Tài liệu hạn chế xem trước, để xem đầy đủ mời bạn chọn Tải xuống
1
/ 17 trang
THÔNG TIN TÀI LIỆU
Thông tin cơ bản
Định dạng
Số trang
17
Dung lượng
220,66 KB
Nội dung
F1–StructureandFunctionofMotor Proteins
F1-001
What single-molecule mechanics can tell us
about mitosis?
J. Howard
Max Planck Institute of Molecular Cell Biology & Genetics,
Dresden, Germany. E-mail: howard@mpi-cbg.de
Our laboratory is interested in the biochemical and biophysical
basis of cell structure. The structureof a cell is determined
primarily by its cytoskeleton, which serves as a scaffold to sup-
port the plasma membrane, and as a network of tracks along
which motorproteins transport sub cellular structures. Our
research is therefore focused on the mechanics of the cytoskele-
ton, with a particular emphasis on microtubules and microtu-
bule-based motors. On one hand, we are interested in the
mechanisms by which these proteins work: i.e. how do kinesins
and dyneins convert chemical energy derived from the hydrolysis
of ATP into mechanical work used to move along or to
Abstracts
332
depolymerize microtubules? And on the other hand, we are inter-
ested in the roles that microtubules and their motors play in cell
morphology and motility. In this regard we are particularly inter-
ested in how the dynamic properties of microtubules and motors
drive spindle and chromosome movements in mitosis. To address
these questions we are combining molecular biology techniques
with image processing, modeling, mechanical measurements and
single-molecule techniques.
F1-002
Control of actin assembly in cell motility
M F. Carlier
CNRS, Gif-sur-Yvette, France. E-mail: carlier@lebs.cnrs-gif.fr
Living cells change shape and move in response to environmen-
tal signals. These motile processes play a pivotal role in mor-
phogenesis, migration of embryonic and metastatic cells,
angiogenesis, synaptic plasticity, immune response and interac-
tion of the host cells with pathogens. They are generated by
polarized, spatially directed actin assembly. It is the treadmilling
(dissipative turnover) of actin filaments, regulated by specific
proteins, which is responsible for force and directional move-
ment. Two cellular machineries are responsible for spatially
directed initiation of actin filaments and they operate in distinct
processes, (i) the WASP-Arp2/3 system is at the origin of the
formation of a branched filament array; (ii) formins, in associ-
ation with profilin, catalyze the rapid processive assembly of
non-branched actin filaments. We have combined a biochemical
and a biomimetic approach to understand the molecular mecha-
nisms of these auto-organized processes. We have reconstituted
the sustained actin-based movement of a N-WASP- or formin-
functionalized particle in a biochemically controlled medium,
which enables measurement of force production in correlation
with structureand motility, and we can derive information on
the molecular mechanism of movement by single molecule
measurements.
F1-003
The structureof the myosin VI motor reveals
the mechanism of directionality reversal
J. Me
´
ne
´
trey
1
, A. Bahloul
1
, C. Yengo
2
, A. Wells
2
, C. Morris
2
,
H. L. Sweeney
2
and A. Houdusse
1
1
UMR144 - CNRS, Institut Curie, Paris, France,
2
Departement of
Physiology, Pennsylvania School of Medicine, Philadelphia, PA
19104–6085 United States of America.
E-mail: anne.houdusse@curie.fr
We have solved a 2.4 A
˚
structure of a truncated version of the
reverse direction myosin motor, myosin VI that contains the
motor domain and binding sites for two calmodulins. Surpris-
ingly, the structure reveals only minor differences in the motor
domain as compared to plus-end directed myosins, with the
exception of two unique inserts. The first insert is near the nuc-
leotide-binding pocket, and alters the rates of nucleotide associ-
ation and dissociation. The second unique insert forms an
integral part of the myosin VI converter domain along with a
calmodulin bound to a previously unseen binding motif within
the insert. This serves to redirect the effective ‘‘lever arm’’ of
myosin VI, which includes a second myosin VI calmodulin bound
to an ‘‘IQ motif’’, towards the pointed (–) end of the actin fil-
ament. This repositioning largely accounts for the reverse direc-
tionality of this class of myosin motors. We propose a model
incorporating a kinesin-like uncoupling/docking mechanism to
fully explain the movements of myosin VI.
F1-004
Regulation and mechanics of myosin V
J. R. Sellers
Laboratory of Molecular Physiology, National Heart, Lung and
Blood Institute, National Institute of Health, Bethesda, MD United
States of America. E-mail: sellersj@nhlbi.nih.gov
Myosin V is vesicle motor that moves processively on actin filaments.
In melanocytes it functions in cooperation with microtubule motors
to localize melanosomes to the dendritic tips. Myosin V dimerizes
via a coiled coil motif in its tail region to produce a two-headed
structure. Each head is composed of a compact motor domain and a
long neck formed by the association of calmodulin residues with six
tandem IQ motifs. This structure allows the molecule to take 36 nm
steps along an actin filament corresponding to the helical pitch of the
actin and, in turn, allows the molecule to keep its cargo positioned
above the cytoskeleton. We study the mechanics of the processive
motor using optical trapping and total internal reflection fluores-
cence microscopy (TIRF). These data confirm that the neck of myo-
sin V acts as a rigid lever arm and that myosin V moves in a hand-
over-hand manner. The enzymatic activity of myosin V is regulated
by calcium in vitro. This is coupled to a large conformational change
in the molecule. In the absence of calcium myosin V folds into a
compact triangular-shaped structure in which the heads bend down
and contact the globular tail domain. Under these conditions the
molecule migrates with a sedimentation coefficient of 14S. In the
presence of calcium or at high ionic strength the molecule opens up
to form T- or Y-shaped structures which sediment at 11S. We pro-
pose that in the cell, the transition between the open, active state and
the folded, inactive state is regulated by binding of receptor or dock-
ing proteins to the myosin V globular tail domain. This would not
only target the myosin to the proper cargo, but would also lock the
myosin into an active form. We are currently studying the interac-
tions responsible for the folded, off state of this molecule.
F1-005
F-actin modulates myosin conformational
states by switch I loop movement
B. Kintses
1
, M. Gyimesi
1
, W. Zeng
2
, P. B. Conibear
2
,
C. R. Bagshaw
2
and A. Ma
´
lna
´
si-Csizmadia
1
1
Departement of Biochemistry, Eo
¨
tvo
¨
s Lora
´
nd University, Buda-
pest, Hungary,
2
Department of Biochemistry, University of Leices-
ter, Leicester, United Kingdom. E-mail: amc26@leicester.le.uk
Myosin converts chemical energy to mechanical work. It undergoes
a large conformational change during the ATPase cycle that results
a step on the actin filament. The main question is how the func-
tional regions communicate with each other. Another problem
concerns the development and relaxation of the mechanical strains
during the catalytic cycle and their perturbation by external force.
We have extensively characterized the energetic coupling between
nucleotide binding, the so-called open/closed transition and actin
binding using different single tryptophans located at the relay loop
(W501, Dictyostelium sequence), the nucleotide binding region
(W129) and the switch 1 region (W239 and W241). The fluores-
cence from native actin tryptophan residues is not significantly per-
turbed on binding to myosin, although a fluorescence signal is
detected as a consequence of a light scatter artifact. ATP, ATPa
˜
S
and ADP binding affected less than threefold by actin. The isome-
rization detected by W129 clearly precedes the dissociation of actin
in the case of ADP and ATPa
˜
S binding. The fluorescence from the
conserved W501 residue located at the distal end of the relay helix
is very sensitive to the lever arm disposition and the observed fluor-
escence emission intensity can be used to estimate the equilibrium
constant between the pre and post power-stroke conformations.
Actin modulates this equilibrium by no more than twofold. These
data suggest that actin activates another process in the mechanism,
Abstracts
333
such as switch 1 movement, rather than influencing the switch 2
equilibrium. Consequently, despite actin does not have major effect
on nucleotide binding and the equilibriums of the pre- and post-
power-stroke the fluxes of the kinetic routes change fundamentally
by actin binding: in the absence of actin phosphate release precedes
the closed-open transition while in ternary complex the main route
is that products releases follow the power stroke.
