Bariloche protein symposium argentine society for biochemistry and molecular biology

33
BIOCELL, 27 (Suppl. I), 2003
S5.
STRUCTURAL STUDIES OF MELANOCORTIN PEPTIDES
IN AQUEOUS AND LIPID MEDIA
Roberto M. Fernandez
1
, Clovis R. Nakaie
2
, Helgi B. Schiöth
3
,
Amando S. Ito
4
 and M. Teresa Lamy
1
1
Instituto de Física, Universidade de São Paulo, São Paulo, SP,
Brazil; 
2
Departamento de Biofísica, Universidade Federal de São
Paulo, São Paulo, SP, Brazil; 
3
Department of Neuroscience,
Uppsala University, Sweden; 
4
Faculdade de Filosofia Ciências e
Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão
Preto, SP, Brazil. E-mail: [e-mail]
In most vertebrates the cationic linear tridecapeptide 
α-melanocyte
stimulating hormone (
α-MSH) is known as the most relevant
physiological hormone regulating skin darkening. It is also involved
in a variety of other physiological and neurological processes. The
hormone 
α-MSH, and several of its derivatives will be discussed
regarding the peptides biological activities, their partition and
interaction with lipid bilayers, and their structures in aqueous and
lipid media. For such studies, the peptides natural fluorescent probe
Trp
9
 was monitored, via steady state and time-resolved fluorescence
spectroscopy. Besides Trp, some of the derivatives contain the
aromatic fluorescent amino acid 
β-(2-naphtyl)-D-alanine (D-Nal)
and the paramagnetic amino acid probe 2,2,6,6-
tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac).
Hence, the fluorescence of Trp and D-Nal and the electron
paramagnetic resonance (EPR) signal of Toac were used to analyze
structural properties of the peptides in different media. To monitor
the effect the peptides cause in the lipid bilayer, the ESR signal of
spin labels intercalated in the membrane were investigated.
S6.
LIPID-PROTEIN INTERACTIONS AT THE OUTER AND
MIDDLE RINGS OF THE ACETYLCHOLINE RECEPTOR
TRANSMEMBRANE DOMAINS
F. J. Barrantes.
UNESCO chair of biophysics & mol. neurobiology and INIBIBB,
Universidad Nacional del Sur-CONICET, Bahía Blanca.
Argentina. E-mail:  [e-mail]
Fluorescence spectroscopy and structural studies are beginning to
delineate a topographic map of the nicotinic acetylcholine receptor
(AChR) transmembrane (TM) region. Site-directed mutagenesis
and single-channel electrophysiology help dissect the involvement
of lipid-sensitive residues in channel gating, opening and closing
mechanisms. Recent structural data reveal that the TM segments
form three concentric layers around the ion channel. An inner ring,
shaped by five M2 segments (one from each subunit) excluded
from contact with the lipid, forms the walls of the pore proper.
The middle ring is formed by M1 and M3 segments, which exhibit
contact with lipids, and the M4 TM domains constitute an outer
ring, distant from the channel and loosely separated from the
middle ring. Although they are not part of the ion conduction
pathway, the lipid-exposed middle and outer rings appreciably
modulate AChR function. Establishing structural-functional
correlations of the lipid-exposed TM rings requires the
identification of sites for lipid modulation and more precise
knowledge of the TM structure. Examples of current strategies
used in our laboratory will be discussed in the presentation.
Work supported by grants from the UNS, FONCYT, Argentina,
and FIRCA 1-RO3-TW01225-01 (NIH).
S7.
PROTEIN-PROTEIN AND  PROTEIN-LIGAND
INTERACTIONS OF THE  GTPase DYNAMIN
Jameson, David M.
Dept. Cell and Mol. Biol., University of Hawaii, Honolulu, Hawaii,
USA. E-mail: [e-mail]
Dynamins are closely related GTPases of approximately 100,000
MW that are essential for receptor-mediated endocytosis and
synaptic vesicle recycling. Two forms of dynamin have been
extensively characterized: a neuron-specific form called dynamin
I and an ubiquitously expressed form called dynamin II. These
two proteins have a similar organization of functional domains
which include an N-terminal GTPase domain (residues 1-300), a
pleckstrin-homology (PH) domain that interacts with lipids
(residues 510-620) a GTPase effector domain (GED) which
stimulates dynamin GTPase activity (residues 620-750) and a C-
terminal proline/arginine-rich domain (PRD) which targets
dynamin to the clathrin-coated pit and binds to a subset of SH3-
domain containing proteins, negatively-charged phospholipids, and
microtubules. Dynamin’s role in endocytosis has been variously
attributed to being a force-generating enzyme or a signalling
protein. Here we review our studies on the oligomeric state of
dynamin at high ionic strength conditions. We also review our
work on the elementary processes of the dynamin GTPase at high
ionic strength and compare these to the ATPase of the force
generating protein myosin and the GTPase of the signalling protein
Ras. Data on the interaction of dynamin with a giant unilamellar
vesicles, studied using two-photon fluorescence microscopy, will
also be discussed.
S8.
PROTEIN-LIPID AND PROTEIN-PROTEIN INTER-
ACTIONS: DIRECT VISUALIZATION BY 2-PHOTON
MICROSCOPY
Sánchez, Susana A.
Laboratory for Fluorescence Dynamics. University of Illinois at
Urbana-Champaign. 1110 W Green St, Urbana, IL 61801. USA.
E-mail: [e-mail]
We have used two-photon excitation fluorescent microscopy to
study the interaction of Phospholipase A2 (PLA2) and HDL
particles with lipids. The interaction of PLA2 with Giant
Unilamellar Vesicles of different lipid composition show
morphological and fluidity changes as a manifestation of the
enzymatic activity. Our studies on the monomer-dimer equilibrium
of PLA2 using micelles and Fluorescence Correlation Spectroscopy
indicate a very strong dimer that will not dissociate until 10
-11
 M.
A second system we have studied is the interaction between
reconstituted particles of HDL with membranes; in this case we
have used the microscopy version of Laurdan Generalized
Polarization technique to study the capability of these particles to
remove cholesterol from Giant Unilamellar Vesicles of different
lipid composition.

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