Bariloche protein symposium argentine society for biochemistry and molecular biology

27
BIOCELL, 27 (Suppl. I), 2003
18:15 - 18:30
PL-C10
POLYAMINE METABOLISM IN NODULES AND ROOTS OF
SOYBEAN PLANTS UNDER CADMIUM STRESS
Balestrasse KB, Benavides MP and Tomaro ML
18:30 - 18:45
PL-C11
FUNCTIONAL ANALYSIS OF THE MAIZE PHOTOSYNTHETIC
NADP-MALIC ENZYME BY SITE DIRECTED MUTAGENESIS
Detarsio E, Andreo CS and Drincovich MF
18:45 - 19:00
PL-C12
PURIFICATION AND ANTIFUNGAL ACTIVITY OF A SUNFLOWER
LIPID TRANSFER PROTEIN EXPRESSED IN Escherichia coli AS A
GST-FUSION
Espinosa Vidal, Esteban; Martín, Mariana; de la Canal, Laura.
19:00 - 19:15
MI-C15
THE SPECIFICITY AND ARCHITECTURE OF ACYL-COA
CARBOXYLASE 
βββββ SUBUNIT IN Streptomyces coelicolor A3(2)
Diacovich L, Gago G, Tsai S-C.(S), Khosla C, and Gramajo H.
19:15 - 19:30
MI-C16
GENETIC VARIABILITY AND RECOMBINATION IN ARENAVIRUSES
Goñi S, Posik D, Romanowski V, Ghiringhelli PD, and Lozano ME.
19:30 - 19:45
MI-C17
GALACTOSIDES METABOLISM OF L. plantarum, GENES AND
THEIR FUNCTION: REGULATION IS THE KEY
Silvestroni A, Connes C, LeBlanc J-G., Piard J-C, Sesma F, Savoy de Giori G..
19:45 - 20:00
MI-C18
REGULATION OF EXPRESSION OF THE TWO-COMPONENT
SYSTEM CitST IN Bacillus subtilis
Sender, Pablo D.; Blancato, Victor S.; Lolkema, Juke; and Magni, Christian.
18:00 - 20:00 (Salón Los Jardines)
POSTER SESSION III
Microbiology:
MI-P49 to MI-P95
Cell Biology:
BC-P20 to BC-P63
Structural Biology:
BE-P39 to BE-P77
Bioenergetic
BG-P1 to BG-P4
New Technologies
NT-P1 to NT-P3
Plants
PL-P56

---

28
BIOCELL, 27 (Suppl. I), 2003

---

29
BIOCELL, 27 (Suppl. I), 2003
L1.
MOLECULAR GENETICS OF CANCER: LESSONS FROM B CELL LYMPHOMA
Riccardo Dalla-Favera (USA)
L2.
FOLDING OF A PREDOMINANTLY 
β-SHEET PROTEIN IN VITRO AND IN VIVO
Lila M. Gierasch.
Departments of Biochemistry & Molecular Biology and Chemistry. University of Massachusetts at Amherst. USA. E-mail:
[e-mail]
This talk will describe ongoing studies of the mechanism by which cellular retinoic acid binding protein I (CRABP I) takes up its native
structure, and how local and global sequence information specifies its fold. Additionally, we seek to observe the folding of CRABP I in
cells, including assessing its thermodynamic stability, kinetics of folding, the nature of folding intermediates and the energy landscape
of folding, and the effects of mutations, and recent progress in this area will be described. CRABP I is a member of the large family of
intracellular lipid binding proteins, whose structures are comprised of a short helix-turn-helix and two nearly orthogonal five-strand 
β-
sheets wrapped around a central cavity. Kinetic analysis by stopped-flow fluorescence and CD, hydrogen exchange, and probing of
ligand binding has provided a description of the landscape of refolding of CRABP I. In a 250 
µs kinetic phase, upon dilution from urea
into folding conditions, the ensemble of CRABP I conformers is hydrophobically collapsed and contains significant local secondary
structure. Native-like topology, as indicated by ligand binding, develops in a ca. 100 ms kinetic phase. Strikingly, stable hydrogen
bonding in the 
β-sheets forms in a fully cooperative manner in a later (1 s) phase, during which specific packing interactions also
develop. The presence of significant secondary structure along with hydrophobic collapse suggests that both global and local forces are
acting in the earliest folding events. We observed previously that the formation of the helix-turn-helix sub-domain of CRABP I is
dictated by local sequence, and more recently examination of peptides corresponding to the turns in CRABP I reveals that two (turns III
and IV) are strongly biased to native structure by local sequence. Hence, local sequence may limit the conformational space available
to CRABP I in the early folding phases. Subsequent kinetic phases likely arise as the conformational ensemble forms longer-range
contacts that specify native topology (~100 ms) and finally native interstrand hydrogen bonds and tertiary structure (1 s). Analysis of
sequences and structures for CRABP I and homologues has identified a network of conserved pairwise hydrophobic interactions that is
likely to specify native-like global topology. Recently, CRABP I was mutated to incorporate in a surface-exposed 
Ω-loop the sequence
CCGPCC, which binds specifically to a membrane-permeable, biarsenical fluoroscein dye. Unfolding of labeled 'tetra-Cys CRABP I' is
accompanied by enhancement of dye fluorescence, which made it possible to determine the free energy of unfolding by urea titration in
cells and to follow in real time the formation of inclusion bodies by the slow-folding, aggregation-prone mutant. Aggregation in vivo
displayed a concentration-dependent lag time characteristic of protein aggregation in purified in vitro model systems. Carrying out
studies of folding and aggregation with a protein such as CRABP I, whose folding is well-characterized in vitro, will provide insight
into the mechanism of folding in cells and the nature of the species that initiate the cellular aggregation process.