F1-006
Calmodulin and calmodulin-like protein as
light chains for myosin-10: Specificity and role
in protein stabilization leading to prolonged
function
E. E. Strehler, A. J. Caride, A. S. Mauer and R. D. Bennett
Biochemistry and Molecular Biology, Mayo Clinic College of
Medicine, Rochester, MN, United States of America.
E-mail: strehler.emanuel@mayo.edu
Vertebrate myosin-10 (MYO10) is involved in filopodial motility,
phagocytosis, and microtubule-F-actin interactions. Each MYO10
heavy chain contains three IQ motifs, which are light chain binding
sites. Previous work has shown that the first two IQ domains of
MYO10 bind calmodulin (CaM) whereas IQ3 alternatively binds
calmodulin-like protein (CLP). We performed stopped-flow experi-
ments with fluorescent derivatives of CaM (TA-CaM) and CLP
(TA-CLP) to analyze their binding to the IQ3 peptide. TA-CaM
bound to IQ3 in the absence and in the presence of 100 lm Ca
2+
.
In the absence of Ca
2+
the binding was faster, albeit with lower
affinity, suggesting an even faster dissociation rate. In the presence
of Ca
2+
, the time course of the TA-CaM-IQ3 reaction was best
described by an exponential function. CLP was able to reduce the
amplitude of this exponential in the presence, but not in the
absence, of Ca
2+
. These results suggest a novel IQ domain interac-
tion, in which Ca
2+
regulates binding of CaM to IQ3 by modula-
ting competition with CLP. In HeLa cells over-expressing CLP,
endogenous MYO10 was strongly upregulated. Likewise, transfec-
tion with GFP-MYO10 resulted in increased fluorescence in cells
that co-expressed CLP or excess CaM. MYO10 upregulation resul-
ted in an increase in the size and number of filopodia. Cells expres-
sing CLP displayed increased motility as indicated by their
shortened wound-healing time compared to control cells lacking
CLP. CLP-dependent upregulation of MYO10 was due to
increased protein stability. CLP expression in specific epithelial
cells may thus prolong MYO10 function in conditions of elevated
intracellular Ca
2+
and limiting CaM. Acknowledgment: Suppor-
ted by grants from the Susan G. Komen Breast Cancer Foundation
(EES) and the American Heart Association (AJC).
F1-007P
Crystal structureof the portal protein from
bacteriophage SPP1 and model for DNA
translocation
A. A. Lebedev
1
, M. H. Krause
2
, A. Vagin
1
, E. V. Orlova
3
,
E. J. Dodson
1
, P. Tavares
4
and A. A. Antson
1
1
YSBL, Chemistry Department, York University, York, United
Kingdom,
2
Max-Planck Institut fu
¨
r Molekulare Genetik, Berlin,
Germany,
3
Birkbeck College, Department of Crystallography, Uni-
versity of London, London, United Kingdom,
4
Unite
´
de Virologie
Mole
´
culaire et Structurale, Gif-sur-Yvette, France.
E-mail: fred@ysbl.york.ac.uk
The mechanism of DNA translocation into a viral procapsid
remains one of the most intriguing questions of viral particle
assembly [1]. Tailed bacteriophages and herpes viruses have a
specialized vertex for double-stranded DNA (dsDNA) entry
into the procapsid during viral chromosome packaging [2, 3].
The main component of this specific doorway is the portal pro-
tein, a circular oligomer with a central tunnel through which
the DNA transfer occurs. Together with viral ATPase (termi-
nase) the portal protein forms a molecular motor that is able
to translocate DNA against high internal pressure [4]. We
determined the X-ray structureof the SPP1 portal protein
(gp6) in its 13-subunit oligomeric form where the tunnel residue
segments, not visible in the previously determined structures of
bacteriophage phi-29 portal protein [5, 6], are well defined and
form a DNA-transfer arm. Our X-ray and electron microscopy
data suggest that DNA translocation is driven by a novel
mechanism involving mechanical movements of arms along the
inner walls of the tunnel. We propose a model for DNA trans-
location where such movements propagate around the double
helix of DNA similar to a ‘‘Mexican wave’’ moving across a
stadium.
References
1. Hendrix RW. Proc Natl Acad Sci USA 1978; 75: 4779–47783.
2. Bazinet C, King, J. Annu Rev Microbiol 1985; 39: 109–129.
3. Droge A, Tavares P. J Mol Biol 2000; 296: 103–115.
4. Smith DE, et al. Nature 2001; 413: 748–752.
5. Simpson AA, et al. Nature 2000; 408: 745–750.
6. Guasch A, et al. J Mol Biol 2002; 315: 663–676.
F1-008P
Unraveling the catalytic mechanism of the
bacteriophage T7 gene 4 helicase
D. J. Crampton, S. Mukherjee, A. van Oijen and
C. C. Richardson
Department of Biological Chemistry and Molecular Pharmacology,
Harvard Medical School, Boston, MA, USA.
E-mail: dcrampton@hms.harvard.edu
The gene 4 helicase of bacteriophage T7 is a molecular motor
that unwinds double-stranded DNA using the energy derived
from the hydrolysis of deoxynucleoside 5-triphosphates. Here
we present data pertaining to four essential activities of the heli-
case that act cooperatively to unwind DNA. (i) Oligomer For-
mation. The T7 gene 4 helicase forms both heptamers and
hexamers dependent upon the presence of nucleoside di- or tri-
phosphates respectively. We find that the heptamer cannot bind
DNA but rather, in the presence of single-stranded DNA, hep-
tamer converts to hexamer when both nucleoside di- and tri-
phosphates are present together. This conversion between
oligomers is regulated by histidine 465 through differentiation
of the absence or presence of a c-phosphate on the bound nuc-
leotide. (ii) DNA Binding. The loop comprised of residues 466–
475 has been postulated to be the major site for the binding of
single-stranded DNA. Lysines 471 and 473 of this loop were
altered to determine their contribution to the overall binding of
single-stranded DNA. (iii) dTTP Hydrolysis. We find that all
subunits of the hexamer are active in the hydrolysis of dTTP.
Changing the catalytic base glutamate 343 to glutamine creates
a non-catalytic subunit that responds to dTTP in a manner sim-
ilar to the effect of non-hydrolyzable analog b,cc-methylene
dTTP on wild-type helicase. The presence of a single non-cata-
lytic subunit per hexameric unit abolishes all dTTPase activity.
(iv) Translocation. We are developing methods of observing
movement along DNA by the gene 4 helicase using single-mole-
cule fluorescence microscopy.
Abstracts
334
F1-009P
Myosin VI in chromaffin cells
M. Dominik
1
, L. Kiljanek
2
, and M. J. Redowicz
1
1
Laboratory of Cell Motility, Department of Muscle Biochemistry,
Nencki Institute of Experimental Biology, Warsaw, Poland,
2
Laboratory of Confocal Microscopy, Nencki Institute of Experi-
mental Biology, Warsaw, Poland.
E-mail: mdominik@nencki.gov.pl
Myosin VI (MVI), the member of one of at least 18 different
myosin families, is ubiquitously expressed in multicellular eukary-
otes. It is an unusual actin-based motor since it walks towards
the minus end of actin filaments, in the opposite direction to
other myosins. MVI is involved in subcellular transport of mem-
brane-containing structures, it has been found to be engaged in
clathrin-based endocytosis and cell spreading and migration.
Here, we aimed at testing the involvement of MVI in chromaffin
granule trafficking within chromaffin cells. MVI, but not myosin
V, has been found in vesicular fractions isolated from bovine
adrenal medulla and rat pheochromocytoma cells (PC12). MVI is
on the apical side of the vesicle and its association with the gran-
ule seems to be very tight as the stripping with high salt concen-
tration or high pH does not remove it from the granule surface;
MVI is stripped from the granules only after addition of Triton
X100. MVI colocalizes with dopamine-b-hydroxylase (DbH),
chromaffin granules marker, both in PC12 cells and primary cell
culture of adrenal medulla. It has been observed that MVI and
DbH were still found on the same granules after their isolation
from PC12 cells, confirming strong association of MVI with the
granules. Five minute stimulation of PC12 cells with 56 mm KCl
seems to affect MVI localization as the fluorescence intensity cor-
responding to MVI was enhanced within the perinuclear area
only in stimulated cells. These preliminary data seem to indicate
the important role of MVI in secretory cells.
