Each one teaches one: characterizing active forms of proteins by single molecule force spectroscopy
Creators: name: Sandal, Massimo <1981>
Publisher: name: Alma Mater Studiorum - Università di Bologna
Year: 2008
Subjects: CHIM/06 Chimica organica

description: This Ph.D. candidate thesis collects the research work I conducted under
the supervision of Prof.Bruno Samor´ı in 2005,2006 and 2007. Some parts of
this work included in the Part III have been begun by myself during my undergraduate
thesis in the same laboratory and then completed during the initial
part of my Ph.D. thesis: the whole results have been included for the sake of
understanding and completeness.
During my graduate studies I worked on two very different protein systems.
The theorical trait d’union between these studies, at the biological level, is
the acknowledgement that protein biophysical and structural studies must, in
many cases, take into account the dynamical states of protein conformational
equilibria and of local physico-chemical conditions where the system studied
actually performs its function. This is introducted in the introductory part
in Chapter 2. Two different examples of this are presented: the structural
significance deriving from the action of mechanical forces in vivo (Chapter 3)
and the complexity of conformational equilibria in intrinsically unstructured
proteins and amyloid formation (Chapter 4).
My experimental work investigated both these examples by using in both
cases the single molecule force spectroscopy technique (described in Chapter 5
and Chapter 6). The work conducted on angiostatin focused on the characterization
of the relationships between the mechanochemical properties and the
mechanism of action of the angiostatin protein, and most importantly their intertwining
with the further layer of complexity due to disulfide redox equilibria
(Part III). These studies were accompanied concurrently by the elaboration of
a theorical model for a novel signalling pathway that may be relevant in the
extracellular space, detailed in Chapter 7.2.
The work conducted on -synuclein (Part IV) instead brought a whole new
twist to the single molecule force spectroscopy methodology, applying it as a
structural technique to elucidate the conformational equilibria present in intrinsically
unstructured proteins. These equilibria are of utmost interest from
a biophysical point of view, but most importantly because of their direct relationship
with amyloid aggregation and, consequently, the aetiology of relevant
pathologies like Parkinson’s disease. The work characterized, for the first time,
conformational equilibria in an intrinsically unstructured protein at the single molecule level and, again for the first time, identified a monomeric folded conformation
that is correlated with conditions leading to -synuclein and, ultimately,
Parkinson’s disease.
Also, during the research work, I found myself in the need of a generalpurpose
data analysis application for single molecule force spectroscopy data
analysis that could solve some common logistic and data analysis problems that
are common in this technique. I developed an application that addresses some of
these problems, herein presented (Part V), and that aims to be publicly released
soon.