Understanding the molecular basis of protein function is a challenging task that
lays the foundation for the pharmacological intervention in many diseases originating
in altered structural states of the involved proteins. Dissecting a complex functional
machinery into modules is a promising approach to protein function. The motivation
for this work was to identify minimal requirements for “local” switching processes in
the function of multidomain proteins that can adopt a variety of structural substates
of different biological activity or representing intermediates of a complex reaction
path. For example, modular switches are involved in signal transduction, where
receptors respond to ligand-activation by specific conformational changes that are
allosterically transmitted to “effector recognition sites” distant from the actual
ligand-binding site. Heptahelical receptors have attracted particular attention due to
their ubiquitous role in a large variety of pharmacologically relevant processes.
Although constituting switches in their own right, it has become clear through
mutagenesis and functional studies that receptors exhibit substates of partial
active/inactive structure that can explain biological phenotypes of different levels of
activity. Here, the notion that microdomains undergo individual switching processes
that are integrated in the overall response of structurally regulated proteins is
addressed by studies on the molecular basis of proton-dependent (chemical) and
force-dependent (mechanical) conformational transitions.
A combination of peptide synthesis, biochemical analysis, and secondary
structure sensitive spectroscopy (Infrared, Circular dichroism, Fluorescence) was
used to prove the switching capability of putative functional modules derived from
three selected proteins, in which conformational transitions determine their function
in transmembrane signaling (rhodopsin), transmembrane transport
(bacteriorhodopsin) and chemical force generation (kinesin-1). The data are then
related to the phenotypes of the corresponding full length-systems. In the first two
systems the chemical potential of protons is crucial in linking proton exchange
reactions to transmembrane protein conformation. This work addresses the
hypothesized involvement of lipid protein interactions in this linkage (1). It is shown
here that the lipidic phase is a key player in coupling proton uptake at a highly
conserved carboxylic acid (DRY motif located at the C-terminus of helix 3) to conformation during activation of class-1 G protein coupled receptors (GPCRs)
independently from ligand protein interactions and interhelical contacts. The data
rationalize how evolutionary diversity underlying ligand-specifity can be reconciled
with the conservation of a cytosolic ‘proton switch’, that is adapted to the general
physical constraints of a lipidic bilayer described here for the prototypical class-1
GPCR rhodopsin (2).
Whereas the exact sequence of modular switching events is of minor
importance for rhodopsin as long as the final overall active conformation is reached,
the related heptahelical light-transducing proton pump bacteriorhodopsin (bR),
requires the precise relative timing in coupling protonation events to
conformationtional switching at the cytosolic, transmembrane, and extracellular
domains to guarantee vectorial proton transport. This study has focused on the
cytosolic proton uptake site of this retinal protein whose proton exchange reactions at
the cytosolic halfchannel resemble that of rhodopsin. It was a prime task in this work
to monitor in real time the allosteric coupling between different protein regions. A
novel powerful method based on the correlation of simultaneously recorded infrared
absorption and fluorescence emission changes during bR function was established
here (3), to study the switching kinetics in the cytosolic proton uptake domain
relative to internal proton transfer reactions at the retinal and its counter ion. Using
an uptake-impaired bR mutant the data proves the modular nature of domain
couplings and shows that the energy barrier of the conformational transition in the
cytosolic half but not its detailed structure is under the control of proton transfer
reactions at the retinal Schiff base and its counter ion Asp85 (4).
Despite the different functions of the two studied retinal proteins, the
protonation is coupled to local switching mechanisms studied here at two levels of
complexity, [a] a single carboxylic acid side chain acting as a lipid-dependent proton
switch [b] a full-length system, where concerted modular regions orchestrate the
functional coupling of proton translocation reactions. Switching on the level of an
individual amino acid is shown to rely on localizable chemical properties (charge
state, hydrophobicity, rotamer state). In contrast, switching processes involving
longer stretches of amino acids are less understood, less generalizable, and can
constitute switches of mechanical, rather than chemical nature. This applies
particularly to molecular motors, where local structural switching processes are directly involved in force generation. A controversy exists with respect to the
structural requirements for the cooperation of many molecular motors attached to a
single cargo. The mechanical properties of the Hinge 1 domain of kinesin-1 linking
the “neck” and motor domain to the “tail” were addressed here to complement single
molecule data on torsional flexibility with secondary structure analysis and thermal
stability of peptides derived from Hinge 1 (5). It is shown that the Hinge 1 exhibits
an unexpected helix-forming propensity that resists thermal forces but unfolds under
load. The data resolve the paradox that the hinge is required for motor cooperation,
whereas it is dispensable for single motor processivity, clearly emphasizing the
modular function of the holoprotein. However, the secondary-structural data reveal
the functional importance of providing high compliance by force-dependent
unfolding, i.e. in a fundamentally different way than disordered domains that are
flexible but yet do not support cooperativity.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa.de:bsz:14-qucosa-25977 |
| Date | 05 January 2010 |
| Creators | Madathil, Sineej |
| Contributors | Technische Universität Dresden, Fakultät Mathematik und Naturwissenschaften, Prof. Dr. Herwig Gutzeit, Prof. Dr. Gert Bernhard, Prof. Dr. Werner Mäntele |
| Publisher | Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis |
| Format | application/pdf |
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