F1-010P
Enzyme kinetics above denaturation
temperature
M. Gyimesi, Z. Simon and A. Ma
´
lna
´
si-Csizmadia
Department of Biochemistry, Eo
¨
tvo
¨
s Lora
´
nd University, Budapest,
Hungary. E-mail: gyimesimate@cerberus.elte.hu
Wide range of temperature dependence of enzyme kinetics meas-
urements enables more precise determination of reaction energetics
and accurate separation of reaction steps. We have developed a
novel temperature-jump/stopped flow method to measure enzyme
transient kinetics at high temperature even above denaturation
temperature of enzymes. This method enables us to increase the
temperature in a millisecond time scale parallel with mixing of the
reactants. Heat denaturation normally occurs in a second time
scale. If the temperature is increased above the enzyme denatura-
tion temperature, essentially all of the events can be followed that
are faster than heat denaturation reaction. We have tested the new
method on myosin ATPase and GFP folding/unfolding reactions.
F1-011P
Changes of inter-subunit contacts in growing
filaments during salt-induced polymerization
of actin
A. Galinska-Rakoczy, B. Wawro and H. Strzelecka-Golaszewska
Laboratory of Structural Muscle Proteins, Department of Muscle
Biochemistry, Nencki Institute of Experimental Biology, Warsaw,
Poland. E-mail: galinska@nencki.gov.pl
Polymerization/depolymerization of actin underlies numerous
motile events in eukaryotic cells. It is well established that
conformational changes in G-actin generated by exchange of its
tightly bound Ca for Mg accelerate the nucleation reaction but
do not influence substantially the rate of filament growth during
salt-induced polymerization of actin. We used N, N’’-1,4-phenyl-
enebismaleimide (PBM) to probe the conformation of F-actin at
various stages of filament growth during polymerization of
CaATP-, MgATP-, and MgADP-G-actin. Effects of the type of
polymerizing salt, filament stabilization by phalloidin, and pro-
teolytic cleavage of actin at Gly-42 within D-loop were also
investigated. The earlier described accumulation of ’upper dimer ’
(UD), a product of cross-linking neighboring protomers from
two long-pitch F-actin strands, concomitant with disappearance
of initially formed ‘‘lower dimmers’’ (LD) with subunits in a
non-filamentous, antiparallel orientation, was observed under all
conditions except for a polymer of cleaved actin that required
phalloidin binding to yield UD. The increase in yield of UD clo-
sely followed filament assembly from MgATP-G-actin, whereas it
lagged behind polymerization of CaATP- and MgADP-G-actin.
Phalloidin accelerated the filament assembly but did not eliminate
the delay in UD formation. These results, along with changes in
morphology of growing filaments visualized by electron micros-
copy, suggest that the tightly bound Mg and initially bound ATP
not only accelerate the nucleation reaction but also promote a
rearrangement of F-actin structure that eliminates LD’s from
growing filaments. They also confirm the suggested role of
D-loop in stabilization of both the longitudinal subdomain 2/1
contacts andof the cross-strand contacts in F-actin.
F1-012P
The dynein light chain binds to a non-coiled-
coil tail domain of myosin-Va that includes an
alternatively spliced exon coding for three
amino acid residues
Z. Ho
´
di
1
,A.Ne
´
meth
1
, E. Kova
´
cs
1
, C. Hete
´
nyi
1
, A. Bodor
2
,
A. Perczel
2
and L. Nyitray
1
1
Deptartment of Biochemistry, Eo
¨
tvo
¨
s Lora
´
nd University, Buda-
pest, Hungary,
2
Department of Organic Chemistry, Eo
¨
tvo
¨
s Lora
´
nd
University, Budapest, Hungary. E-mail: nyitray@cerberus.elte.hu
Class V myosins are involved in short-range intracellular trans-
port along actin filaments. Of the three mammalian myosin-V
heavy chain genes, mutations of MYO5A are responsible for the
dilute phenotype and Griscelli syndrome type1 in mice and
humans respectively. A dynein light chain (DLC) has been identi-
fied as a tail domain light chain of myosin-Va (myo5a). DLC
may function as a cargo-binding and/or regulatory subunit of
both motor proteins. Our goal was to identify and characterize
the binding site of DLC on myo5a. Various fragments of myo5a
tail and DLC were expressed in E. coli and human cells. Forma-
tion of their complex was analyzed by pull-down assays, gel fil-
tration, and spectroscopic methods. DLC was found to bind as a
homodimer to a 12 residues segment (Pro1282-Thr1293) locali-
zed between the medial and distal coiled-coil predicted domains
of the tail. The binding region contains 3 residues, coded by the
alternatively spliced exon B that is essential for DLC binding.
Using CD spectroscopy, we demonstrate that binding of DLC to
the intrinsically unstructured DLC binding domain (DBD) stabil-
izes the neighboring coiled-coil domain. NMR spectroscopy and
molecular docking simulations show that a short synthetic pep-
tide of DBD binds to a surface groove on DLC, as has been
found with other known binding partners of DLC. We hypothes-
ize that the second binding site of the DLC dimer is either occu-
pied by the DBD of the other heavy chain, or by a cargo, or
interacts with other regulatory proteins.
Acknowledgment: Supported by OTKA T43746
Abstracts
335
F1-013P
Mutagenic analysis of the HsdR motor subunit
of type IC restriction modification enzyme
EcoR124I
E. Sisakova and M. Weiserova
Laboratory of Molecular Genetics of Bacteria, Division of Cell
and Molecular Microbiology, Institute of Microbiology,
Czech Academy of Sciences, Prague, Czech Republic.
E-mail: sisak@biomed.cas.cz
Enzyme EcoR124I belongs to the IC family of restriction modifi-
cation enzymes, intelligent molecular motors. It is able to detect
the methylation status of its DNA target sequence and respond
with alternative activities, methylation or translocation of DNA.
While bound to its target site, it translocates DNA towards itself
simultaneously in both directions (500bp/sec). It uses the free
energy associated with ATP hydrolysis to translocate DNA so
that DNA cleavage occurs remote from the asymmetric recogni-
tion site. The enzyme EcoR124I is multifunctional, multi-subunit
enzyme, composed of three different subunits, which are encoded
by the genes hsdR, hsdM and hsdS. Products of all three genes
are required for DNA cleavage, producing the endonuclease.
HsdR subunit is a multifunctional motor protein, which has been
shown to posses ATPase, helicase and restriction activity. To
provide a fully functional molecular motor, which can never
cleave DNA, the amino acid motif X, the active site of the endo-
nuclease domain of the HsdR subunit, was subjected to site-
directed mutagenesis. The complementation analysis proved that
the substitutions D151A, E165A, E165D, E165H, K167A in the
HsdR subunit fully removed the restriction activity in vivo of the
EcoR124I enzyme. The mutant subunits were separately overpro-
duced, purified and mixed with purified methylase to reconstitute
the EcoR124I endonuclease in vitro. As a substrate for DNA
cleavage in vitro we used the plasmid pCFD30 containing a sin-
gle site for EcoR124I. The test of restriction activity showed that
reconstituted endonucleases were not able to cleave covalently
closed plasmid DNA to linear DNA in contrast to the wild-type
enzyme.
F1-014P
Dielectric and fluoroscopic study on the
dynamic effects of myosin-S1 with/without
ATP on the hyper-mobile water around actin
filaments
M. Suzuki
1
, M. P. Siddique
1
, T. Miyazaki
1
, J. Mogami
1
,
E. Katayama
2
, T. Kodama
3
and T. Q. P. Uyeda
4
1
Laboratory of Physicochemistry of Biomolecular Systems, Depart-
ment of Materials Science and Engineering, Tohoku University,
Sendai, Japan,
2
Division of Fine Morphology, The Institute of
Medical Science, The University of Tokyo, Tokyo, Japan,
3
Molecular Enzymology, Department of Bioscience and Bioinfor-
matics, Kyushu Institute of Technology, Ihzuka, Japan,
4
Gene
Function Research Center, AIST, Tsukuba, Japan.
