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

INTRODUCTION: LIPID-PROTEIN INTERACTIONS
ability of some organic solvents to stimulate hydrogen bonding and alpha-helical
structure (Killian et al., 1994). This is particularly important when working with TM
alpha-helical peptides, since this treatment can prevent aggregation and/or transition to
different secondary structures that can be irreversible in some cases. Examples of such
solvents are trifluoroethanol (TFE) and hexafluoroisopropanol.
Sonication of mixed solutions of proteins in buffer and lipids can also affect the
transfer of proteins to liposomes. The downfall of this technique is that it requires
solubility of the protein in water, it can induce protein denaturation and always results
in SUV’s, which can be problematic since some proteins reconstituted in high curvature
liposomes have been shown to underwent changes in their activities. Freeze-thawing of
mixtures of sonicated liposomes and proteins has also been used in cases where the
protein was sensitive to both detergents and sonication (Seddon et al., 2004).
For a protein to be considered as correctly reconstituted, some requirements must be
fulfilled. The characteristic function or activity of the protein in vivo must be regained
(at least partially) after reconstitution. Ideally the process should result in a defined lipid
environment for the protein with an also defined lipid to protein ratio (Yeagle, 1993).
2.2. Peptides as models
Membrane proteins are extremely complex entities. In addition to protein-lipid
interactions, protein-protein interactions are crucial in dictating final properties as
protein domains interact between themselves in the final structure. In order to study
protein-lipid interactions an alternative is the use of peptide models, either TM or
peripherically associated with the membrane. An additional advantage of this
minimalist approach is that synthetic peptides can be obtained in large quantities, which
is not feasible for many membrane proteins. The possibility of synthesis also allows an
easy control over the primary sequence and wide mutation possibilities, and this is
essential in understating the role of particular amino acids (Wimley and White, 1996)
and peptide segments on the interactions with the lipid environment, and on the function
of the protein itself. For large membrane proteins the range of possible mutations is
quite limited due to problems related to cell viability of mutants.
Putative TM alpha-helices can be estimated from hydropathy plots as described in
Section 1.8, and hydrophobic moment estimations can assist on the search for
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