Eichelbaum, Steven

Fulcrand, Geraldine

Leng, Fenfei

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Tuesday, February 5, 2013 417acreation by Gag remain controversial. Here we reveal that Gag adsorption and
polymerization is modulated by membrane curvature. Negative curvatures
stimulate formation of distinct fluid-like membrane domains tightly packed
with Gag molecules, which further polymerizes into a stable protein shell.
The nucleation of these domains happens at physiologically relevant high cur-
vatures and Gag polymerization leads to stabilization of these highly bent
membrane configurations. Our findings indicate a novel mechanism of negative
curvature creation based upon curvature-driven polymerization of Gag and in-
volving curvature-polymerization feedback.
2140-Pos Board B159
Lentiviral Vectors Nano-Engineered with ‘Marker of Self’ CD47 to avoid
Immune Surveillance
Nisha Sosale1, Irena I. Ivanovska1, Richard K. Tsai1, Philip W. Zoltick2,
Yale Goldman1, Dennis E. Discher1.
1University of Pennsylvania, Philadelphia, PA, USA, 2Harvard University,
Boston, MA, USA.
Macrophages and dendritic cells take up foreign microbes from the circula-
tion and other tissues and often present microbial components to the adaptive
immune system. Uptake of viruses including Lentiviral Vectors (LVs) by
macrophages not only makes viral delivery inefficient, but also contributes
to an unwanted immune response to vector components as well as delivered
gene products. Macrophage uptake of micron-size particles and cells is in-
hibited by CD47 display on the surface of target particles by an interaction
with macrophage receptor SIRPa. A novel LV was engineered to present
an oriented human CD47-GFP fusion protein on the vector envelope with
the aim of reducing uptake by macrophages without affecting transduction
of other cells – which is indeed demonstrated. While fluorescent microscopy,
flow cytometry, and western blotting confirm that virus as well as the produc-
ing cells indeed express CD47-GFP, the key physical question is whether
virus displays CD47-GFP in the proper orientation. AFM-coupled nano-fluo-
rescence imaging of viral vectors displaying envelope CD47 demonstrated
colocalization of the GFP tagged protein with acridine orange stained
RNA, and AFM imaging also showed viral vectors were rigid and quasi-
spherical, ranging in diameter from 100 to 300 nanometers as expected.
Kinetics of lentiviral vector binding specifically to anti-CD47 coverslips
established the proper orientation of CD47-GFP on the viral surface. This
result confirms the development of a novel lentiviral vector that properly dis-
plays CD47 to specifically minimize macrophage uptake and subsequent
immune activation to virus.2141-Pos Board B160
Calcium-Mediated Fusion between Endo-Lysosomal Compartments
Enhances Virus-Like Particles Release
Andrea Gramatica1, Maik J. Lehmann2, Roland Schwarzer1,
Andreas Herrmann1.
1Humboldt University of Berlin, Department of Biology/Molecular
Biophysics, Berlin, Germany, 2Humboldt University of Berlin, Department
of Biology/Molecular Parasitology, Berlin, Germany.
Assembly of the human immunodeficiency virus (HIV) is governed by the
structural polyprotein Gag, which is necessary and sufficient for the release
of virus-like particles (VLPs) from the host cell. Although the plasma mem-
brane has been recognized as the major site for the production of VLPs, in
some cell-types Gag is targeted to late endosomes/multivesicular bodies (LE/
MVBs), where assembly and budding take place. Virus release into the extra-
cellular space then occurs after regulated exocytosis. It is well accepted that the
release of VLPs requires participation of different host cell components. In par-
ticular, it was shown that induction of a transient rise in cytoplasmic Ca2þ in-
creased the amounts of VLPs in MVBs, and resulted in a dramatic enhancement
of VLPs release (Perlman M. et al., 2006). However, although cellular factors
have been already proposed as mediators of Ca2þ provision (Ehrlich L. et al.,
2010), how Ca2þ can promote the release of VLPs remains to be determined.
With FACS analyses on live cells, we could identify variations of intracellular
Ca2þ in Gag-expressing cells treated with Ca2þ fluorescent indicators. High-
resolution confocal and electron microscopy have confirmed that Gag can as-
semble and bud into VLPs in lysosomes (Ly) and LE. Furthermore, we could
show for the first time that Ca2þ released specifically from those compartments
causes formation of Ly/LE hybrid organelles, which in turn fuse with the PM
and release VLPs into the extracellular space. This heterotypic fusion process
requires components of the SNARE complex and the Ca2þ sensor protein Syn-
aptotagmin VII, which regulates Ly exocytosis. All these elements constitute
a productive pathway for virus assembly and release. We believe that the
Gag protein itself, or a cellular factor recruited by Gag, might promote the in-
crease of Ca2þ required for this process to function.Protein-Nucleic Acid Interactions II
2142-Pos Board B161
Single Molecule Tracking of Lac Repressor Diffusing on Stretched DNA
Gionata Belcastro1, Carina Mo´nico1, Marco Capitanio1,2,
Francesco Vanzi1,3, Francesco Saverio Pavone1,2.