E-mail: msuzuki@material.tohoku.ac.jp
Using high resolution microwave dielectric spectroscopy, we have
recently shown hyper-mobile water molecules around actin fila-
ments (F-actin) which have a much higher rotational mobility
than that of bulk water [1] and its marked volume increase with-
out significant change in the ordinary hydration shell when the
myosin motor-domain (S1) binds to F-actin [2]. Here, we report
that (i) hyper-mobile water around actin filaments has been
detected with the fluoroscopic technique based on the fact that
the fluorescence intensity of fluorophore molecules, such as ribo-
flavin and pyrene-derivatives in caotropic salt aqueous solutions
was in clear correlation with those viscosity B-coefficients, (ii) the
fluorescence intensity from pyrenyl actin markedly increased
when myosin-S1 binds to actin filaments, which is consistent with
the dielectric result (BBRC, 2004), (iii) in the solution of acto-S1
fused chimera protein and skeletal actin copolymers the fluores-
cent intensity from pyrenyl-group (linked at Cys374 of actin
SD-I) increased in the presence of ATP indicating a marked
decrease of hyper-mobile water around actin filaments. An
important implication of the present result is that the S1/F-actin
interaction forms an asymmetric field of viscosity-gradient along
the filament axis, which drives the unidirectional sliding of S1
hydrolyzing ATP by means of thermal brownian movements.
References
1. Kabir SR. et al. Biophys J 2003; 85: 3154–3161.
2. Suzuki M. et al. Biochem Biophys Res Comm 2004; 322: 340–
346.
F1-015P
Drosophila myosin V: solution kinetics and
motile properties
J. To
´
th
1,2
, M. Kova
´
cs
1
, F. Wang
1
, L. Nyitray
2
and J. R. Sellers
1
1
Laboratory of Molecular Physiology, NHLBI, National Institutes
of Health, Bethesda, MD, United States of America,
2
Bioke
´
miai
Tansze
´
k, ELTE, Budapest, Hungary. E-mail: tothj@mail.nih.gov
Myosin V is the best characterized vesicle transporter myosin
motor in vertebrates but it is unknown whether all members of
the myosin V family share a common, evolutionarily conserved,
mechanism. To address this question, we performed steady state
and transient kinetic measurements on a recombinant Drosophila
myosin V S1 fragment. Interestingly, none of the product release
steps can be identified as a single rate-limiting step. The ADP
release rate constant from acto-S1, which is the bottleneck in the
vertebrate myosin V ATPase cycle, appears to be 5-fold higher
than the maximal steady-state ATPase activity. The Pi release
rate constant from the acto-S1-products complex is even faster,
implying that Drosophila myosin V does not follow a myosin
II-type kinetic pattern, either. The key features of the ATPase
cycle are the low ATP hydrolysis equilibrium constant (0.33) and
the high steady-state actin attachment (96% at 5 m actin). The
behavior of double-headed Drosophila myosin V in an in vitro
motility assay suggests that a single molecule of myosin V cannot
take multiple steps on the actin filament while being attached to
it. We propose a mechanism whereby the ATP hydrolysis and
the so-called weak actin-binding equilibria define the steady-state
ATPase rate rather than the product release process. This mech-
anism suggests that Drosophila myosin V will not act as a single
molecule vesicle transporter, in contrast to vertebrate myosin Vs.
However, it may be able to perform processive transport if pre-
sent in small ensembles on the surface of its cargo.
F1-016P
A novel centrosomal protein, Cep57 stabilizes
microtubules in vivo and binds FGF-2 in vitro.
Y. Yoshitake, N. Tsuruoka and A. Iwai
Department of Biochemistry, Kanazawa Medical University, Uchi-
nada, Ishikawa Japan. E-mail: yositake@kanazawa-med.ac.jp
We have identified a gene encoding a novel protein that binds
microtubules (MT) and fibroblast growth factor-2 (FGF-2). It
was found as FGF-2 associated protein in the course of studying
the functionof the 24kDa isoform of FGF-2 in nuclei by yeast
two-hybrid screening. This gene shares a sequence similarity with
an uncharacterized cDNA reported in GenBank as KIAA0092
and the encoded protein is identical to Cep57, a coiled-coil pro-
Abstracts
336
tein in the centrosomes. The occurrence of Cep57 in human cen-
trosomes has been recently reported but its function is unknown.
GFP-fused protein over-expressed in HeLa cells was predomin-
antly observed as filamentous structures and co-localized with
MT. Weekly-expressed tagged proteins were co-localized with
MT as spindle, spindle pole and MT in the midbody of the mito-
tic cells and also localized in the centrosomes of interphase cells.
Treatments disturbing the MT network did not have any effect
on this protein distribution in the cells. Over-expressed Cep 57
protected the MT structure against those treatments. Therefore,
Cep 57 stabilizes MT. Recombinant Cep57 protein bound both
isoforms of FGF-2 (24kDa and 18kDa) in vitro. However, the
localization patterns of endogenous Cep57 observed by immuno-
fluorescent staining using the antibodies differed in both patterns
of FGF-2 isoforms. siRNA to Cep57 induced a morphological
change (spindle-like shape with a poor cytoskeletal MT network)
and decreased cell growth in HeLa cells. Cep57 may work as a
MT stabilizing protein in the centrosomes and spindle pole, and
then play a role on MT network formation and cell division.
F1-017P
A novel dynamic model for actin
polymerization based on the idea of
self-catalysis
J. Zhang
1,2
and Y. L. Wang
2
1
Department Biology, Chongqing University of Medical Sciences,
Chongqing City, PR China,
2
Key Lab of Biomechanics & Tissue
Engineering, Institute of Bioengineering, Chongqing University,
Chongqing City, PR China. E-mail: zhangjun1017@sohu.com
More and more researches have recently demonstrated that there
exist multiple pathways in actin polymerization. In order to eluci-
date the macroscopic dynamics of actin assembly, a novel phe-
nomenological theory for actin polymerizing under simple
thermodynamic conditions has been put forward. The theoretical
model, called self-catalytic dynamics, emphasizes that the process
of actin polymerizing can be thought of an autocatalytic chain
reactions. To set up the quantified kinetic equations group, three
basic postulations is employed, which are in good agreement with
the fundamental physical rules and published literatures: (i) the
actin monomers driven by Brownian motion in solution can
interact with each other, and further form actin dimer via non-
covalent association under certain molecular colliding conditions;
(ii) the formed actin dimer and higher polymer spontaneously
own catalyzing ability and can catalyze free actin monomer or
dimer changing into actin residues (that refer to the G-actin
monomers existing in actin filaments); (iii) the polymerization/
depolymerization processes can go on synchronously at the both
ends (generally, the barbed and pointed ends, sometimes, also at
the side of the elongating filaments) with different polymeriza-
tion/depolymerization rates. According to the analyses and basic
enzyme catalysis principles, a scheme of kinetic differential equa-
tions can be established with definite initial conditions. By strug-
gling out the integral curves of the equations group, we get a
series of dynamic functions, via which we can accurately calculate
the length distribution of filaments, the concentration of free
G-actin and polymerized filaments versus the polymerizing time,
and also quantificationally explain the treadmilling phenomenon
of actin turnover.
F2–Single Molecule Biochemistry and Mechanics
F2-001
Recent Advances in Single Molecule
Biophysics
C. Bustamante
Department of Physics, University of California, Berkeley, CA,
USA. E-mail: carlos@alice.berkeley.edu
I will present our recent results on the packaging of DNA by the
connector motor at the base of the head of bacteriophage Ø29.
As part of their infection cycle, many viruses must package their
newly replicated genomes inside a protein capsid to insure its
proper transport and delivery to other host cells. Bacteriophage
Ø29 packages its 6.6 mm long double-stranded DNA into a
42 nm dia · 54 nm high capsid via a portal complex that hydro-
lyses ATP. This process is remarkable because entropic, electro-
static, and bending energies of the DNA must be overcome to
package the DNA to near-crystalline density. We have used opti-
cal tweezers to pull on single DNA molecules as they are pack-
aged, thus demonstrating that the portal complex is a force
generating motor. We find that this motor can work against
loads of up to 57 picoNewtons on average, making it one of the
strongest molecular motors ever reported. Movements of over
5 mm are observed, indicating high processivity. Pauses and slips
also occur, particularly at higher forces. We establish the force–
velocity relationship of the motorand find that the rate-limiting
step of the motor’s cycle is force dependent even at low loads.
