Bariloche protein symposium argentine society for biochemistry and molecular biology

50
BIOCELL, 27 (Suppl. I), 2003
BE-C6.
THE HPV16 E7 ONCOPROTEIN CAN FORM SPHERE-
LIKE PARTICLES THAT RESEMBLE RING PROTEINS
Alonso, Leonardo
1
; García Alai, Maria
1
; Smal, Clara
1
; Iacono,
Rubén
2
; Castaño, Eduardo
2
 and Prat Gay, Gonzalo
1
.
1
Instituto Leloir, FCEyN, UBA, Buenos Aires. 
2
IQUIFyB, UBA.
E-mail: [e-mail]
High risk HPV16 E7 oncoprotein is an extended dimer capable of
undergoing pH dependent conformational transitions that expose
hydrophobic surfaces to the solvent. The dimer shares some
properties with intrinsically disordered proteins but it is clearly
structured.  We found that it is capable of forming highly ordered
soluble aggregates that elute in the void volume of a gel filtration
column. Dynamic light scattering showed monodispersity and
molecular weight of 790 kDa, with a radius of 26 nm assuming a
globular architecture. Indeed, electron microscopy and atomic force
microscopy showed regular and apparently homogeneous sphere-
like particles of 40 to 50 nm diameter. The protein undergoes a
conformational transition upon formation of these particles, with
a substantial increase in 
β-sheet content as judged by circular
dichroism. We introduced a tryptophan residue at position 98,
present in the HPV18 E7 protein, and used it as probe for
fluorescence studies that indicate an increase in tertiary (and
quaternary) structure, also supported by near-UV CD spectra.
These structures bind and shift congo red spectra and bind
thioflavin T, the standard probes for amyloid structures. However,
no insoluble material is formed under any condition tested. The
assembly is very slow (t1/2 20 min), which will eventually allow
the dissection of the mechanism.
BE-C7.
STUDYING INTERFACES IN PROTEIN-PROTEIN
COMPLEXES WITH A PHOTOCHEMICAL PROBE
Gómez GE
1
, Cauerhff AA
2
, Craig PO
1
, Goldbaum FA
2
, Delfino
JM
1
1
IQUIFIB (Universidad de Buenos Aires-CONICET).
  2
Instituto
Leloir. E-mail: [e-mail]
The importance of interactions between proteins in biology has
made the molecular recognition process an area of considerable
interest. A consequence of complex formation is a reduction in
the solvent accessible surface area (SASA). Our approach is based
on a general photochemical modification of the polypeptide chain,
useful for addressing changes in SASA occurring upon protein
interaction. 
3
H-diazirine (
3
H-DZN) is a photoreactive gas similar
in size to water. By irradiation at 
λ>300nm 
3
H-DZN generates
methylene carbene (
[
3
H
]:CH
2
), which reacts with its immediate
molecular cage, inserting even into C-H bonds. As a model system
we used this reagent to probe the complex formed by HEL (hen
egg white lysozyme) and the monoclonal antibody IgG
1
D1.3. HEL
was labeled free or complexed with IgG
1
D1.3 yielding 2.76 and
2.32 mmol CH
2
/ mol protein at 1mM DZN, respectively. This
agrees with the observed decrement in SASA occurring because
of complex formation. Tryptic digests derived from labeled HEL -
free or complexed - were separated by size exclusion
chromatography and RP-HPLC. The measurement of radioactivity
and the identification of each isolated peptide showed that those
implicated in the area of interaction had the highest differential
labeling (H
15
GLDNYR
21
; G
117
TDVQAWIR
125
;
G
22
YSLGNWVCAAK
33
). Thus, 
′protein footprinting′ with DZN
emerges as a feasible methodology useful for mapping contact
regions of protein domains involved in macromolecular assemblies
BE-C8.
ANALIZYNG LOCAL PROTEIN STRUCTURE
PERTURBATIONS WITH COLORES
Lema, Martin A. and Echave, Julian.
Universidad Nacional de Quilmes, Argentina. E-mail:
[e-mail].
COLORES (Comparison of LOcal Residue Environment
Structures) is a computer program for performing analysis of local
differences among similar protein structures. It is suitable for
comparison of different protein mutants or alternate structural
predictions for a given protein sequence. Here we introduce the
basic algorithms behind the program, we detail its multiple options
and we give examples of use. The program is available from the
authors for academic purposes for Unix and Windows platforms
BE-C9.
NMR CHARACTERIZATION OF POLYAMINE
COMPLEXES WITH 
α-SYNUCLEIN
Fernández, C.O.
1
; Hoyer, W.
2
; Zweckstetter, M.
2
; Jares-Erijman,
E.A.
3
; Subramaniam, V.
4
; Griesinger, C.
2
 and Jovin, T.M.
2
1
LANAIS RMN 300 (CONICET-UBA), Buenos Aires, Argentina.
2
Max Planck Institute for Biophysical Chemistry, Goettingen,
Germany. 
3
Facultad de Ciencias Exactas y Naturales, Buenos
Aires, Argentina. 
4
AstraZeneca R&D Charnwood, Loughborough,
United Kingdom. E-mail:[e-mail]
The aggregation (fibrillation) of 
α-synuclein
 
is characteristic of
Parkinson's disease (PD) and other neurodegenerative
synucleinopathies. The 140 amino-acid protein is natively
unstructured; thus ligands binding to the monomeric form are of
potential therapeutic interest. Biogenic polyamines promote the
aggregation of 
α-synuclein and may constitute naturally occurring
agents modulating the pathogenesis of PD. We characterized the
complexes of natural and synthetic polyamines with 
α-synuclein
by NMR at 15°C.
1
 The perturbations of the 
13
C/
15
N backbone
chemical shifts in the region aa109-140 identified the binding site
in the C-terminal domain and yielded dissociation constants for
the different polyamines. Greater molecular charge (+2 
→ +5)
correlates with greater affinity and enhancement of fibrillation at
37°C, for which we propose a simple kinetic mechanism involving
a dimeric nucleation center. From the derived parameters, the
polyamines increase the extent of nucleation by 10
3 
and the rate of
monomer addition 
∼40-fold. Significant secondary structure is not
induced at 15°C. Instead, NMR reveals changes in a region (aa22-
93) far removed from the polyamine binding motif and presumed
to adopt the 
β-sheet conformation characteristic of fibrillar α-
synuclein.
1
Fernández CO, Hoyer W, Zweckstetter M, Jares-Erijman EA,
Subramaniam V, Griesinger C, Jovin TM. Submitted (2003).

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