NMR: Structural biology in Solution
Structure, dynamics and interactions of proteins/complexes up
to 100 kDa.
Laboratory head: Erik R.P.
Zuiderweg, Ph.D.
Professor of Biological Chemistry
(734) 936 3850 zuiderwe@umich.edu
General support: Alexander
V. Kurochkin, Ph.D.
Research
Engineer, LSA Biophysics
(734) 763 0329
akur@umich.edu
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 (45 kDa), substrate binding
domain (15 kDa) and C-terminal domain (10 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 full-length chaperone constructs in order to decipher the allosteric
mechanism (see pic) and to help develop drugs (together with the Jason
Gestwicki lab) 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 substrate, transition state 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 equipped with a cryo-probe. We have a
4 channel Bruker Avance 500 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. We have access to the Michigan Life sciences Corridor
Bruker 900 MHz with cryoprobe. Thus, there is much instrument time available.
The lab is heavily computerized with UNIX systems of the PC, Sun and Mac-type.
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 and instruments are located in the Chemistry building in
downtown Ann Arbor. In addition to our group there is a another solution NMR
group (Al-Hashimi) working on structure and dynamics of RNA, and there is a
Biomolecular solid-state NMR group (Ramamoorthy). The NMR groups share
instrumentation, group meetings, seminars and parties.
Its location
Ann Arbor is a friendly and hilly town with much culture and local recreational
possibilities in the parks along the Huron riverIt has recently been
distingiushed as the USA’s most “livable” town. The town is extremely
cosmopolitan with large city amenities but without large city problems. The
population is around 150,000, from which roughly 40,000 are associated with the
University of Michigan -- so, it is a student town, but there are plenty of
"normal" people too. Michigan's international
airport, a Northwest hub, is within 40 minutes drive. Michigan weather is
moderated by the Great Lakes - never really cold, never really hot. Summer,
fall and winter are very nice. Michigan's "up-north" on
Lake Michigan offers beautiful scenery, and has plenty of summer and winter
recreation. Chicago is a four-hour drive by either train or car.
Biomolecular
NMR tutorial. chembiol_2009.pdf
Nmr relaxation pulse sequences for quick assessment
of conformational exchange
Varian sequences in simple
programming style