ayout 1 - EMBL Grenoble

EMBL Research at a Glance 2009
Edward Lemke
PhD, MPI for Biophysical
Chemistry, Göttingen.
Research Associate, the
Scripps Research Institute.
Group leader at EMBL since
2009. Joint appointment with
Cell Biology and Biophysics
Unit.
Structural light microscopy/single molecule
spectroscopy
Previous and current research
Research in our laboratory combines modern chemical biology and biochemistry/molecular biology
methods with advanced fluorescence and single molecule techniques to elucidate the nature
of protein disorder in biological systems and disease mechanisms.
Currently, more than 50,000 protein structures with atomic resolution are available from the protein
databank and due to large efforts (mainly crystallography and NMR) their number is rapidly
growing. However, even if all 3D protein structures were available, our view of the molecular building
blocks of cellular function will still be rather incomplete, as we now know that many proteins
are intrinsically disordered, which means that they are unfolded in their native state. Interestingly,
the estimated percentage of intrinsically disordered proteins (IDPs) grows with the complexity of
the organism (prokaryotes ≈ 5% and eukaryotes ≈ 50%). In a modern view of systems biology, these
disordered proteins are believed to be multi-functional signalling hubs central to the interactome
(the whole set of molecular interactions in the cell). Their ability to adopt multiple conformations
is considered a major driving force behind their evolution and enrichment in eukaryotes.
While the importance of IDPs in biology is now well established, many common strategies for probing protein structure are incompatible with
molecular disorder and the highly dynamic nature of those systems. In addition, a mosaic of molecular states and reaction pathways can exist
in parallel in any complex biological system, further complicating the situation to measure these systems. For example, some proteins might
behave differently than the average, giving rise to new and unexpected phenotypes. One such example are the infamous Prion proteins, where
misfolding of only subpopulations of proteins can trigger a drastic signalling cascade leading to completely new phenotypes. Conventional
ensemble experiments are only able to measure the average behaviour of a system, discounting such coexisting populations and rare events.
Ignoring such information can easily lead to generation of false or insufficient models, which may further impede our understanding of the
biological processes and disease mechanisms.
In contrast, single molecule techniques, which probe the distribution of behaviours, can shed light on important mechanisms that otherwise remain
masked. In particular, single molecule fluorescence (smF) studies allow probing of molecular structures and dynamics on the nanometer
scale with high time resolution. Although not inherently limited by
the size of a macromolecule, smF studies require site-specific labelling
with special fluorescent dyes which still hampers the broad application
and general use of this technique. It was recently demonstrated
that amber nonsense suppression technology of genetically reprogrammed
hosts is an especially powerful approach to overcome this
limitation (Brustad et al., 2008). Here, unnatural amino acids with
unique chemical properties are conveniently site-specifically introduced
into any protein site by the host organism itself, serving as manipulation
sites. Our lab also continues to develop and apply such
protein engineering tools to facilitate fluorescence studies of complex
biological mechanisms.
Labelled proteins are excited using advanced laser techniques and
emitted fluorescence photons are detected using home-built highly
sensitive equipment. This strategy allows to study structure and
Future projects and goals
dynamics of even heterogeneous biological systems.
Recent studies have shown that even the building blocks of some of
the most complex and precise machines with an absolute critical role to survival of the cell, such as DNA packing and many transport processes,
are largely built from IDPs. We aim to explore the physical and molecular rationale behind the fundamental role of IDPs by combining molecular
biology and protein engineering tools with single molecule biophysics. Our long-term goal is to develop general strategies to study structure
and dynamics of IDPs within their natural complex environments.
Selected references
Ferreon, A.C.M., Gambin, Y, Lemke, E.A., Deniz A.A. (2009). Single-
Molecule Fluorescence illuminates a multi-conformational switch in
α-synuclein. Proc. Natl. Acad. Sci. USA, doi:10.1073/
pnas.0809232106
Brustad, E.M., Lemke, E.A., Schultz, P.G. & Deniz, A.A. (2008). A
general and efficient method for the site-specific dual-labeling of
proteins for single molecule fluorescence resonance energy transfer.
J. Am. Chem. Soc., 130, 1766-5
8
Deniz, A.A., Mukhopadhyay, S. & Lemke, E.A. (2008). Singlemolecule
biophysics: at the interface of biology, physics and
chemistry. J. R. Soc. Interface, 5, 15-5
Lemke, E.A., Summerer, D., Geierstanger, B.H., Brittain, S.M. &
Schultz, P.G. (2007). Control of protein phosphorylation with a
genetically encoded photocaged amino acid. Nat. Chem. Biol., 3,
769-72