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Biological Chemistry
Structural Biology and Biophysics by NMR
Professor Gottfried Otting
http://rsc.anu.edu.au/research/otting.php
The group, started in April 2002,
works primarily with biomolecular applications of NMR spectroscopy.
Besides 3D structure determinations of proteins and protein domains,
methods are developed for rapid identification of ligand binding
sites, including protein-protein interaction sites, with the aim of
pharmaceutical applications. From the beginning of 2004, projects
will be supported by an 800 Mhz NMR spectrometer with cryoprobe.
This goal is approached from several angles:
- Development of an algorithm for the
rapid resonance assignment of uniformly 15N labelled
proteins for which the 3D structures have been determined by X-ray
crystallography. The goal is to assign the 15N-HSQC
spectrum of the protein without expensive 13C labelling.
High-yield in vitro protein expression techniques are
developed in collaboration with Dr N.E. Dixon to allow inexpensive
residue-selective labelling of proteins.
- Development and implementation of NMR
experiments suitable for 3D structure determination of small regions
in a protein at high resolution. The goal is to "zoom" in
on a region of a protein, selected by segmental isotope labelling or
a paramagnetic spin label, and determine its structure quickly and
with high accuracy. The approach further relies on
residue-selectively labelled protein samples which allow the
assignment of NMR signals of strategically chosen amino acids without
having to assign or measure the resonances of the rest of the
protein.
- Identification of protein-protein and
protein-ligand binding surfaces by chemical modification. This
includes the development of a variety of experimental strategies
based on OH-radical triggered H/D exchange, chemical cross-linking
and paramagnetic spin labelling techniques. The aim is to develop
practical techniques which identify intermolecular binding surfaces
rapidly either by NMR spectroscopy or mass spectrometry.
Highlights
of the year were the completion of 3D structure determinations for
several proteins and conserved protein domains, and the demonstration
of an in vitro protein expression system with yields
sufficiently high that NMR spectra (15N-HSQC spectra)
could be recorded straight from the reaction medium without any
protein purification or concentration step.
In
September and October Professor Gottfried Otting visited his former
laboratory at the Karolinska Institute in Stockholm for experiments
on an NMR spectrometer with cryoprobe. Continuing major
collaborations are with Dr Nicholas Dixon (in-house), Dr Edvards
Liepinsh (Karolinska Institute), Dr Anatoly Sharipo (Latvian
University), Dr Laszlo Patthy (Hungarian Academy of Sciences) and an
EU network on cross-correlation effects in NMR led by Professor
Geoffrey Bodenhausen (Paris).
Protein Structure Determinations
3D structures were completed of Citrobacter freundii AmpD, a
187-residue protein involved in the evolution of constitutive
antibiotic resistance; the R3H domain from human Sµbp-2,
a protein domain conserved in over 100 proteins with specificity for
binding to single-stranded 5' -phosphorylated
DNA; the PYRIN domain, also called DAPIN or PAAD domain, which turned
out to present a fourth class of death domain type proteins. All
three proteins belong to large families of proteins with amino-acid
sequence homology. Our structures were the first for any member of
these families. An unexpected structural relationship between AmpD
and the previously reported structure T7 lysozyme allowed us to
predict accurate models of a novel class of proteins, the
peptidoglycan recognition proteins (PGRP), and enzymatic activity for
several members of these proteins which play important roles in human
immune defence. (with E. Liepinsh, L. Guignard
[Karolinska Institute, Stockholm], C. Généreux,
D. Dehareng, B. Joris [U. Liège, Belgium],
A. Leonchiks, A. Sharipo [Latvian U.], E. Staub
[metaGen Pharmaceuticals, Berlin])
In vitro Expression of Residue-selectively Isotope Labelled Samples
The
cell-free expression system available in Dr Nicholas Dixon's
laboratory was used to express samples of selectively 15N
and 13C-labelled PpiB, a 163-residue
prolyl-cis-trans isomerase from E. coli. The yields
were sufficiently high that NMR spectra (15N-HSQC spectra)
could be recorded straight from the reaction medium without any
protein purification or concentration step. The spectra were
sufficiently clean to enable an interaction study with substrate
added to the NMR sample. The system allowed the identification of an
amino-acid residue in the active site of the enzyme in less than
twenty-four hours, including in vitro protein synthesis and
NMR data recording and analysis. (with K. Ozawa, N.E. Dixon)
Dipole-Curie-Spin Cross-correlation
Dipole-CSR
cross-correlation effects between the Curie spin of the iron and the
amide protons in 15N-labelled myoglobin were measured in a
quantitative way for high-spin and low-spin complexes with F-
and CN-, respectively, by subtraction of the dipole-CSA
cross-correlation effect measured with diamagnetic CO-myoglobin. The
data contain accurate long-range structural information about the
angle formed between the 15N-1HN
bond and the vector connecting the 1HN spins
with the electron spin of the iron. (with G. Pintacuda
[Karolinska Institute, Stockholm], K. Hoenthanner, N. Müller
[U. Linz, Austria])
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