Browsing by Subject "Membrane proteins"

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    Biophysical characterization of membrane proteins and antimicrobial peptides by solution and solid-state NMR spectroscopy.
    (2011-03) Verardi, Raffaello
    Membrane proteins and antimicrobial peptides represent two diverse and challenging classes of macromolecules to characterize at the molecular level. They are linked by the interaction with the lipid bilayer of the cell membrane. Within the lipid bilayer, membrane proteins are involved in vital biochemical processes such as ion transport, signal transduction and cell adhesion. Antimicrobial peptides are a broad class of polypeptides produced by all living organisms, representing the first line of defense against bacterial infections. They work by selectively targeting the bacterial membranes and subsequently killing the cell by a variety of mechanisms such as membrane disruption, membrane potential dissipation and enzyme inactivation. Although very important, membrane proteins and antimicrobial peptides are underrepresented in terms of available high-resolution structural information compared to water-soluble proteins and this limits the current understanding of how they work in living cells. In this thesis I summarize my contribution towards the elucidation of the high-resolution structures of the integral membrane protein phospholamban and the mechanism of action of two important antimicrobial peptides (LL37 and distinctin) by a hybrid solution and solid-state nuclear magnetic resonance spectroscopy approach. These results provide new insights and methodologies to study and understand how key membrane proteins and antimicrobial peptides elicit their function.
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    Rational design of loss-of-function phospholamban mutants to tune SERCA function.
    (2012-04) Ha, Kim N.
    Unphosphorylated phospholamban (PLN) is the endogenous inhibitor of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA), the enzyme that regulates cardiac muscle relaxation in humans. In its phosphorylated state, PLN (pS16-PLN, pT17-PLN, and pS16pT17-PLN) does not inhibit SERCA. Dysfunctions in SERCA:PLN interactions and in the PLN phosphorylation mechanism have been implicated in cardiac disease and targeting PLN is becoming a viable avenue for treating heart disease. Specifically, innovative genetic treatments using recombinant adeno-associated virus (rAAV) with S16E-PLN, a pseudo-phosphorylated form of PLN, have shown a remarkable efficacy in reducing the progression of cardiac failure in both small and large animals. The following thesis summarizes efforts to rationally design PLN mutants to tune SERCA function. Using a combination of NMR spectroscopy and biochemical assays, we have built a structure-dynamics-function correlation that shows PLN can be tuned to augment SERCA function by acting on the conformational coupling between the cytoplasmic and transmembrane domain and by pseudo-phosphorylation. Additionally, to better understand the role of mutation in PLN:SERCA interactions, we also investigated a mutant of PLN (R9C) known to be linked to hereditary dilated cardiomyopathy, showing that the mutation disrupts the pentamer-monomer equilibrium, and that these effects are exacerbated under oxidizing conditions. Insights to these issues will provide better paradigms with which to design therapeutic mutants of PLN for treatment of heart failure.