1LENS, Sesto Fiorentino, Italy, 2Department of Physics, Sesto Fiorentino,
Italy, 3Department of evolutionary biology ‘Leo Pardi’, Florence, Italy.
Lac Repressor (LacI) is a DNA-binding protein that regulates genes expression
by specific binding to its target sequence on DNA. This sequence is found rap-
idly among millions of base pairs in the genome, presumably via a combination
of 3D and 1D-diffusion (following the interaction with non-specific DNA).
Here we report diffusion analysis through the combination of single molecule
localization and manipulation. For single molecule localization, Atto532 dye
has been covalently linked to a single-cysteine mutant of LacI, LacIQ231C.
A double optical tweezers system is used for trapping and stretching a single
DNA molecule. All components of the experiment (protein, DNA, beads and
buffer) are assembled (and/or rapidly exchanged) with a flow-system. This in-
tegrated configuration has several advantages: biomolecules are not in the prox-
imity of the glass surface thus preventing possible electrostatic effects;
controlled forces can be measured or applied. We characterized LacI 1D-diffu-
sion under different forces applied to the DNA. The figure shows an example of
the data obtained in the form of a kymogram.
Acknowledgments:The research leading to the results has received funding from
Italian Ministry for Education, Univer-
sity and Research in the framework of
the Flagship Project NANOMAX
2143-Pos Board B162
Lac Repressor-DNA Interactions assessed by Ultrafast Force-Clamp
Spectroscopy
Carina Monico1, Marco Capitanio1,2, Gionata Belcastro1,
Francesco Vanzi1,3, Francesco S. Pavone1,2.
1LENS_ European Laboratory For Non Linear Spectroscopy, Sesto
Fiorentino (FI), Italy, 2Department of Physics and Astronomy, University of
Florence, Sesto Fiorentino (FI), Italy, 3Department of Evolutionary Biology,
University of Florence, Florence, Italy.
We recently developed an ultrafast force-clamp laser trap technique [Capitanio
et al., Nature Methods 9,1013-1019(2012)] that allows probing, under con-
trolled force, both long- and short-lived biomolecular interactions (100ms to
tens/hundreds of seconds), as well as sub-nanometer conformational changes
occurring upon bond formation. Here, we show the application of our method
to the study of lactose repressor (LacI). Our results show two kinetically well-
distinct populations of interactions, which clearly represent strong interactions
(targeting the two operators located 100nm apart from each other: long events
in the figure) and fast scanning of LacI along non-cognate DNA (during target-
search: short events in the figure). Our results demonstrate the effectiveness of
the method to study the sequence-dependent affinity of DNA-binding proteins
along the DNA molecule and the effects of force on a wide range of interaction
durations, including ms time scales not accessible to other methods. This im-
provement in time resolution provides
also important means of investigation on
the long-puzzled mechanism of target
search on DNA and possible protein con-
formational changes occurring upon target
recognition.
This research is funded by the Italian Min-
istry for Education, University and Re-
search in the framework of the Flagship
Project NANOMAX.2144-Pos Board B163
Lactose Repressor Functions as a DNA Topological Barrier in Escherichia
Coli Lactose Operon
Geraldine Fulcrand, Steven Eichelbaum, Fenfei Leng.
Florida International University, Miami, FL, USA.
Escherichia coli lac repressor (LacI) is a paradigm transcriptional factor that
controls the expression of three genes in the lac operon. It is a tetrameric protein,
specifically binds to lac operators, such asO1,O2 andO3, and forms aDNA loop
to negatively control transcription initiation. Previously, we found that LacI
upon binding tomultiple lacO1 operators is capable of acting as a DNA topolog-
ical barrier to block DNA supercoil diffusion and dividing a supercoiled DNA
molecule into two independent topological domains (Leng et al. (2011) Proc
Natl Acad Sci USA 108: 19973-78). In this study, we showed that LacI is able
to function as a topological barrier and block supercoil diffusion upon binding
418a Tuesday, February 5, 2013toO1,O2, andO3 operators in the lac operon. Specifically, we demonstrated that
LacI kept two supercoils within the 401 bp DNA-loop between O1 and O2 op-
erators. Additionally, We carried out time course studies to determine the stabil-
ity of the topological barriers that are produced by the different LacI-operator
complexes. Our results showed that the stability of the topological barriers cor-
relates with the DNA-binding affinity of LacI to the different operators i.e., O1,
O2, O3, and Os operators. Furthermore, we confirmed our previous observation
in which LacI is able to ‘‘keep’’ certain superhelical energy to stabilize LacI-
lacO1 complexes. Our results can be explained by a model in which LacI be-
haves as a topological barrier in the lac operon to regulate the expression of lac-
ZYA genes in Escherichia coli cells.
