NMR comes of age: Structure, dynamics
and interactions of proteins/complexes up to 100 kDa.
Laboratory head: Erik
R.P.
Zuiderweg, Ph.D.
Professor, Biophysics
Research
Division, Dept. Biological Chemistry and Dept. Chemistry
(734) 936 3850
zuiderwe@umich.edu
General support: Alexander
V. Kurochkin, Ph.D.
Assistant Research Scientist,
Biophysics Research Division
(734) 763 0329
akur@umich.edu
Link to
Biophysics Link to
Biological Chemistry Link to Chemistry
Structure,
dynamics, allosterics and function of Hsp70 Chaperones
Hsp70
proteins mediate trafficking,
folding and refolding of proteins
in all known cellular structures. Damage to these proteins is fatal;
overexpression of these proteins is observed in stressed cells, such as
in cancerous tissues. As such Hsp70's are a target for the
treatment of especially breast cancer. These Heat Shock Proteins are 70
kDa, and have a three domain
structure: nucleotide-binding domain (44kDa), substrate binding domain
(15-20 kDa)
and C-terminal domain (15-20 kDa). The chaperone helps the refolding of
proteins by binding and release cycles (see pic) driven by
an allosteric mechanism. We use high field NMR in solution
(600 - 900 MHz) to study its structure, dynamics and
interaction as a function of substrate, nucleotides and
co-chaperones. We are working on large 60 kDa
chaperone
constructs in order to decipher the allosteric mechanism (see
pic) and to help
develop drugs to suppress its activity as an aid in cancer therapy.
PICTORIAL
INTRODUCTION TO THE CHAPERONES
Methodology
Development in NMR detection of Molecular Dynamics
The understanding of protein function is incomplete without considering
entropy, that is, dynamics. In enzymes, the active site is often
dynamic to be able to adept to ubstrate, transition conformation and
product; for protein
complexes, the intermolecular interface sites are particularly
dynamic to accomodate induced fits. Binding processes often involve
perturbation of fast dynamical
components, contributing (sometimes to a dominant extent)
to the
ligand binding entropy and hence ligand binding free energy (=
affinity). The rate determining step of enzymatic catalysis has
in several cases found to be set by milli-micro-second dynamical
processes of opening and closing the active sites (see pic).
NMR plays an important role in experimentally measuring dynamics in
proteins at time scales ranging from seconds to pico seconds. Our
mission is to develop and apply methods to describe what the
motions
actually are. It is of importance to distinguish, e.g., between
concerted
and non-concerted motion: these different modes have very different
entropic and hence functional
consequences. Wo do this by obtaining a multitude of
dynamic parameters on a restricted number of
sites.
The increased information density allows us to develop models
for the dynamics and their entropic content. Our long-term aim is to
achieve a better design of pharmaceuticals by incorporating dynamical
information.
INTRODUCTION
TO NMR RELAXATION MEASUREMENTS FOR DYNAMICS
Research
infrastructure
The group currently has 5 grad students and post-docs. We are the main
users of a 4 channel Varian Inova 800 (January 1999) which will b
eequipped with a cryo-probe in summer 2005. We have
a 4 channel Bruker Avance 500 (December 1999) for the group's
exclusive use and an old Bruker AMX 600. We have partial access
to a Bruker Avance 600 MHz system with cryo-probe. Up
and coming: access to the Michigan Life sciences Corridor Bruker 900
MHz with cryoprobe in summer 2005 (located at MSU).Thus, there is
much instrument time available. We have a
cluster of Silicon Graphics and Linux-PC systems. We have
two labs for protein expression and purification, and have access
to PCR equipment, shakers, and a New Brunswick fermentor.
The Lab
The laboratory is in the Biophysics Research Division, located in the
Chemistry building in downtown Ann Arbor. The Division counts roughly
15 faculty and 60 students / postdocs, mostly interested in the
physical descriptions of biological phenomena. The NMR
infrastructure in Biophysics is second to none: in addition to our
group there is a new solution NMR group (Al-Hashimi) working on nucleic
acids mainly using RDC's, and there is a Biomolecular solid-state NMR
group (Ramamoorthy) working on peptide-membrane interactions. Further,
there
are two X-ray groups, two single-molecule spectrocopy / manipulation
groups, and several computational groups.
Many
groups are involved in interdisciplinary collaborations.
Nmr
relaxation pulse
sequences
for quick assessment of conformational exchange
Varian
sequences in simple programming style