Bariloche protein symposium argentine society for biochemistry and molecular biology

49
BIOCELL, 27 (Suppl. I), 2003
BE-C2.
MULTIPLE PARTIALLY-FOLDED STATES OF
β-LACTAMASE AT EQUILIBRIUM
Javier Santos
1,2
, Valeria A. Risso
1
, Raúl G. Ferreyra
1,2
, Leopoldo
G. Gebhard
1,2
 and Mario R. Ermácora
1,2
1
Universidad Nacional de Quilmes, Argentina. 
2
Consejo Nacional
de Investigaciones Científicas y Técnicas. E-mail:
[e-mail]
Site-directed mutagenesis was used to introduce single cysteine
residues into 
β-lactamase (ES-βL), an α+β protein that naturally
does not contains any cysteine residue. Two mutants, S126C and
S265C, in which a single cysteine residue substitutes the isosteric
residue serine, were prepared. The new thiol moieties were used
as a chemical probes to map the two domains of ES-
βL in
urea-induced unfolding experiments. Fluorescence and CD were
also measured. In terms of protein stability, the S126C substitution
is well tolerated, but S265C ES-
βL is significantly less stable than
wild-type ES-
βL. The single Cys of the two variants is unreactive
in the native state. In the urea-unfolded state Cys reacts (100%),
with the same rate as free 
β-mercaptoethanol. Under partially
denaturat conditions, the stability, thiol accessibility, and secondary
structure suggests multiple partially folded states at equilibrium.
In order to compare the stability of the two subdomains in a globally
isoenergetic unfolding process, we built a double mutant, S126C,
S265C. Our results suggests that folding of proteins with complex
architecture involves a hierarchical.
BE-C3.
UDP-GLC:GLYCOPROTEIN GLUCOSYLTRANSFERASE
RECOGNIZES SUBSTRATES WITH MINOR
STRUCTURAL PERTURBATIONS
Julio J. Caramelo
1
, Andrea A. Meras
2
, Olga C. Castro
1
 and
Armando J. Parodi
1
1
Fundación Instituto Leloir, Ciudad de Buenos Aires, Argentina.
2
Instituto de Investigaciones Biotecnológicas (UNSAM), San
Martín, Argentina. E-mail: [e-mail]
Protein folding in the cell involves the action of different molecular
chaperones and folding-facilitating enzymes. In the
 
endoplasmic
reticulum (ER), the folding status of glycoproteins
 
is stringently
controlled by a glucosyltranferase enzyme (GT)
 
that creates
monoglucosylated structures recognized by ER resident
 
lectins
(calnexin/calreticulin, CNX/CRT). GT serves as a folding
 
sensor
because it only glucosylates misfolded or partly folded
glycoproteins. Nevertheless, the molecular mechanism behind this
recognition process remains largely unknown. We addressed this
issue by using a family of chemically glycosylated proteins derived
from chymotrypsin
 
inhibitor-2. By progressive truncation from the
c-terminus we generated a family of 7 neoglycoproteins, ranging
from 53 to 64 residues long, aimed to simulate the last stages of
the folding process. Structural characterization of species
 
showing
higher glucose acceptor capacity suggests that GT recognizes
solvent accessible hydrophobic patches in molten globule-like
conformers, mimicking intermediate and advanced folding stages
of nascent glycoproteins.
 
Surprisingly, deletion of only one residue
from the full-length molecule (the C-terminal one, that was
required for attaining the native conformation) triggered GT
recognition, pointing to the high sensitivity of GT as a folding
sensor.
BE-C4.
A SIALIDASE MUTANT DISPLAYING TRANS-
SIALYLATION ACTIVITY
Paris, Gastón; Ratier, Laura and Frasch, Alberto C.C.
IIB-INTECH. CONICET-UNSAM. Buenos Aires. Argentina. E-
mail: [e-mail]
Trypanosoma cruzi, the agent of Chagas disease, unable to synthe-
size sialic acid, requires this monosaccharide on surface molecules
for biological processes like protection and cell infection. To in-
corporate sialic acid, the parasite expresses a surface trans-sialida-
se (TS) that transfers sialic acid from host glycoconjugantes to
ter-minal ß-galactoses present in parasite mucins. Another
American trypanosmatid, T.rangeli, expresses a homologous
protein that has sialidase (TrSA) activity but is devoid of transfer
activity. Recently, the 3D structure of TcTS and TrSA proteins
had been determined. The comparison between both structures
reveals that few amino acids close to the active center might explain
the trans-glycosylation activity of TcTS. We constructed TrSA
exchange mutant proteins with the aim of obtaining TS activity
from a sialidase scaffold. TrSA containing only five point mutations
at M96V, A98P, S120Y, G249Y and G283P, displayed about 1%
of the trans-sialidase activity present in the recombinant TcTS.
Inclusion of another mutation at position I37L or G342A in the
above framework increased the transfer activity up to 10% of TcTS.
2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA), a
sialidase inhibitor that has to be used ten times more concentrated
to inhibit TS, was used as a probe of the structure of the active
site of mutant enzymes. TrSA mutants displaying TS activity loose
their sensitivity to DANA. The results show that few amino acid
posi-tions in the active site cleft are the ones essential to achieve
trans-sialylation activity from a sialidase scaffold.
BE-C5.
CHARACTERIZATION OF A TRANSCRIPTION FACTOR-
ANTIBODY INTERACTION
Cerutti, M. Laura; Ferreiro, Diego U.; Prat Gay, Gonzalo and
Goldbaum, Fernando A.
Fundacion Instituto Leloir. Buenos Aires, Argentina.  E-mail:
[e-mail]
We have previously generated and characterized a set of
monoclonal antibodies (mAbs) against the C-terminal domain of
the E2 transcriptional activator (E2C) of the human papillomavirus.
Functional epitope mapping analysis revealed that two separate
antibodies populations were obtained: those able to form a stable
ternary complex with E2C and DNA, and those which recognize
the DNA-binding surface of the transcription factor, interfering
with its binding to DNA.  In order to characterize in more detail
the interaction involved in the different antibody:E2C complexes,
the affinity constant and kinetic parameters of the reactions were
determined.  In addition, the effect of the ionic strength on these
parameters was analyzed.  Experiments were carried out with mAb
ED15 (directed towards an epitope in the DNA-binding surface)
and mAb ED23 (recognizing an epitope in the opposite surface of
the protein).  Results show that both antibodies recognize the E2C
antigen with high affinity (nanomolar range) and that their
association reactions are highly sensitive to ionic strength, being
the interactions completely abrogated at NaCl concentrations close
to 0.3 M.  Furthermore, both mAbs present a high proportion of
acidic residues in their variable region sequences, pointing out
that these interactions are essentially mediated by electrostatic
forces.  We present a new system model useful to understand the
role of electrostatic interactions in antibody-protein and protein-
protein recognition.

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