to apply cutting edge measurement science to biological systems.
INTENDED OUTCOME AND BACKGROUND
The Quantum Physics Division investigates
important biological systems at the single molecule level, thus leveraging our measurement expertise and experience
with atomic and quantum systems. Accordingly, we are evolving a part of our research program in this direction to help NIST contribute to the scientific revolution
taking place in the biosciences.
The Division’s strengths are the foundation for our efforts to contribute to ground-breaking research on biological systems. These strengths include our ability to build institutional bridges to renowned university
departments, a superlative infrastructure,
experience in manipulating and measuring atomic and quantum mechanical
systems, and a reputation that allows us to attract and hire the best and brightest
of today’s young scientists. Our new biophysics program is being implemented in close collaboration with the Department of Molecular, Cellular, and Developmental Biology and the Biochemistry Division of the Chemistry Department at the University
of Colorado. We expect that bringing additional departments into JILA will enhance the very productive interdisciplinary
character of the institute. Most importantly,
because of our existing expertise, NIST’s bioscience program should rapidly acquire the high stature achieved by our other programs.
Accomplishments
Subnanometer Optical Tweezers Measurements of DNA Molecular Motor
Figure 8. An optical trap stretches a DnA molecule. One detector laser measures the trapped bead position, while the other one measures
the sample drift. This drift is then actively suppressed by moving a piezoelectric stage in three dimensions.
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Single molecule experiments are revolutionizing
biophysics. Optical traps, also known as optical tweezers, can hold micrometer-sized beads in three dimensions,
apply calibrated piconewton-scale forces, and measure displacement with atomic precision.
Atomic-scale sensitivity is most useful for biology if it can be maintained over periods
sufficient to average Brownian motion, and thus reveal the underlying protein motion.
The most widely used single molecule experiments are anchored to surfaces, but drift of this surface prevents them from being atomically precise. To address this shortcoming, the Division has developed an actively stabilized, optical-trapping microscope based upon improved laser stability and the introduction of a fiducial mark firmly attached to a cover slip.
To demonstrate the capabilities of this microscope, measurements were made on a DNA helicase. DNA helicases are molecular
motors that unwind DNA, which is critical to the replication and repair of the genome. As they hydrolyze adenosine-triphosphate (ATP), they unwind the DNA duplex.
Pioneering work on a super-family one (SF1) helicase provided the first direct evidence
for the step size of a helicase. These single-turnover biochemical unwinding experiments showed a rate limiting step every 4 to 5 base pairs (bp). This large “kinetic”
step size has been seen in other SF1 helicases for unwinding double stranded DNA (dsDNA). In contrast, recent crystallographic
studies of SF1 helicases bound to DNA, in different nucleotide states, suggested one base pair motion per ATP.
Fluorescent ATPase assays also show 1 ATP/bp. Thus, the mechanism and step size of SF1 helicases (if a single step size is even correct) remains unresolved.
To address this ongoing uncertainty, we directly measured the physical step size of a SF1 helicase (RecBCD) along DNA using a single-molecule, optical-trapping assay with atomic-scale resolution. With this enhanced resolution, we note three significant results. First, RecBCD takes distinct steps of variable size (2 bp to 7 bp, occasionally larger). Second, the average observed step size is 4.1 bp, which agrees with the previously determined kinetic step size (3.9 bp). Third, we see an unexpected backward motion of a few base pairs at moderate force and low ATP levels, consistent
with a kinetic competition between
reannealing of the unwound DNA andforward helicase activity.
Femtosecond Spectroscopy of Heme-proteins
Heme proteins fulfill diverse roles, important in both human health and in biotechnology. In the former, they serve as carriers and storage molecules for oxygen and other small molecules in tissues, as electron transfer mediators in mitochon
drial respiration, and as drug metabolizers in the liver. In technological applications, heme proteins are being employed in toxic waste remediation and in hybrid bio-nanoelectronics
systems.
Research on the function of heme proteins has largely focused on structure. However, paralleling the growth in structural data, a large body of evidence has accumulated showing that proteins are flexible molecules,
continuously undergoing structural
fluctuations on timescales ranging from femtoseconds to seconds or longer. Since protein motions are critical to their kinetics (e.g. the access of molecules to their interiors) and thermodynamics (e.g. entropy), there is a compelling need to quantify the relation between structure, dynamics, and function.
We have performed photon echo spectroscopy
on heme proteins, and have used a combination of experiments and theory to demonstrate that, with an understanding of the symmetry properties of the heme, this popular measurement technique can be used to provide information on protein motions. The development of this method sets the stage for detailed studies of physiologically
and industrially relevant heme proteins.
Figure 9. The P4-P6 domain of the Tetrahymena thermophyla group 1 Intron. The “tetraloop/tetraloop receptor” and “A-rich bulge” are ubiquitously present in ribozymal RnA.
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Real Time Folding Kinetics of Single RNA Molecules
The ability to image fluorescence from single biomolecules represents one of the major scientific achievements of the last decade.
In combination with time-correlated
single photon counting, these methods now offer an unprecedented window into kinetics and dynamics of single biomolecules.
They completely avoid the “blurring” that unavoidably occurs in any collection of molecules due to ensemble averaging.
The biomolecules are labeled with donor and acceptor dyes, where the efficiency of fluorescence resonance energy transfer (FRET) from initially excited donor to acceptor
is a strong function of the biomolecular
conformation. For donor/acceptor dye pairs bound to a folding biopolymer, the FRET efficiency
(i.e., Ired/ (Ired+Igreen))
and polarization anisotropy information are monitored in real time and used to provide conformational information from the well known
distance and alignment dependence for dipole-dipole energy transfer.
Work in the Division has focused on real time folding dynamics of small RNA sequences, using single molecule FRET methods to explore tertiary
“loop-loop” interactions responsible for making larger ribozymal RNA fold into biochemically active conformation. The specific system of interest is
the P4-P6 domain of the Tetrahymena thermophyla group 1 Intron, in particular the presence of two classic tertiary binding motifs
(the “tetraloop/ tetraloop receptor” and “A-rich bulge”) ubiquitously present in ribozymal RNA. This system is an interesting target from a
biophysical perspective because we know the exact RNA sequence, and therefore can successfully design relatively simple constructs
that recapitulate a specific tertiary motif in isolation.
Recently, we have focused on developing new experimental “burst” capabilities for studying single RNA constructs freely diffusing in solution, which
permit a rigorous
control for ruling out possible surface tethering effects on the kinetics. Data reveal “bursts” of fluorescence as single RNA molecules diffuse into
and out of the focused laser excitation volume (≈0.1 fL). Analysis of the “red” vs. “green” photon arrival
statistics permits us to infer the FRET value and therefore the folded vs. unfolded conformation of an individual RNA as a function of external
stimulus (e.g., [Mg+2]).
First strategic focus |
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Fourth strategic focus
"Technical Activities 2005-2007" - Table of Contents |
