Bariloche protein symposium argentine society for biochemistry and molecular biology

51
BIOCELL, 27 (Suppl. I), 2003
BE-C10.
MOLECULAR DYNAMICS SIMULATIONS OF LIVER
BASIC FATTY-ACID BINDING PROTEIN (LB-FABP) IN
LIPID MEMBRANES
Guillermo G. Montich
1
, Veronica Nolan
1
, Massimiliano Perduca
2
,
Hugo L. Monaco
2
, and Marcos A. Villarreal
1
.
1
Dpto. Qca. Biológica-CIQUIBIC- Fac. Cs. Qcas., UNC, Córdoba,
Argentina, and 
2
Facolta di Scienze, Universita di Verona, Italia.
E-mail: [e-mail]
We have performed several molecular dynamics simulations of
Lb-FABP in the vicinity of lipid membranes. The different
simulations were started with the protein located in the water
phase, with none or a few contacts with the lipids. When the
membrane was made of the anionic lipid DLPS it was observed
that Lb-FABP approaches the membrane and establishes contacts
with the phospholpid head groups. At the same time, a
conformational change was observed: a beta-hairpin which is distal
from the domain that interact with the membrane undergo a partial
unfolding with loss of secondary structure. When the membrane
was composed of the zwitterionic lipid DPPC, Lb-FABP did not
establish contacts with the membrane and no conformational
changes were observed as compared with the protein in solution.
The partial unfolding induced by the interaction with anionic
membranes was in agreement with previous experimental
observations [Nolan V, Perduca M, Monaco HL, Maggio B and
Montich GG. (2003). Biochim. Biophys. Acta, 1611 98-106].
BE-C11.
ANALYSIS OF THE CHOLESTEROL-DEPENDENT
INTERACTION OF PERFRINGOLYSIN O WITH
MEMBRANES USING FLUORESCENCE SPECTROSCOPY
Heuck A.P., Ramachandran R., Johnson A.E.
Texas A&M University, College Station, TX 77843-1114, USA.
E-mail: [e-mail]
Perfringolysin O (PFO) is a 
β-barrel pore-forming toxin secreted
by the pathogen Clostridium perfringens. Upon encountering a
cholesterol-containing membrane, the toxin oligomerizes and
spontaneously inserts to form a large pore. In recent years, we
have used fluorescence spectroscopy to analyze the structure of
the membrane-inserted oligomer of PFO (Nat. Struc. Biol. 2002,
9, 823), and also to examine the mechanism of pore formation (J.
Biol. Chem. 2003, 278, 31218).
Despite significant progress made on the structural aspects of these
bacterial toxins, how cholesterol mediates PFO binding remains
one of the main unsolved issues in the field. Which cytolytic step
requires cholesterol? Which region of PFO is involved in
cholesterol recognition?
Fluorescence resonance energy transfer studies reveal that binding
of PFO to the membrane is the critical step that requires
cholesterol. We have prepared different derivatives of PFO that
have been site-specifically mutated in the membrane-exposed loops
of domain 4 of the molecule. The cholesterol-dependent binding
properties of these mutants show that the region located close to
the unique cysteine residue of the toxin substantially alters the
interaction of PFO with the bilayer. In contrast, modification of
membrane-exposed residues that are located further away from
this conserved loop does not affect PFO binding to the bilayer.
These results suggest that the cholesterol-sensing region of PFO
is located near the unique cysteine residue.
BE-C12.
DIRECTED MOLECULAR EVOLUTION OF A METALLO-
β-LACTAMASE: DEVELOPING BAD MANNERS
Tomatis, Pablo E. and Vila, Alejandro J.
Instituto de Biología Molecular y Celular de Rosario (IBR) Area
Biofísica (UNR) E-mail: [e-mail]
Metallo-
β-lactamases (MBLs) are zinc-dependent enzymes able
to hydrolyze 
β-lactam antibiotics. Their active sites are largely
conserved in enzymes from various sources, even if they display
different metal binding affinities. BcII from Bacillus cereus (a
non-pathogenic soil bacterium) is a MBL with low specific activity
against different substrates. We have proposed that BcII could be
an evolutionary precursor of more efficient 
β-lactamases from
pathogenic bacteria.
In order to test this hypothesis, we have employed the iterative
method of in vitro molecular evolution called DNA shuffling to
try to find BcII variants with increased activities. In particular, we
decided to improve the enzymatic activity towards cephalexin (a
poor substrate of BcII). Cephalexin also displays a low MIC
towards  E. coli cells expressing BcII in the periplasm. After 4
rounds of DNA shuffling and clonal selection of variants being
able to confer higher resistance levels to cephalexin, we isolated
several clones displaying  MIC 64-fold increased. When we
analized the sequence of 18 clones, we found that 4 residues
showed a high frequency of mutation. Interestingly, one of these
is naturally present in some MBLs. The BcIIm5 mutant, which
harbours these 4 mutations, was purified and its hydrolytic
activities towards different substrates were determined. We found
a selective increase in  the specific activity towards cephalexin.
BE-C13.
PURIFICATION, CRYSTALLIZATION AND
PRELLIMINARY X-RAY ANALYSIS OF TRIATOMA
VIRUS (TrV) FROM TRIATOMA INFESTANS
Marcelo D. Costabel
1
, Gabriela S. Rozas-Dennis
1
, Diego M.A.
Guérin
1
, Gaëlle Squires
2
, Jean Lepault
2
, Jorge Navaza
2
 and Félix
A. Rey
2
.
1
Grupo de Biofísica, Departamento de Física U.N.S, Bahía Blanca,
Argentina. 
2  
Laboratoire de Génétique des Virus, CNRS-UPR9053,
Gif-sur-Yvette, France. E-mail: [e-mail]
Objetive:  TrV is a viral pathogen of T. infestans and  T.
Patagonica, and after TrV genome came out this virus was
considered as a possible member of Cricket paralysis-like viruses
genus.
Methods: Live insects of T. infestans experimentally infected with
TrV were used to purify TrV and viral particles observed under
TEM. Hanging-drop vapour-diffusion technique was used to obtain
virus crystals and X-ray diffraction data collected  at a synchrotron
beamline under  cryogenic conditions.
Results: Here we report the purification, crystallization and data
collection of TrV crystals. Hexagonal crystals of  about 200ìm
long were obtained after 5 days. The unit-cell parameters are
a=b=306.6Å, c=788.4Å, á=â= 90°, ã=120° and their diffraction
reached 3.2Å resolution.
Conclusions: Chagas disease is caused in humans by the infection
of the protozoan Trypanosoma Cruzi and T. infestans is the main
vector of this parasite. TrV was proposed as a potential control
agent against triatomines and to belong to the Cricket paralysis-
like virus genus can reinforce this hypothesis. TrV capside is being
determined and the structure can give substantial information for
the virus classification.

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