2145-Pos Board B164
Flanking DNAMatters in DNALoop Formation and BreakdownMediated
by Lac Repressor Protein
Joel D. Revalee, Jens-Christian Meiners.
University of Michigan - Ann Arbor, Ann Arbor, MI, USA.
The lac Repressor protein (LacI) is a paradigm for the study of protein-
mediated DNA loops in bacteria. When it binds to two distant operator sites
on substrate DNA, it causes the formation of DNA loops. Although past
in vitro and in vivo experiments have shown that changes in the inter-loop
DNA can affect loop formation and breakdown, here I will present in vitro ex-
perimental evidence that the DNA outside the loop that is flanking the operators
can also affect looping kinetics. Observing loop formation and breakdown in
single molecules with Tethered Particle Motion (TPM) we found fluctuations
in how much DNA is bound to the protein, and these fluctuations depend on
the AT- or GC-content of the flanking regions outside the loop. This suggests
an interaction between the looped complex and the flanking region which has
not been previously observed. The studies complement previous experiments
which found that DNA between the operator sites interacts with LacI while
in the looped state. These results suggest that the presence or absence of flank-
ing DNA interactions with the protein complex could give rise to additional
states in TPM experiments that are not caused by conformational changes of
the protein complex or topologic variations of the loop.
2146-Pos Board B165
DNA Competition Experiments Reveal the Importance of Operator
Binding Strength and Inter-Operator Sequence in Protein-Mediated
DNA Looping
Joel D. Revalee, Henry D. Wilson, Jens-Christian Meiners.
Univeristy of Michigan, Ann Arbor, MI, USA.
DNA loopingmediated by the lacRepressor protein (LacI) is a paradigmof study
for protein-DNA interaction as well as DNAmechanics on the nanoscale. Loop
formation in this systemoccurs by the spontaneous binding of LacI to two distant
operators on its DNA substrate. We employ single-molecule Tethered Particle
Motion (TPM) to observe loop formation and breakdown in various DNA sub-
strates.We are discussing and aiming to explain substantial differences between
loop lifetimemeasurements in bulk and single-molecule experiments, namely an
unexpected difficulty in competing bound protein off in the presence of excess
DNA even though loops continued to form and break down rapidly. For this
aim, we conduct single-molecule DNA competition experiments using DNAs
with different operator strengths and intra-operator sequences giving rise to in-
trinsic bends.Wefind thatwe are able to compete off LacI that is bound to unbent
DNA constructs with non-ideal operators in amatter of minutes, in line with typ-
ical loop breakdown rates. In contrast, competing off LacI bound to unbent or
intrinsically curved DNA substrates with ideal operators took at least hours up
to days in both single-molecule and bulk experiments, even though loops con-
tinue to break down and form repeatedly within minutes. To explain this resis-
tance to competition of the LacI-DNA complex in DNA substrates with ideal
operators while loops continue to break down, we posit a weak binding of
LacI to non-operator DNA in the unlooped state; this hypothesis is supported
by recent complementary experiments that reveal interactions between LacI
and non-operator DNA outside the looping region.
2147-Pos Board B166
Analysis of DNALoopingKinetics in TetheredParticleMotionExperiments
using Hidden Markov Models
Martin Linde´n1,2, Stephanie Johnson2,3, Jan-Willem van de Meent4,
Rob Phillips2, Chris H. Wiggins4.
1Stockholm University, Stockholm, Sweden, 2California Institute of
Technology, Pasadena, CA, USA, 3University of California, San Fransisco,
CA, USA, 4Columbia University, New York, NY, USA.
Tethered particle motion (TPM) is a powerful method for measuring DNA-
protein interactions at the single molecule level. TPM experiments monitorthe Brownian motion of beads tethered to a microscope cover slip. The Brow-
nian motion changes when a protein binds to and deforms the DNA ’leash’, for
example in the formation of a DNA loop. A complicating factor in the interpre-
tation of TPM data is that the number of observable states, corresponding to
different conformations of the DNA-protein complex, is often not known in ad-
vance. Moreover, conformational transitions that occur on time scales compa-
rable to the diffusive motion of the bead are difficult to extract from the data.
