Chen, Yan

Jia, Menghui

Knutson, Jay

Pan, Haifeng

Tao, Zhandong

Xu, Jianhua

Zhang, Sanjun

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684a Wednesday, February 6, 20133514-Pos Board B669
MdDQm Calculations Explore the Origins of Differences amongst the
Red Fluorescent Proteins
J. Nathan Scott1, Patrik R. Callis2.
1Saint Francis University, Loretto, PA, USA, 2Montana State University,
Bozeman, MT, USA.
We present a number of theoretical results concerning the properties of and dif-
ferences amongst several red fluorescent proteins (RFPs). RFPs are an extraor-
dinarily useful group of fluorescent proteins that fluoresce at red-shifted
wavelengths compared to GFP, thereby extending the color palette of fluores-
cent proteins for use in applications like FRET and multi-channel imaging. This
redder fluorescence is caused by the extension of the pi-conjugated chain
through oxidation of a second backbone bond of either Phenylalanine or Isoleu-
cine, depending on the particular RFP. Our work has focused on the RFPs
mCherry, mPlum, and DsRed, which share a common chromophore. Since
these RFPs share a chromophore, differences in their photophysical properties
must be due to differences in their chromophore environments. We performed
a series of 100 ns molecular dynamics simulations followed by ZINDO semi-
empirical quantum mechanical calculations in an effort to explore variables
such as excitation wavelength, quantum yield, and photostability in these
RFPs. The ZINDO calculations were performed on MD simulation frames at
1 ps resolution. This fine resolution allowed for the observation of rare confor-
mational states that suggest reasons for the known differences in quantum yield
amongst the proteins. Notably, the electric field contributions from every non-
chromophore protein atom and ion, and the ~14,000 simulation cell water mol-
ecules, were used as a perturbation in the ZINDO calculations, which allowed
us to query the electrostatic environmental influence on the chromophore.
3515-Pos Board B670
Femtosecond Fluorescence Dynamics of Trp-Trp Dipeptide in Water and
its Interaction with DNA
Yan Chen1, Haifeng Pan2, Zhandong Tao2, Menghui Jia2, Sanjun Zhang2,
Jianhua Xu2, Jay Knutson3.
1Tongji Hospital Affiliated to Tongji University, shanghai, China, 2East
China Normal University, shanghai, China, 3National Institutes of Health,
Bethesda, MD, USA.
We reported here the systematic measurements of fluorescence decay and
quenching of tryptophan dipeptide Trp-Trp in water, and its interaction with
DNA by using an ultraviolet upconversion fluorescence measurement system
with time resolution better than 100 fs together with a time correlated single
photon counting apparatus on the 100 ps to 30 ns time scale. We analyzed
the effect of acidity and phosphate ion etc upon Trp-Trp fluorescence decay
profiles. Fluorescence data reveal multiexponential decay of Trp-Trp dipeptide
in water, and Trp-Trp can interact with DNA. It is shown that Trp-Trp is subject
to ultrafast quenching with DNA and also senses nearby water dynamics. The
time constant for the bulk water ‘‘solvent relaxation’’ remains near 1 ~ 2 ps, but
a new ultrafast decay, fit to an exponential time constant of ~ 10 ps with asso-
ciated amplitude positive or negative (>380 nm) depending on the emission
wavelength, has been found. Candidate mechanisms (including ET to nearby
acceptors and/or collisional quenching etc.) and the analysis of time resolved
emission spectra will be discussed.
3516-Pos Board B671
Phospholipase Cb Binds to C3PO and its Components that Orchestrates
RNA Interference
Shriya Sahu, Finly Philip, Suzanne Scarlata.
Stony Brook University, Stony Brook, NY, USA.
Phospholipase Cb (PLCb1-4) is a family of signaling enzymes coupled to G
protein Gaq. When activated by Gaq, PLCb hydrolyses phosphatidylinositol
4,5 bisphosphate to produce two second messengers that lead to activation of
protein kinase C and an increase in intracellular calcium. While the majority
of PLCb resides on the plasma membrane, a small but significant population
is present in the cytosol and nucleus. Recently, our lab identified translin-
associated factor X (TRAX) to be a cellular binding partner of PLCb with
a binding constant only 10 fold lower than Gaq. TRAX is known to bind to
translin forming the complex C3PO which assists in RNA interference, along
with other potential nuclease functions. Recently, our lab showed that PLCb
can reverse the down regulation of specific genes (GAPDH) presumably
through its interaction with TRAX thus linking PLCb with gene regulation.
Here, we have characterized the complexes formed between PLCb and
TRAX, translin and C3PO. Using FRET, we show that PLCb binds to C3PO
with Kd similar to PLCb and Trax (8nM) whereas the Kd for binding to translin
is much weaker (40 nM). We also found that translin, Trax and C3PO occur in
multiple oligomeric states as shown by FCS and in native gels. Anisotropy
studies using labeled oligonucleotides and translin and C3PO show thatPLCb inhibits a nucleotide binding mode of the proteins. Studies to character-