Interestingly, the packaging rate decreases as the prohead is
filled, indicating that an internal pressure builds up due to DNA
compression. We estimate that at the end of the packaging the
capsid pressure is 6 MegaPascals, corresponding to an internal
force of 50 pN acting on the motor. The biological implications
of this internal pressure and the mechano-chemical efficiency of
the engine are discussed.
F2-002
The complete folding/unfolding trajectory
of a protein captured with single molecule
force-clamp spectroscopy
J. M. Fernandez
Biological Sciences, Columbia University, New York, NY 10027,
USA. E-mail: jfernandez@columbia.edu
A dense network of interconnected proteinsand carbohydrates
forms the complex mechanical scaffold of living tissues. The
recently developed technique of single molecule force spectro-
scopy has enabled a detailed analysis of the force-induced confor-
mations of these molecules and the determinants of their
mechanical stability. These studies provide some of the basic
knowledge required to understand the mechanical interactions
that define all biological organisms. The application of mechanical
force to biological polymers produces conformations that are dif-
ferent than those that have been investigated by chemical or ther-
mal denaturation, and are inaccessible to conventional methods of
measurement such as NMR spectroscopy and X-ray crystallogra-
phy. Force-induced conformational transitions are physiologically
Abstracts
337
important, and offer novel perspectives on the structureof bio-
molecules. Recent developments in single molecule force spectros-
copy have enabled study of the full unfolding and refolding
pathways of a protein under force-clamp conditions. Mechanical
unfolding of a protein placed under a constant stretching force
allows us to obtain precise information about the pathway and
kinetics of unfolding. Force-quench experiments capture for the
first time the full folding pathway of a protein. Due to the force
of gravity and the need of living organisms to perform mechan-
ical work, mechanical stretching is most likely to have played a
role in the evolution of proteins. By contrast, the large changes
in temperature or chemical denaturants commonly employed in
protein folding studies are not found in living cells. Hence, the
mechanical unfolding/folding trajectories captured by force-clamp
spectroscopy reflect much more closely the conformations and
pathways ofproteins in vivo, compared to those obtained by
means of thermal or chemical manipulations. This lecture will
focus mostly on the use of these novel techniques to study the
dynamic changes that proteins undergo in response to a mechan-
ical stretching force.
References
1. Fernandez JM, Li HB. Force-clamp spectroscopy monitors the
folding trajectory of a single protein. Science 2004; 303: 1674–
1678.
2. Carrion-Vazquez M, Li H, Lu H, Marszalek PE, Oberhauser
AF, Fernandez JM. The mechanical stability of ubiquitin is
linkage dependent. Nature Structural Biology 2003; 10(9): 738–
743.
3. Li HB, Linke WA, Oberhauser AF, Carrion-Vazquez M,
Kerkvliet JG, Lu H, Marszalek PE, Fernandez JM. Reverse
engineering of the giant muscle protein titin. Nature 2002; 418:
998–1002.
F2-003
Tethering single protein concatamers to
enable measurement of thermal noise
response by Atomic Force Microscopy (AFM)
K. Byrne, M. Kawakami, B. Khatri and A. Smith
Laboratory of Molecular and Nanoscale Physics, Department of
Physics and Astronomy, University of Leeds, Leeds,
West Yorkshire, UK. E-mail: phykb2@phys-irc.leeds.ac.uk
The thermal noise response of single molecules under tension can
be measured using AFM and used to reveal both the conserva-
tive and dissipative response of the molecule. This recently devel-
oped method has been applied successfully to single sugar
molecules tethered via non-specific interactions between the
AFM cantilever and the base substrate. In order to apply the
method to single protein concatamers we have found that a more
durable means of tethering the molecules is required. Single pro-
tein molecules can be suspended between a cantilever and sub-
strate using non-specific tethering but the tether breaks before
sufficient data can be captured to give an accurate noise spec-
trum in all cases observed thus far. Tethering molecules through
covalent rather than non-specific bonds should enable longer dur-
ation experiments to be carried out with ease and has the added
advantage that the exact location of the tether point is known. A
protocol to tether a modified 5-domain concatamer of I27
between a gold coated cantilever and gold substrate via distinct
covalent links is being developed. The gold binding functionality
of the C-terminal cysteine residue and the ability of N-terminal
histadine residues to bind succinamide will be exploited. The
lysine residues have been removed from the concatamer so that
succinamide-amine bonds should only form at the N-terminal
end of the concatamer. A self assembled monolayer of the short
linker molecule Di-thio-bis-succinamidyl-propionate (DSP) on
gold will be used to tether the N-terminus. The short linker will
interfere only minimally in the thermal noise spectrum molecule
of interest.
F2-004
Single molecule and single paritcle imaging
in solution and in live cells
X. Zhuang
Department of Chemistry and Chemical Biology,
Harvard University, Cambridge, MA, USA.
E-mail: zhuang@chemistry.harvard.edu
Understanding the molecular mechanisms of complex biological
processes is one of the major goals in modern biology. As
molecular and cell biology get increasingly quantitative, a com-
prehensive understanding of biological processes at the molecular
level is becoming more readily accessible. However, roadblocks
still exist, among which is the challenge that we face in character-
izing the complex dynamics of biological processes. The existence
of multiple kinetic paths and transient intermediate states often
makes these processes difficult to dissect, as individual steps of a
multi-step process are typically not synchronized among mole-
cules. To tackle this problem, we are exploring optical imaging
techniques to monitor, in real-time, the behavior of individual
biological molecules and complexes, in vitro and in live cells. In
this talk, I will report our recent progress in the following two
areas. (i) Molecular mechanisms of viral infection: our single-
virus tracking experiments allow us to visualize the viral infection
process in real time, dissect individual stages of the viral entry
pathway, and obtain a better understanding of the molecular
mechanisms governing the influenza infection. (ii) Structural
dynamics of RNA and ribonucleoprotein enzymes: our single-
molecule studies provide critical insights into the molecular
mechanisms governing RNA structural dynamics, and the effects
of proteins on the structural dynamics of RNA enzymes.
F2-005
Single molecule analysis of protein
aggregation and prions by SIFT
U. Bertsch
1
, K. F. Winklhofer
2
, T. Hirschberger
3
, J. Bieschke
1
,
P. Weber
1
, F. U. Hartl
2
, P. Tavan
3
, J. Tatzelt
2
,
H. A. Kretzschmar
1
and A. Giese
1
1
ZNP, University of Munich, Munich, Germany,
2
Department of
Cellular Biochemistry, Max-Planck-Institute for Biochemistry,
Martinsried, Germany,
3
Department of BioMolecular Optics,
University of Munich, Munich, Germany.
E-mail: armin.giese@med.uni-muenchen.de
Protein aggregation is a key event in a number of diseases such
as Alzheimer’s disease, Parkinson’s disease and prion diseases.
We present a general method to quantify and characterize pro-
tein aggregates by dual-colour scanning for intensely fluorescent
targets (SIFT). In addition to high sensitivity, this approach
offers a unique opportunity to study co-aggregation processes at
the single particle level in complex mixtures containing different
types of aggregates. In the case of prion diseases the prion pro-
tein (PrPC), a neuronal glycoprotein, undergoes a conformational
change from the normal, mainly alpha-helical conformation to a
disease-associated, mainly beta-sheeted scrapie-isoform (PrPSc),
which forms amyloid aggregates. This conversion, which is cru-
cial for disease progression, depends on direct PrPC/PrPSc inter-
action. We developed a high-throughput SIFT assay for the
identification of drugs, which interfere with this interaction at the
molecular level. Screening a library of 10 000 drug-like com-
pounds yielded 256 primary hits, 80 of which were confirmed by
Abstracts
338
dose–response curves with half-maximal inhibitory effects ranging
from 0.3 to 60 lm. Among these, six compounds displayed an
inhibitory effect on PrPSc propagation in scrapie-infected N2a
cells. Four of these candidate drugs share a N
´
-benzylidene-ben-
zohydrazide (NBB) core microstructure. Thus the combination of
high-throughput in vitro assay with the established cell culture
system provides a rapid and efficient method to identify new
anti-prion drugs, which corroborates that interaction of PrPC
and PrPSc is a crucial molecular step in the propagation of pri-
ons. Moreover, SIFT-based screening may facilitate the search
for drugs against other diseases linked to protein aggregation.
