My research is driven by a search for answers to two questions: how do proteins cross membranes, and how do they insert into lipid bilayers? In the cell, these vital functions are achieved by complex multi-protein assemblies. But the diphtheria toxin T-domain (DTT) by itself inserts into the membrane under acidic conditions and translocates the catalytic domain across the lipid bilayer. Low pH is also involved in transbilayer insertion of annexin 12 (ANX), a representative of a large protein family that has been implicated in a variety of membrane functions and in a number of human diseases. The pH-triggered refolding-insertion-translocation of DTT and other proteins (e.g., botulinum toxin) is not only of inherent importance, but also can reveal general physicochemical principles underlying membrane protein assembly and stability. The objectives of this grant are to refine these principles, develop experimental tools for following protein folding transitions in membranes, and apply these principles and tools to the problem of pH-induced membrane insertion of DTT and ANX. These new tools will take advantage of the lifetime fluorescence methodology, uniquely suited to analyzing heterogeneous conformational states. Model helical peptides will be used to determine the energetics of protonation and folding on membrane interfaces. Various spectroscopic approaches, chiefly the site-selective fluorescence labeling of DTT and ANX, will be used to test the following hypothesis: Membrane insertion pathways for non-constitutive membrane proteins contain an obligatory interfacial intermediate state; formation of this intermediate and subsequent transbilayer insertion is mediated by a subtle balance of hydrophobic and electrostatic interactions between proteins and the membrane interface.
The specific aims are: (1) Characterize folding intermediates on the membrane insertion pathway of DTT and ANX. (2) Elucidate the mechanism of pH-induced refolding of DTT on membranes. (3) Refine the physicochemical rules for predicting membrane protein insertion/folding using model helical peptides. (4) Advance the development of fluorescence methods for insertion/folding studies of membrane proteins. ? ?
|Gnanasambandam, Radhakrishnan; Ghatak, Chiranjib; Yasmann, Anthony et al. (2017) GsMTx4: Mechanism of Inhibiting Mechanosensitive Ion Channels. Biophys J 112:31-45|
|Rodnin, Mykola V; Li, Jing; Gross, Michael L et al. (2016) The pH-Dependent Trigger in Diphtheria Toxin T Domain Comes with a Safety Latch. Biophys J 111:1946-1953|
|Ladokhin, Alexey S (2015) Membrane Protein Folding & Lipid Interactions: Theory & Experiment. J Membr Biol 248:369-70|
|Ghatak, Chiranjib; Rodnin, Mykola V; Vargas-Uribe, Mauricio et al. (2015) Role of acidic residues in helices TH8-TH9 in membrane interactions of the diphtheria toxin T domain. Toxins (Basel) 7:1303-23|
|Flores-Canales, Jose C; Vargas-Uribe, Mauricio; Ladokhin, Alexey S et al. (2015) Membrane Association of the Diphtheria Toxin Translocation Domain Studied by Coarse-Grained Simulations and Experiment. J Membr Biol 248:529-43|
|Vargas-Uribe, Mauricio; Rodnin, Mykola V; Öjemalm, Karin et al. (2015) Thermodynamics of Membrane Insertion and Refolding of the Diphtheria Toxin T-Domain. J Membr Biol 248:383-94|
|Flores-Canales, Jose C; Kurnikova, Maria (2015) Targeting electrostatic interactions in accelerated molecular dynamics with application to protein partial unfolding. J Chem Theory Comput 11:2550-9|
|Flores-Canales, Jose C; Kurnikova, Maria (2015) Microsecond Simulations of the Diphtheria Toxin Translocation Domain in Association with Anionic Lipid Bilayers. J Phys Chem B 119:12074-85|
|Kyrychenko, Alexander; Vasquez-Montes, Victor; Ulmschneider, Martin B et al. (2015) Lipid headgroups modulate membrane insertion of pHLIP peptide. Biophys J 108:791-794|
|Rodnin, Mykola V; Ladokhin, Alexey S (2015) Membrane translocation assay based on proteolytic cleavage: application to diphtheria toxin T domain. Biochim Biophys Acta 1848:35-40|
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