Biophysical studies of membrane proteins/peptides. Interaction with ...

was used in binding studies for the V-ATPase inhibitors bafilomycin and SB242784.
This transmembrane domain is expected to comprise the binding site for the studied
inhibitors. SB242784 was developed to be used as a potential drug for treatment of
osteoporosis. This inhibitor presents selectivity for the osteoclastic form of the enzyme,
while bafilomycin is extremely toxic due to its lack of selectivity. FRET was again used
(a tyrosine residue of the peptide was used as a donor and the inhibitors as acceptors) to
determine binding, and the results indicate a weak binding of the chosen peptide with
bafilomycin whereas binding of the peptide to SB242784 was not detected. Overall, the
results indicate that the V-ATPase inhibitor binding site is likely not formed only by the
4 th transmembrane segment of the c-subunit of V-ATPase, but is the result of
contributions from other transmembrane domains, reflecting a more complex view of
the inhibitory mechanism of V-ATPase than originally proposed.
In Chapter IV, a binding study was conducted between ciprofloxacin (CP), a
quinolone antibiotic, and a purified trimer of the outer membrane porin, OmpF. CP
requires interactions with OmpF for efficient entry into the cell. In this case, the native
protein structure was used instead of the minimalist approach described in Chapter III.
FRET was again applied, this time making use of the presence of two types of
tryptophans of OmpF (as donors) and the UV absorbing properties of CP (acceptor).
Fluorescence from intrinsic amino acids of large proteins in FRET can be difficult to
analyze, since each donor (fluorescent amino acid) population is expected to sense a
different population of acceptors. Donors closer to the protein periphery, can e.g., be in
closer proximity to acceptors than donors in the core of the protein. With this in mind, a
FRET methodology suitable for application to FRET studies with multi-donor proteins
was developed according to the geometric restrictions of the OmpF system. This model
allowed the recovery of a range for the binding constants of the OmpF-CP association
process. Due to the large dimensions of the OmpF trimer, depending on the site of
inhibitor binding assumed in the model for FRET data analysis, the retrieved binding
efficiency can be different. Two limiting binding sites were considered, and comparing
the results from our analysis with the results obtained with an independent method, it
was possible to conclude that the binding site for CP in OmpF is likely to be displaced
from the center of the trimer and closer to the periphery.
In Chapter V, the interaction of the N-terminal amphipatic peptide of a N-BAR
domain with model membranes was studied. Several authors hypothesized that this
segment of N-BAR domain contributed to the membrane remodelling properties of N-