F2-006
Studying protein reaction kinetics by
fluorescence correlation spectroscopy in
microfluidic mixers
P. Galajda
1
, J. Puchalla
2
, R. Riehn
1
and R. H. Austin
1
1
Department of Physics, Princeton University, Princeton, NJ,
USA, ,
2
Department of Molecular Biology, Princeton University,
Princeton, NJ, USA. E-mail: pgalajda@princeton.edu
Fluorescence correlation spectroscopy is a powerful method to
gather information about the motion and interactions of mole-
cules and particles in solution. The autocorrelation of the fluores-
cence intensity fluctuation emerging from fluorophores traversing
a confined excitation/observation volume is used for analysis.
Usually either diffusion or convection dominates on the time-
scales of the fluctuations. Measurements lasts for seconds making
it inadequate for kinetic studies of most biomolecular reactions.
Here we present a method to overcome this limitation. Reactants
meet in a microfluidic diffusional mixer. A steady-state flow
establishes a direct mapping of the temporal evolution of the
reaction to the spatial position along the channel opening a way
for kinetic studies. The experimental conditions are set so that
we can extract both the characteristics of diffusion and flow from
the data. A variety of biological reactions might be studied by
the above technique from protein folding to DNA-protein inter-
actions.
F2-007P
Changes in cell morphology, viability,
proliferation and cytotoxicity associated with
helium–neon laser irradiation of diabetic
wounded human skin fibroblasts
H. Abrahamse, N. Houreld and D. Hawkins
Laser Research Unit, Faculty of Health, University of
Johannesburg, Johannesburg, Gauteng South Africa.
E-mail: heidi@twr.ac.za
Low Level Laser Therapy (LLLT) is a form of phototherapy
used to promote wound healing in different clinical conditions.
Laser radiation has the capability to inhibit or stimulate cellular
activity in the absence of significant heating. Currently, no uni-
versally accepted theory has explained the mechanism of laser
biostimulation. Although not established, an alternative treat-
ment modality for diabetic wound healing includes LLLT. Laser
biostimulation of such wounds may be of benefit to patients by
reducing healing time. Structural, cellular and genetic events in
normal, wounded and unwounded diabetic induced human skin
fibroblasts was evaluated after exposing cells to increasing doses
of helium–neon (632.8 nm) laser irradiation. Structural changes
were evaluated by assessing colony formation, haptotaxis and
chemotaxis. Cellular changes were evaluated using cell viability,
(ATP), proliferation, (ALP), cytotoxicity (LDH) while the Comet
assay evaluated genetic integrity. Morphologically, wounded
diabetic fibroblasts responded to single exposure of 5 J/cm
2
with
the highest rate of haptotaxis and chemotaxis indicating a stimu-
latory effect while 16 J/cm
2
showed the lowest rate indicating an
inhibitory effect. Cell viability and proliferation changes indicated
a decrease at higher doses while a dose of 5.0 J/cm
2
appeared to
stimulate mitochondrial activity, which leads to normalization of
cell function with an increase in cell viability. A dose of 5 J/cm
2
showed the highest rate of cell viability and proliferation with a
minimal amount of damage to the cell membrane or DNA, sup-
porting morphological evidence. In conclusion, LLLT of 5 J/cm
2
stimulates migration, proliferation, and metabolism of wounded
fibroblasts to accelerate wound closure.
F2-008P
Methionine sulfoxide reductase from class A:
aminoacids involved in catalysis of the
reductase step and in substrate binding
S. Boschi-Muller, M. Antoine, A. Gand and G. Branlant
Laboratoire MAEM UMR 7567 CNRS-UHP, Universite
´
Henri
Poincare
´
Nancy I, vandoeuvre-le
`
s-Nancy, France.
E-mail: Sandrine.Boschi@maem.uhp-nancy.fr
Methionine sulfoxide reductases (Msrs) are ubiquitous enzymes
that reduce protein-bound methionine sulfoxide back to Met in
the presence of thioredoxine. In vivo, the role of Msrs is des-
cribed as essential in protecting cells against oxidative damages,
and to play a role in infection of cells by pathogenic bacteria.
There exist two structurally-unrelated classes of Msrs, called
MsrA and MsrB. MsrA is specific for the S epimer of MetSO,
whereas MsrB reduces the R epimer. Both Msrs present a similar
catalytic mechanism of sulfoxide reduction by thiols via the sulf-
enic acid chemistry. The rate of the reductase step that leads to
the formation of the sulfenic acid intermediate is fast. This sup-
ports an activation of the catalytic Cys and an increase of the
electrophilic character of the sulfur atom of the sulfoxide sub-
strate via an acid catalyst from the active site. Both activations
should favor the efficiency of the reductase process. Data will be
presented which illustrate the role of some amino acids involved
in the catalytic mechanism of the reductase step and in the sub-
strate recognition of MsrAs, specifically in the formation of the
hydrophobic pocket responsible for the methyl group recogni-
tion.
F2-009P
Role of Induced cAMP Early Repressors in B/K
Gene Transcription
M H. Choi, Y M. Jang, Y S. Jang and O J. Kwon
Department of Biochemistry, The Catholic University of Korea,
College of Medicine, Seoul, South Korea.
E-mail: cmh0912@hanmail.net
B/K protein, structurally classified as a member of double C2
domain proteins, is highly expressed in PC12 cells. We previously
found that forskolin decreased the expression of both B/K
mRNA and protein by PKA-dependent mechanism, and that
inducible cAMP early repressor (ICER) might be involved in that
process via the interaction with a CRE-like domain that is
located in the promoter region of B/K gene. Here, we showed
the direct involvement of ICER in the mechanism of forskolin-
induced down-regulation of B/K gene expression in PC12 cells.
To investigate the role of ICER, we’ve successfully cloned four
types of rat ICER (I, Ig, II and IIg). Transient transfection
(24 h) of each construct resulted in the successful expression of
ICERs, and the expression level was even higher than that
induced by 3-h treatment of forskolin, the time when the expres-
Abstracts
339
sion of ICER increased to the highest level. In promoter assay,
ICER suppressed the promoter activity of B/K in CHO-K1 cells
that were cotransfected with the construct containing B/K CRE-
like sequence. Inhibition level might not be related with the iso-
types of ICER but with the expression level in the cells, suggest-
ing the absence of isotype specificity of ICER in the repression of
B/K gene transcription. Effect of ICER did not observed in the
cells cotransfected with the B/K construct having mutated CRE-
like sequence (AC:TG). Direct binding of B/K CRE-like
sequence to ICER was also observed in chromatin immunopre-
cipitation experiment. These results strongly suggest that ICER
may be the principal suppressor molecule in the process of B/K
gene expression.
F2-010P
Comparative structural and conformational
studies on two forms of beta lactoglobulin
(A and B) upon interaction with lead ion
A. Divsalar and A. A. Saboury
Laboratory of Biophysical Chemistry, Institute of Biochemistry
and Biophysics, University of Tehran, Tehran, Iran.
E-mail: divsalar@ibb.ut.ac.ir
One of the most widely studied and fascinating proteinsof animal
origin is Beta lactoglobulin (blg). This polypeptide, with 150–170
amino acids (depending on the species), is an abundant compo-
nent of the milk of most animals, although it has not been detec-
ted in human milk. Bovine blg consists of 162 amino acid residues
and contains two disulfide bonds and a free thiol. Its interaction
with a great variety of hydrophobic ligands has led to its inclusion
in the lipocalin (transport protein) superfamily. blg is a dimeric
native conformation at neutral pH, while the conformation at pH
2 is a monomeric but still native. Thermodynamic studies have
been made on the effect of Pb
2+
on the structureof two forms of
blg (A and B) in 50 mm sodium chloride at 27 °C using UV spec-
trophotometry, circular dichroism (CD) and fluorescence spectros-
copy. UV spectrophotometry studies show that the protein
absorbance changes due to the increase concentration of ion lead
are a biphasic curve for blg-A form and a monophasic sigmoid
curve for blg-B form. Far CD studies do not show any change on
the secondary structureof blg-A upon interaction with concentra-
tions of 15.54 and 35.8 mm of Pb
2+
, while far CD studies show
considerable change in the secondary structureof blg-B by
increasing the concentration of Pb
2+
. Near CD and fluorescence
spectroscopy studies show considerable change in the tertiary
structure of Blg-A related to the interaction with lead ion, but
these studies do not show any change in the tertiary structure of
blg-B. Different behaviors of blg (A and B) upon interaction with
lead ion, related to different native structureof both proteins.
