download report - Istituto Pasteur

P a r t i c i p a n t s :
Carlo Travaglini-Allocatelli, professor; Stefano Gianni, CNR
researcher; Ylva Ivarsson, post-doc fellow; Nicoletta Calosci,
PhD student.
C o l l a b o r a t i o n s :
University of Uppsala, Sweden (Prof. Per Jemth); University of
Cambridge, UK (Prof. Michele Vendruscolo).
Report of activity
The main goal of the present project is to understand
if and how the topological properties and the
sequence connectivity between different elements of
secondary structure affect the folding pathway and
the molecular recognition process mediated by proteins.
The model system employed is the PDZ
domain, small globular protein of 90 - 100 a.a.
residues, involved in a variety of cellular processes,
from the organization of macromolecular complexes
to the regulation of signalling cascades. We plan to
take advantage of topological mutations (both engineered
or naturally evolved circular permutants) to
test the role of sequence connectivity between different
elements of secondary structure in the
(de)stabilization of metastable species, such as intermediate
and transition states. The task is to unveil
the molecular events involved in the folding and the
binding reactions of PDZ domains, and the correlation
between the two processes. Furthermore, the
results are compared with the folding and binding
reaction of recently identified naturally evolved circularly
permutated variants (i.e. PDZ domains from
bacteria and plants). The folding pathway of each
protein is studied by employing an array of experimental
methods, including protein engineering,
innovative ultra-rapid mixing instruments in combination
with stopped-flow equipment, and molecular
dynamics simulations. Particular attention is devoted
to the identification and characterization of mis-fold-
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Molecular recognition in biomolecules - AREA 4
How proteins recognize their biochemical partners: ligand binding
and folding pathways of PDZ domains
Principal investigator: Maurizio Brunori
Professor of Chemistry and Biochemistry
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”
Tel: (+39) 06 49910544; Fax: (+39) 06 4440062
maurizio.brunori@uniroma1.it
ed states, such as off-pathway intermediates.
Molecular dynamics simulations allow to model the
structure of such mis-folded states, in an attempt to
obtain structural information on species which are
prone to aggregation, leading to the formation of
amyloids, often implicated in neurodegenerative
human diseases.
The process of intermolecular recognition involving
PDZ domains and their target proteins may be investigated
to determine the mechanism of the complex
formation and the regions of the PDZ domains
involved in the control, over-and-above the binding
pocket. By employing this overall strategy we aim at
the identification of both the residues crucial in the
protein folding process and those that occupy functionally
important positions in molecular recognition
events. In addition, the role of internal protein
dynamics in controlling the folding mechanism and
the specific recognition of protein targets of biological
relevance is investigated.
Results and Perspectives
The energy landscape theory provides a general
framework for describing protein folding reactions.
However, since a large number of studies have
focused on two-state proteins with single welldefined
folding pathways and without detectable
intermediates, the extent to which free energy landscapes
are shaped up by the native topology at the
early stages of the folding process has not been fully
characterized experimentally.
To obtain a glimpse of the width of the free energy
landscape at the early stages of the folding, we compared
the folding pathways of two homologous
three-state proteins. The study of homologous proteins
represents a powerful approach to obtain
insight into the process of protein folding, especially
when combined with structural information on intermediate
events. Here we present an original illustration
of the width of the upper regions of a free energy
funnel by comparing the early and late transition