR.=20
ENVIRONMENT & LOCATION:
AECOM provides an interactive, collaborative, friendly, and stimulating
environment. The Biochemistry Department is particularly strong in
mechanistic and structural biochemistry. The Albert Einstein College of
Medicine campus is located in a "pleasant residential neighborhood" of the
North Bronx. Many faculty and staff live in the surrounding neighborhoods.
Others live in Manhattan, or in Westchester County, which is an easy 10-30
minute commute from AECOM, depending on the location. Check us out at:=20
http://leper1.ca.aecom.yu.edu/sggd/pages/overview/bulltext.htm
APPLICATION:
The preferred starting date is September to November 1996. Please send
Curriculum Vitae and three letters of reference to: Mark Girvin,
Biochemistry Department, Albert Einstein College of Medicine, Jack and Pearl
Resnick Campus, 1300 Morris Park Ave., Bronx, NY 10461.
AECOM is an Equal Opportunity Employer.
______________________________________________________________
Mark Girvin =20
Biochemistry Department =20
Albert Einstein College of Medicine girvin@aecom.yu.edu
1300 Morris Park Ave. Tel:(718) 430-2025/2021
Bronx, NY 10461 FAX:(718) 430-8565
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