F2-011P
Strong specific binding forces between
adhesive extracellular proteoglycan
carbohydrates
I. Bucior
1
, S. Garcia-Manyes
2
, R. Ros
3
, F. Sanz
2
,
D. Anselmetti
3
, M. M. Burger
1
and X. Ferna
`
ndez-Busquets
4
1
Friedrich Miescher-Institut, Basel, Switzerland,
2
Department of
Physical Chemistry, University of Barcelona, Barcelona, Spain,
3
Experimental Biophysics, University of Bielefeld, Bielefeld,
Germany,
4
Laboratory of Nanobioengineering, Barcelona Science
Park, University of Barcelona, Barcelona, Spain.
E-mail: busquets@qf.ub.es
Specific carbohydrate-carbohydrate interactions are rarely repor-
ted in biologically relevant situations such as cell recognition.
However, carbohydrate structures have immense structural diver-
sity, a ubiquitous distribution in vertebrate and invertebrate tis-
sues, and are associated with the cell surface, as required of cell
recognition molecules. Carbohydrate–carbohydrate interactions
are characterized by relatively weak forces which, when multi-
merized, can be easily potentiated by orders of magnitude, repre-
senting a highly versatile form of cell recognition and adhesion
given the extraordinary plasticity of their structures. Sponge cells
associate in a species-specific process through multivalent interac-
tions of carbohydrate structures on a type of extracellular bifunc-
tional proteoglycan molecules termed spongicans. In the marine
sponge Microciona prolifera the spongican molecule, Microciona
aggregation factor (MAF), self-interacts via a Ca
2+
-dependent
interaction. We have dissected MAF into its components, track-
ing the individual self-binding units down to the circular core of
the sunburst-like molecule and further down to a 200-kDa glycan
(g200). Force spectroscopy data and surface plasmon resonance
measurements reveal that the carbohydrate self adhesion is highly
specific when compared with the binding to other sulfated carbo-
hydrates such as chondroitin sulfate. The strength of the binding
per surface area between two spongican molecules is comparable
to that of focal contacts between vertebrate cells. The results
obtained reveal surprisingly high forces and selectivity for this
most ancient cell adhesion system, and the existence of poly-
morphism between g200 purified from different individuals, thus
representing the first indication of specific carbohydrate–carbohy-
drate interactions implicated in the discrimination between cells
of different individual origin within a single species.
Acknowledgments: This work was supported by grant
BIO2002-00128 from the Ministerio de Ciencia y Tecnologı
´
a,
Spain, that included FEDER funds.
F2-012P
Scanning probe microscopy as tool to study
the stability of azurin in air
V. Frascerra, G. Maruccio, V. Arima, L. del Mercato,
P. P. Pompa, F. Calabi, R. Cingolani and R. Rinaldi
National Nanotechnology Laboratory of INFM, Ingegneria dell’
Innovazione, University of Lecce, Lecce, Italy.
E-mail: vanessa.frascerra@unile.it
Scanning probe microscopy (SPM) comprises a family of tech-
niques that can be used as tool to study biomolecules morphol-
ogy and properties on the atomic scale. We focus on the use of
the SPM to study the azurin stability in air. Azurin is a metallo-
protein involved in electron transfer during denitrification path-
way of the bacterium Pseudomonas aeruginosa. Thanks to its
electron transfer properties, azurin and its physiological partners
could be employed for the implementation of planar biosensors,
with enhanced sensitivity and selectivity, exploiting a monolayer
of proteins that should act as an optimal surface for the analyte
molecules to react with. For this purpose it is fundamental that
the immobilized proteins preserve their native conformation and
properties, especially in ambient conditions. First, Scanning
Tunneling Microscopy (STM) allows us to achieve a very high
resolution and to probe the electronic properties of Au (111)-
immobilized Azurin, both in buffer solution and in air. Our
results demonstrates that immobilized proteins preserve their
electron transfer function. Secondly, the force distance mode of
atomic force microscopy has been used to measure the interac-
tion between tip and SiO2-immobilized azurin by mercaptosi-
lanes. The force curves show that the adhesion of the tip to the
azurin is weaker than to the SiO2 and silanized surface. The
mechanical properties of azurin could be deduced by analyzing
these curves and correlated with the native conformation of the
protein.
Abstracts
340
F2-013P
Hydrogen peroxide mediates Rac1 activation
of S6K1
J W. Han
1
, Y G. Chung
1
, S. J. Paek
1
, S. H. Park
1
, J S. You
1
,
J K. Kang
1
, Y. M. Kim
2
, H. Y. Lee
3
, Y. K. Kim
4
and
H. W. Lee
1
1
Lab of Biochem & Mol Biol, College of Pharmacy, Sungkyunk-
wan University, Suwon, Gyeonggi-do South Korea,
2
College of
Pharmacy, Duksungsung Women’s University, Seoul, South Korea,
3
College of Medicine, Konyang University, Nonsan, Chungcheong-
nam-do South Korea,
4
College of Medicine, Kwandong University,
Gangneung, Gangwon-do South Korea.
E-mail: jhhan551@skku.edu
We previously reported that hydrogen peroxide (H
2
O
2
) mediates
mitogen activation of ribosomal protein S6 kinase 1 (S6K1)
which plays an important role in cell proliferation and growth.
In this study, we investigated a possible role of hydrogen perox-
ide as a molecular linker in Rac1 activation of S6K1. Overexpres-
sion of recombinant catalase in NIH-3T3 cells led to the drastic
inhibition of H
2
O
2
production by PDGF, which was accompan-
ied by a decrease in S6K1 activity. Similarly, PDGF activation of
S6K1 was significantly inhibited by transient transfection or sta-
ble transfection of the cells with a dominant-negative Rac1
(Rac1N17), while overexpression of constitutively active Rac1
(Rac1V12) in the cells led to an increase in basal activity of
S6K1. In addition, stable transfection of Rat2 cells with
Rac1N17 dramatically attenuated the H
2
O
2
production by
PDGF as compared with that in the control cells. In contrast,
Rat2 cells stably transfected with Rac1V12 produced high level
of H
2
O
2
in the absence of PDGF, comparable to that in the con-
trol cells stimulated with PDGF. More importantly, elimination
of H
2
O
2
produced in Rat2 cells overexpressing Rac1V12 inhib-
ited the Rac1V12 activation of S6K1, indicating the possible role
of H
2
O
2
as a mediator in the activation of S6K1 by Rac1. How-
ever, H
2
O
2
could be also produced via other pathway which is
independent of Rac1 or PI3K, because in Rat2 cells stably trans-
fected with Rac1N17, H
2
O
2
could be produced by arsenite which
has been shown to be a stimulator of H
2
O
2
production. Taken
together, these results suggest that H
2
O
2
plays a pivotal role as a
mediator in Rac1 activation of S6K1.
F2-014P
Reaction of hydroxymethylarginine and
endogenous formaldehyde with
tetrahydrofolate producing N
5
,N
10
-methylene
tetrahydrofolate coenzyme
L. Hulla
´
n
1
, L. Tre
´
zl
2
,Z.M.Ja
´
szay
3
, M. Tejeda
4
, A. Csiba
5
,
J. Bariska
6
and T. Szarvas
7
1
Department of Biochemistry, National Institute of Oncology,
Budapest, Hungary,
2
Department of Organic Chemical Technol-
ogy, Budapest University of Technology and Economics, Budapest,
Hungary,
3
Organic Chemical Technological Research Group, Hun-
garian Academy of Science, Budapest, Hungary,
4
Department of
Experimental Pharmacology, National Institute of Oncology,
Budapest, Hungary,
5
Veterinary and Food Control Station, Buda-
pest, Hungary,
6
Central Laboratory, National Institute of Rheuma-
tology and Physiotherapy, Budapest, Hungary,
7
Institute of
Isotopes Co. Ltd., Budapest, Hungary. E-mail: hullan@oncol.hu
The endogenous formaldehyde (E-CH
2
O) means the always exist-
ing CH
2
O content of various biological samples. E-CH
2
O is sup-
posed to be bound to biomolecules, because it has no toxic
effect. N
G
-hydroxymethyl derivatives of arginine (HMA) synthes-
ized by Tre
´
zl and co-workers from CH
2
O and L-arginine may
belong to that type of molecules. HMA was found in human
blood and urine, and in plants (leaves, fruits and vegetables) in
higher concentration than in human samples since HMA is one
of the products of photosynthesis. We have proved that the reac-
tion of HMA and THF produces – under physiological condi-
tions – N
5
,N
10
-methylene tetrahydrofolate (CH
2
THF), the
coenzyme of thymidylate synthase (TS). In recent work
13
C-HMA was synthesized and the reaction of
13
C-HMA and
THF was compared to that of
13
C-CH
2
O and THF by
13
C-NMR
in the reaction mixture for producing of CH
2
-THF according to
the Roberts-method which is used for the determination of TS
activities. The two reactions were also compared to the reaction
between E-CH
2
O and THF. To test the CH
2
-THF product meas-
urement of TS activities was applied using either cytosol of P388
lymphoid leukemia tumor, or TS purified from Lactobacillus ca-
sei. Both the NMR spectra and the enzyme assays proved that
the reactions produced CH
2
-THF, however, the rate of reaction
by free CH
2
O is higher than those of the two other reactions.
