Proposta di ricerca:
Introduction
Ever since the observation that different concentrations of different salts are required to precipitate a given
protein [1] attempts have been made to provide a theoretical foundation for the phenomenon (see the
comprehensive reviews of Lo Nostro and Ninham. [2], Collins and Washabaugh [3] and Cacace et al. [4]).
Until recently, however, they have only been partially successful, and there was no unifying formalism
covering the entire spectrum of salts from salting out (i.e. precipitants, called also „kosmotrops”) to salting
in (i.e. solubilizers, called „chaotrops”). One approach had been to correlate these attributes with effects
on water structure, in particular the fraction of hydrogen-bonded water molecules: precipitants give a
higher fraction and are therefore called kosmotropes, and solubilizers give a lower fraction and are
therefore called chaotropes. According to another branch of interpretations, dispersion forces had been
suspected to be the main factor responsible for the effects [5].
In our recent work we demonstrated that a unified formalism based on solute-water interfacial tension is
able to account for the entire range of observed behaviour [6]. A crucial divergence from previous similar
treatments was that, instead of the irrelevant air-water interface [7], we used solute- (protein-) water
interfacial tension, as the main parameter to describe the effects.
The main conclusion from the theory is that Hofmeister effects are manifested via the surface dependent
term of the free energy (G) of proteins (or, in general, other colloid disperse systems). Addition of
kosmotropic or chaotropic salts to the solvent is expected to increase or decrease solute-water interfacial
tension, respectively, giving rise to a corresponding change of deltaG. (This is supposed to be the reason for
the observed effects on aggregation and conformational properties of proteins). An important implication
of the theory is that large conformational transitions of proteins involving water-exposed surface changes,
are expected to be highly affected by Hofmeister salts, which can be used as a diagnostic tool for the
investigation of extended conformational changes. We also pointed out that the microscopic interpretation
of the Hofmeister effects concerns salt-induced fluctuation changes in the interfacial water layer and the
adjacent protein groups [6, 8, 9].
According to our working hypothesis based on these investigations, the interfacial water structure has a
determining role in defining protein structure and conformational dynamics. In the framework of the
present project, we are going to address this point by a complex methodological approach involving
powerful experimental techniques, such as force spectroscopy, surface tension measurements and
integrated optical techniques. The range of the target objects will span from model surfaces with different
hydrophobicity (e.g., self-assembled monolayer with thiols on gold, or polymers with different plasma
treatment) to biological macromolecules (e.g., bacteriorhodopsin, intrinsically disordered proteins and
peptides).
The work will take advantage of the collaboration between the group of the Biological Research Center of
the Hungarian Academy of Sciences in Szeged (named MTA in the following), lead by Dr. Der Andras, and
the group of the Institute for the Study of Nanostructured Materials at the National Research Council in
Bologna (named CNR in the following) lead by Dr. Valle Francesco.
MTA is one of the leading European institutes in the field of optically excitable proteins such as
bacteriorhodopsin, as well as electrical and optical properties of proteins and microorganisms. MTA has a
well known expertise in the study of the Hofmeister series; Dr. Der and his coworkers developed the
innovative model based on the protein-solvent interfacial energy to interpret the chaotropic and
kosmotropic effects that is the starting point of the present proposal.
CNR has a long standing experience in nanotechnology applied to living matter, in particular by mean of
micro- and nanofabrication, microfluidics, atomic force microscopy imaging and manipulation and
interfacial properties characterization. In the frame of the present proposal CNR will fabricate surfaces with
controlled surface energy that can mimic the protein surface for contact angle experiments and, in parallel,
it will perform single molecule mechanical unfolding experiments.
1. Hofmeister, F. : Zur Lehre von der Wirkung der Salze. II. Arch. exp. Pathol. Pharmakol. 24 (1888) 247-260.
2. P.L. Nostro, B.W. Ninham, B.W.: An update on ion specific biology. Chem. Rev. (2004) 2286-2322.
3. Collins, K.D. and Washabaugh, M.W.: The Hofmeister effect and the behaviour of water at interfaces. Q.
Rev. Biophys. 18 (1985) 323-422.
4. Cacace, M.G., Landau, E.M. and Ramsden, J.J.: The Hofmeister series: salt and solvent effects on
interfacial phenomena. Q. Rev. Biophys. 30 (1997) 241-278.
5. Gurau, M.C., Lim, S-M., Castellana, E.T., Albertorio, F. Kataoka, S. and Cremer, P.S.: On the mechanism of
Hofmeister effect. J. Am. Chem. Soc. 126 (2004) 10522-10524
6. Dér,A., Kelemen, L. Fábián, L., Taneva, S.G., Fodor, E., Páli, T., Cupane, A., Cacace, M.G. and Ramsden, J.J.:
Interfacial water structure controls protein conformation. J. Phys. Chem. B 111 (2007) 5344-5350.
7. Melander, W. and Horvath, Cs.: Salt effects on hydrophobic interactions in precipitation and
chromatography of proteins. Arch. Biochem. Biophys. 183 (1977) 200-215.
8. Der, A and Ramsden, J.J.: Evidence for loosening of a protein mechanism. Naturwissenschaften 85 (1998)
353-355.
9. Neagu, A., Neagu, M. and Der, A.: Fluctuations and the Hofmeister effect. Biophys. J. 81 (2001) 12851294.
10. Godawat, R., Jamadagni, S.N. and Garde, S.: Characterizing hydrophobicity of interfaces by using cavity
formation, solute binding, and water correlations. Proc. Natl. Acad. Sci. USA 106 (2009) 15119-15124.
Obiettivi:
Objectives
The main objective of the present proposal is to provide an experimental support for the presently
available theory of the Hofmeister salt effect on the protein structure.
In particular the project aims at shedding light on the role of the surface tension at the interface between
the protein surface and salt solution in stabilizing or weakening the three dimensional protein structure; for
this purpose the projects has the following main objectives:
Objective1: assessing the opposite wetting behaviour of chaotropic and kosmotropic salts by mean of
nanostructured surfaces displaying varying and controlled hydrophobicity
Objective2: assessing how the wetting ability of chaotropes and kosmotropes changes when the surfaces
are functionalized with proteins displaying different conformations (globular proteins, membrane proteins,
intrinsically unfolded proteins)
Objecctive3: accurately measuring, by mean of nanomechanical manipulation of multimodular protein
constructs, the effect of the salts placed in different position along the Hofmeister series on the free energy
profile of proteins; in particular how the stabilization/destabilization of their structure takes place (shifting
the position or changing the height of the free energy barriers, altering the shape of the free energy wells)