Structure and Dynamics of
Macromolecules
Our research is focused on the structural characterization of
nucleo-protein assemblies. The structure of chromatin and the
global structure of protein-nucleic acid complexes relevant to
the molecular mechanisms of control of transcription in
prokaryotes are investigated using high resolution scanning force
microscopy (SFM). This microscope, also known as Atomic Force
Microscope (AFM) works by scanning a tip over the sample to sense
the topography of the surface, thus functioning in much the same
way than old record players. In addition, we are studying the
elastic response of long linear polymers, the forces responsible
for maintaining the tertiary structure of proteins, and the
mechanical properties of molecular motors, using methods of
single molecule manipulation such as laser tweezers and the SFM.
Current Projects
Our laboratory is involved in the study of the structural basis
of protein-DNA interactions and their relevance in the processes
of control of gene expression. In prokaryotes, and specially in
eukaryotes, replication and transcription regulation involve the
interaction of many specialized protein factors at regulator
locations on the sequence to insure correct sequence recognition,
initiation, processivity, fidelity, and kinetic control. We wish
to understand the multiple structural, spatial, and functional
relationships among these regulatory factors. We are using the
SFM as a high resolution tool to image initiation and elongation
transcription complexes of E. coli RNA polymerase to characterize
the spatial relationships between the enzyme and the DNA
template.
We are also beginning to investigate what structural changes
are negotiated between RNA polymerase and chromatin during
transcription. To this end, we are using the SFM to image
complexes of nucleosome-containing DNA fragments carrying a
promoter and a terminator upstream and downstream of the
nucleosome positioning sequence, respectively. We plan to compare
the behavior of various prokaryotic and eukaryotic polymerases as
they transcribe through the nucleosome, to investigate whether
transcription through a nucleosome is an inherent property of the
core particle, or a property of each enzyme itself, and to
characterize various intermediates of the translocation process.
Our laboratory is also working actively in the development of
methods of single-molecule manipulation, including the use of SFM
cantilevers, optical or laser tweezers, and magnetic beads to
investigate the mechanical properties of macromolecules. In one
project, we first tether a single protein molecule of T-4
lysozyme between a surface and the end of an SFM cantilever. We
can then separate the surfaces in a controlled fashion to induce
the mechanical unfolding of the molecule to characterize the
nature, range, and strength of the forces that maintain its
three-dimensional structure. Our objective is to carry out the
unfolding of the molecule at equilibrium so as to obtain the
potential energy function of the molecule as a function of the
mechanical extension. This function represents the most complete
description of the folded state of the protein. We plan to
investigate how external conditions in the medium, i.e.
temperature, denaturant concentration, etc., or point-directed
mutations affect the shape of the potential energy function.
Finally, our laboratory is also engaged in the study of
DNA-binding molecular motors (RNA polymerase, DNA polymerase,
etc.) using optical tweezers to investigate the dynamics of these
molecules during translocation, as well as the effect of external
force load and nucleotide tri-phosphate concentration on their
power and force generation. In parallel, we are developing both
microscopic (chemical ratchet-type) and phenomenological models
of molecular motors which will be tested experimentally. We
believe that single molecule experiments can provide a unique
look into the molecular mechanisms responsible for the
mechano-chemical conversion process in these protein machines.
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