---

30
BIOCELL, 27 (Suppl. I), 2003
L3.
DETECTION OF CONFORMATIONAL CHANGES IN
MULTIDRUG TRANSPORTERS
C. Vigano, L. Manciu and J.-M. Ruysschaert*
Structure et Fonction des Membranes Biologiques, Centre de
Biologie Structurale et de Bioinformatique, Université Libre de
Bruxelles. E-mail: [e-mail]
The multidrug resistance is mainly due to the overexpression in
tumor cells of proteins that use the energy derived from ATP
hydrolysis to transport drugs, out of the cell, against a concentration
gradient.These proteins are composed of two homologous halves,
each formed by six putative transmembrane helices and one
nucleotide-binding domain.The mechanism of coupling ATP
hydrolysis, at the cytoplasmic nucleotide binding domain to drug
transport involves conformational changes in the protein structure.
To gain further insight into the mechanism by which multidrug
transporters-mediated drug transport occurs, our group investigates
the different transient conformations adopted by the protein in the
presence of nucleotide ligands and drugs. Multidrug transport
proteins (Pgp, MRP, LmrA) are reconstituted into proteoliposomes
in such a way that they conserve their ATPase and drug transport
activity. Our studies including infrared spectroscopy, tryptophane
quenching, enzymatic proteolysis demonstrate that multidrug
transport proteins change their conformation during the catalytic
cycle and that several conformational states are involved in the
drug transport. Structure changes are transmitted between the
cytosolic domains and the membrane domains. This coupling
between the drug binding site and the catalytic site plays a crucial
role in the transport mechanism. Interestingly, it is differently
affected by drugs which accumulate or do not accumulate in
resistant cells.
L4.
CHOLESTEROL-DEPENDENT STRUCTURAL
TRANSITIONS INITIATE OLIGOMERIZATION AND
BETA-BARREL PORE FORMATION BY A BACTERIAL
PROTEIN TOXIN
Arthur E. Johnson.
The Texas A&M University System Health Sciences Center, College
Station, TX 77843-1114, USA. E-mail: [e-mail]
Perfringolysin O (PFO) is secreted from the Gram-positive
bacterium, Clostridium perfringens, as a water-soluble and stable
monomeric protein. But upon encountering a mammalian cell
membrane that contains cholesterol, PFO binds, oligomerizes, and
forms a very large hole in the bilayer with a diameter near 300 Å.
Using multiple independent fluorescence techniques, we showed
previously that the transition of PFO from a water-soluble monomer
to a membrane-inserted oligomer containing about 50 subunits
involves the conversion of six short 
α-helices in each monomer
into two transmembrane 
β-hairpins in the oligomer. We have now
found that this major structural transition is initiated by the binding
of one end of the PFO molecule to the membrane surface. This
association elicits a conformational change at the other end of the
molecule that exposes an otherwise-protected region of PFO that
forms the interface between adjacent proteins in the oligomer.
The cholesterol-dependent structural changes in PFO therefore
extend throughout the entire molecule and are required to initiate
oligomerization. The oligomerized monomers then act
cooperatively to puncture the membrane. The initial binding of
PFO to cholesterol at the membrane surface therefore triggers a
sophisticated sequence of coupled and cooperative intramolecular
and intermolecular conformational changes that ultimately lead
to pore formation on the appropriate target membrane.
L5.
3-D PARTICLE TRACKING IN A TWO-PHOTON
MICROSCOPE
Gratton, Enrico; Levi, Valeria and Ruan, Qiaoqiao.
Laboratory for Fluorescence Dynamics. University of Illinois at
Urbana-Champaign. 1110 W Green St, Urbana, IL 61801. E-mail:
[e-mail]
Particle tracking in a cell offer the possibility to determine the
diffusive or non-diffusive behavior of a particle over large
distances. The presence of obstacles, flow and particle interaction
with the substrate is recognized by the analysis of the particle
trajectory. Conventionally, particle tracking has been performed
using cameras and reflecting or fluorescing particles. In addition,
interferometric or reflecting techniques have been used to
determine the particle position. In this work, we explore the
possibility to perform particle tracking in 3-D using the 2-photon
excitation microscope. We show that rapid tracking is achievable
over large distances. The tracking bandwidth is essentially limited
by the number of photons that can be collected during the interval
of time used for the feedback of the particle position. For small
movements, it is not necessary to center feedback the particle
position, but the 3-D position can be determined by a simple
algorithm. We present result of particle tracking in the nucleus of
cells.
L6.
INFLUENCE OF VIRGIN OLIVE OIL ON
CARDIOVASCULAR RISK FACTOR
Valentina Ruiz Gutierrez (Spain)

---