The results suggest that TS and probably other proteins are the
most important molecules in keeping of E-CH
2
O in bound form.
Acknowledgment: This work was supported by National Sci-
ence Foundation OTKA Grant No. T034245.
F2-015P
Co-operation of H
2
O
2
-mediated ERK activation
with Smad pathway in TGF-b1 induction of
p21
WAF1/Cip1
Y. K. Kim
1
, E. K. Lee
2
, J. W. Park
2
, J. Y. Lee
2
, H. Y. Jung
2
,
H. J. Kim
2
, J. H. Park
2
, S N. Kim
3
, H. Y. Lee
4
, H. W. Lee
2
and J W. Han
2
1
College of Medicine, Kwandong University, Gangneung, South
Korea,
2
College of Pharmacy, Sungkyunkwan University, Suwon,
South Korea,
3
Skin Research Institute, Amore-Pacific Corporation,
Yong-In, South Korea,
4
College of Medicine, Konyang University,
Nonsan, South Korea. E-mail: yksnbk@kwandong.ac.kr
Although it has been demonstrated that p21
WAF1/Cip1
could be
induced by transforming growth factor-b1 (TGF-b1) in a Smad-
dependent manner, the cross-talk of Smad signaling pathway
with other signaling pathways still remains poorly understood. In
this study, we investigated a possible role of hydrogen peroxide
(H
2
O
2
)-ERK pathway in TGF-b1 induction of p21
WAF1/Cip1
in
human keratinocytes HaCaT cells. Using pharmacological inhibi-
tors specific for MAP kinase family members, we found that
ERK, but not JNK or p38, is required for TGF-b1 induction of
p21
WAF1/Cip1
. ERK activation by TGF-b1 was significantly
attenuated by treatment with N-acetyl-L-cysteine or catalase,
indicating that reactive oxygen species (ROS) generated by TGF-
b1, mainly H
2
O
2
, stimulates ERK signaling pathway to induce
the p21
WAF1/Cip1
expression. In support of this, TGF-b1 stimula-
tion caused an increase in intracellular ROS level, which was
completely abolished by pre-treatment with catalase. ERK activa-
tion does not appear to be associated with nuclear translocation
of Smad-3, because ERK inhibition did not affect nuclear trans-
location of Smads by TGF-b1, and H
2
O
2
treatment alone did
not cause nuclear translocation of Smad-3. On the other hand,
ERK inhibition led to the disruption of interaction between
Smad-3 and Sp1 induced by TGF-b1, indicating that ERK signa-
ling pathway might be necessary for their interaction. Taken
together, these results suggest that activation of H
2
O
2
-mediated
ERK signaling pathway is required for p21
WAF1/Cip1
expression
by TGF-b1 and led us to propose a co-operative model whereby
TGF-b1-induced receptor activation stimulates not only a Smad
pathway but also a parallel H
2
O
2
-mediated ERK pathway that
acts as a key determinant for association between Smads and
Sp1 transcription factor.
Abstracts
341
[...]... E-mail: myoshida@res.titech.ac.jp The ATP synthase can be thought of as a complex of two motors – the ATP-driven F1motorand the proton-driven Fo motor– that rotate in opposite directions Our recent findings are follows 1 Direct observation of reaction sequence: Single molecule imaging ofF1motor has provided direct evidence that all of the three catalytic beta subunits cooperate in sequence during... Laboratory of Biochemistry, School of Medicine, University of Patras, Patras, Greece, 2Laboratory of Biochemistry, Department of Biology, University of Patras, Patras, Greece E-mail: konstantinidis.t@med.upatras.gr The functionof mutations rdn1A, rdn1T and rdn2 in 18S rRNA of Saccharomyces cerevisiae has been examined They correspond to C1054A, C1054U in helix 34 and G517A in loop 530 of helix 18 of 16S... Control of the direction of rotation of these motors is the basis for the chemotactic response, i.e for the ability of cells, such as E coli, to swim up spatial gradients of chemical attractants I will tell you about motor rotation and its modulation by the cell’s signal transduction pathway F3-004 Three-dimensional structures of the bacterial flagellar rotary motor D R Thomas, N R Francis and D J DeRosier... FliN, forming a complex of two rings, the C ring (FliM and FliN), located in the cytoplasm and the M ring (FliF and FliG) found in the membrane The transmembrane proteins MotA and MotB form the proton channel and are anchored to the peptidoglycan layer thus forming the stator of the motor There are many models for how the motor functions but they have been devised in the absence of structural information... invasion potential (MDA-MB-231) and one with low invasion potential (BT-20), and also normal cells MCF-12A were used The expression of PDGF-R and c-Kit, their ligands, MMP-9 and signal transduction pathways PI3/Akt and MAPK were studied by RT-PCT and western blotting and immunocytochemistry The three receptors are expressed in all the breast cancer cell lines that were studied Exogenous SCF and PDGF-BB... identified by RNase H digestion and primer extension ´ analysis at C1400 and C1411 of the 3-minor domain of 16S rRNA, at positions located in the central loop of domain V, and in helices H42-H44 and H95 of 23S rRNA Specifically, in the central loop of domain V of 23S rRNA, ABA-spermine labels nucleosides U2584 and A2602 Nucleoside A2602 has been sug´ gested to propel the spiral rotation of the tRNA-3 end from... complexes are stable It seems likely that strong and stable, but non-productive interaction of the polymers determinates peroxidase inhibition during the reaction by preventing the access of regular substrates to the active center of the enzyme F3–Rotary Motor Complexes F 3–0 01 The rotary motors in F- and V-type ATPases D Stock and R A Bernal MRC Laboratory of Molecular Biology, Cambridge, UK E-mail: stock@mrc-lmb.cam.ac.uk... obtain paths for ligand binding to heme proteins The truncated hemoglobin, trHbN, from Mycobacterium tuberculosis belongs to the hemoglobin superfamily and may have functional roles in NO detoxification, long-term ligand storage, O2/NO chemistry, and O2 delivery under hypoxic conditions Its tertiary structure is based on a 2-on-2 a helical sandwich rather than the 3-on-3 a helical sandwich of the classical... homeostasis and enable transport processes across membranes While most eubacterial ATPases are of the F-type, some eubacteria and all known archaea have ATPases of the A-type, which are close homologues of V-type ATPases, but are used for ATP synthesis Although V- and A-type ATPases are similar in size and shape to F-type ATPases, only the catalytic subunits and the core of the transmembrane motor share... to a relatively rigid structureof the filament to work as a propeller, the hook has a much higher bending flexibility to function as a universal joint We reported the X-ray crystal structureof a 31 kDa core fragment of FlgE named FlgE31 (residues 7 0–3 63) and an atomic model of the straight hook built by docking the FlgE31 model to a density map obtained by electron cryomicroscopy and image analysis Since . F1 – Structure and Function of Motor Proteins
F1- 001
What single-molecule mechanics can tell us
about mitosis?
J. Howard
Max Planck Institute of Molecular. myoshida@res.titech.ac.jp
The ATP synthase can be thought of as a complex of two motors
– the ATP-driven F1 motor and the proton-driven Fo motor –
that rotate in opposite directions.