We present an analysis method for TPM data that overcomes these limitations in
existing approaches. Ourmethod relies on variationalBayesian inference on a var-
iantof theHiddenMarkovmodel.This variational approach allowsus to determine
the number of states directly from the data in a statistically principled manner.
Moreover, by operating directly on the position data, we achieve significantly
better time resolution compared to methods based on running averages of the
bead root-mean-square distance from the tethering point. Finally, we show
that hierarchical techniques developed in the context of single molecule
FRET experiments can be adapted to our TPMmethods to perform pooled anal-
ysis on many trajectories. This increases the accuracy of the method despite
considerable bead-to-bead variability, and allows a more precise characteriza-
tion of rare events.
We apply our method to Lac-mediated loop formation on a short (107 bp)
construct, and demonstrate direct interconversion between two different
looped states, with implications for structual models of the looped Lac-
DNA complex.
2148-Pos Board B167
Binding to Different Homologous DNA Sequences by a Protein alters the
Dynamical Properties of the Bound Protein in a DNA Sequence Dependent
Manner: Operator DNA Induced Allostery in the Structure and Dynamics
of Lambda Repressor
Tanumoy Mondol, Samir Kumar Pal.
SNBNCBS, Kolkata, India.
D-repressor-operator sites interaction, particularly OR1 and OR2, is a key
component of the D-genetic switch. The N-terminal domain of the protein
D-repressor interacts with the DNA, while the C-terminal domain is responsi-
ble for most of the protein–protein interactions, essential for the co-operative
binding and for the functioning of the genetic switch. Frster resonance energy
transfer from the dansyl, bound to the C-terminal domain of the protein, to the
intercalated EtBr in the operator DNA, indicates that the structure of the pro-
tein is more compact in the OR2 complex than in the OR1 complex. We have
explored the photoinduced electron transfer process from the tryptophan moi-
eties of D-repressor to OR1 and OR2 DNA to verify the conformational differ-
ences of the C-terminal domain of the repressor, bound to OR1 and OR2 DNA.
Most importantly, fluorescence anisotropy study reveals enhanced flexibility
of the C-terminal domain of the repressor at ultrafast timescales upon com-
plexation with OR1. In contrast, OR2 bound repressor shows no significant en-
hancement of protein dynamics at these timescales. Moreover, sedimentation
equilibrium study reveals that this differential dynamics is important for cor-
rect protein-protein interactions between two D-repressor dimers bound to
OR1 and OR2, for the functioning of the genetic switch. Hence, we demon-
strate that binding of transcription factors to specific DNA sequences alters
the dynamical properties of the bound protein in a DNA-sequence dependent
manner and the dynamical difference contributes to the formation of correct
regulatory complex.
References:
1. Mondol, T. et al. FEBS Lett. 586 (2012) 258 (Cover Article).
2. Mondol, T. et al.J. Biomol. Struct. Dyn. 30 (2012) 362.
2149-Pos Board B168
TheDifferentFMRPIsoformsBindwithHighAffinity to theG-Quadruplex
formed by the FMRP mRNA
Anna Blice-Baum, Rita Mihailescu.
Duquesne University, Pittsburgh, PA, USA.
Fragile X syndrome, the most common form of inherited mental retardation in
humans, affects about 1 in 3000 males and 1 in 5000 females. It is caused by the
loss of expression of the fragile X mental retardation protein (FMRP) due to
a CGG trinucleotide repeat expansion in the 5’-untranslated region (UTR) of
the fragile x mental retardation-1 (fmr1) gene. FMRP has been shown to use
its arginine-glycine-glycine (RGG) box RNA binding domain to bind with
high affinity and specificity to G quadruplex forming mRNA sequences. The
binding of FMRP to a proposed G quadruplex structure in the coding region
of its own mRNA (100 nucleotide fragment named FBS) has been proposed
to affect mRNA splicing events for isoforms 1 through 3. In this study we trun-
cated the original 100 nt FMRP-FBS to 42 nt and used biophysical methods to
directly demonstrate its folding into a G-quadruplex structure and the binding
affinity of the different FMRP isoforms to it.


Lactose Repressor Functions as a DNA Topological Barrier in Escherichia Coli Lactose Operon 

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Lactose Repressor Functions as a DNA Topological Barrier in Escherichia Coli Lactose Operon

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