ize the change in the composition and stoichiometry of C3PO and translin by
PLCb in the presence and absence of various oligonucleotides using FCS
and Photon Counting Histogram (PCH) analysis are currently underway.
This work was supported by NIH GM053132.
3517-Pos Board B672
Ultrafast Fluorescence Spectroscopy and Cell Imaging Techniques in
Exploring the Caffeine Mediated Dissociation of Mutagen from Biomimet-
ics, Nucleic Acids and Cells
Soma Banerjee, Samir Kumar Pal.
SNBNCBS, Kolkata, India.
We report a systematic investigation of caffeine induced dissociation of ethid-
ium (Et) cation, a potential mutagen from nucleic acids and biomimetic sys-
tems. Time-resolved fluorescence studies are consistent with a mechanism
where caffeine-Et complex formation in bulk solution drives the dissociation
of DNA-bound Et. Caffeine induced extraction of Et from whole cells were
also performed on squamous epithelial cells collected from the inner lining
of the human mouth, A549 (lung carcinoma), A375 (human skin), RAW (mac-
rophage) and Vero (African green monkey kidney epithelium) cell lines. Inter-
estingly, the efficiency of caffeine in extracting Et has been found to be
dependent on cell types. Our steady state and picosecond resolved spectro-
scopic studies on the detachment of Et from various biomimicking micelles
of different charges reveal the specificity of caffeine molecule for carrying
out such dissociation. The picosecond resolved Fo¨rster resonance energy trans-
fer (FRET) studies between a DNA minor groove binder dye Hoeschst 33258
(H258, donor) and Et (acceptor) have been employed to investigate the alter-
ation in their association in presence of caffeine in the molecular level. Finally,
our fluorescence micrographs of epithelial cells validate the alteration of FRET
efficiency between the donor and the acceptor due to the caffeine mediated re-
lease of the latter. Our results both in-vitro as well as ex-vivo provide important
clues about efficiency and mechanism of caffeine as a potential anti-mutagenic
therapeutic agent.
Vibrational Spectroscopy
3518-Pos Board B673
DFT-Level Calculations of IR, VCD and Raman Spectra for 13C-Labeled
Beta Sheets
William R.W. Welch1, Jan Kubelka1, Timothy A. Keiderling2.
1University of Wyoming, Laramie, WY, USA, 2University of Illinois at
Chicago, Chicago, IL, USA.
With the aid of theoretical models, optical spectroscopy has proven valuable for
elucidating secondary structural features in protein systems including b-sheets,
which form a large portion of protein assemblies in amyloid systems. For such
large systems, computational methods must be both efficient and robust in or-
der to achieve accurate predictions in a timely manner. Quantum chemical
methods are in principle the most robust methods available, but suffer from
high computational cost that usually makes them impractical for large bimolec-
ular assemblies. In this study optical spectra for b-sheets composed of five
strands, ten-amides each, 5x10, were obtained by transferring parameters cal-
culated using DFT for smaller 3x3 sheet segments. Sensitivity of spectra to var-
iations in conformations was examined for parallel, anti-parallel, twisted and
flat sheets. Some effects of having strands shifted out of register were also
explored. Predicted amide I IR spectra show classic features for parallel and
anti-parallel sheets and some new possibilities for discerning between the
two are observed, primarily in IR and VCD spectra, while Raman amide I spec-
tra was predicted to be less useful for structural discrimination. Predictions for
particular isotopic labeling schemes show clear IR patterns that might be used
in elucidating shifts in register for anti-parallel sheets.
3519-Pos Board B674
Expanding the Utility of 4-Cyano-L-Phenylalanine as a Vibrational
Reporter of Protein Environments
Christopher Bazewicz, Melanie Liskov, Kevin Hines, Scott Brewer.
Franklin and Marshall College, Lancaster, PA, USA.
The ability to genetically incorporate amino acids modified with spectroscopic
reporters into proteins with high efficiency and fidelity has greatly enhanced the
ability to probe local protein structure and dynamics. We have synthesized the
unnatural amino acid (UAA), 4-cyano-L-phenylalanine (pCNPhe), containing
the nitrile vibrational reporter and three isotopomers (15N, 13C, 13C15N) of
this UAA to enhance the ability of pCNPhe to study local protein environments.
Each pCNPhe isotopic variant was genetically incorporated in an efficient, site-
specific manner into superfolder green fluorescent protein (sfGFP) in response
to an amber codon with high fidelity utilizing an engineered, orthogonal
aminoacyl-tRNA synthetase. The isotopomers of 4-cyano-L-phenylalanine


Femtosecond Fluorescence Dynamics of Trp-Trp Dipeptide in Water and its Interaction with DNA 

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Femtosecond Fluorescence Dynamics of Trp-Trp Dipeptide in Water and its Interaction with DNA

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