Dr. Mikhail V. Bogdanov
The University of Texas Health Science Center at Houston
Department of Biochemistry and Molecular Biology
- Membrane protein folding and topogenesis
- Assisted protein folding
- Lipids as molecular chaperones
- Molecular mechanism of protein insertion into and through biological membrane
- Protein folding and conformational diseases
- Membrane protein biogenesis and topological disorders
Lipid membrane manifests a myriad of unique phospholipid molecules and amazing diverse array of surfaces ranging from a hydrophobic bilayer core to aqueous solutions on either side of the membrane that can fold and orient an integral protein.
The aim of the current project is to elucidate the molecular basis by which phospholipids function as molecular chaperons in facilitating membrane protein folding. The critical importance of molecular chaperones in the folding of proteins has likely ensured that the primary sequence of these proteins evolved to contain molecular chaperone-binding sites in addition to information for attaining the proper tertiary structure. Therefore, individual membrane phospholipids could exhibit affinity for some regions of polytopic membrane proteins, thus helping the polypeptides avoid intramolecular interaction which is unfavorable for proper folding.
Experimental questions we focus on:
• How do phospholipids facilitate membrane protein folding?
• What structural features of lipid account for the ability of lipid act a molecular chaperone?
• What is the minimum structural feature of membrane protein folded with and without phospholipid assistance?
• What is and where is a crucial "folding" and "insertion" domain of the polytopic membrane protein? Are they the same or not?
• How do protein and non-protein molecular chaperones cooperate in vivo?
• How do individual lipids and components of "insertase" contribute to membrane protein insertion?
• How widely might phospholipid-assistance apply to membrane protein folding?
• Protein misfolding and diseases: phospholipids as molecular anti-chaperones?
• How do healthy protein commit "deadly" misfolding and self-aggregation?
An arsenal of genetic methods, biochemical and biophysical techniques including protein engineering by site-directed and incremental truncation, chemical labeling and photo-cross linking, NMR and surface plasmon resonance (BIAcore system) will be used to address these very important questions. A tutorial also would provide experience with in vitro refolding of membrane proteins (by novel Eastern-Western blotting technique) and de novo folding and insertion of membrane protein being transcribed and translated in cell-free system protein and phospholipid biosynthesis system.
Membrane protein topogenesis problem to understand and predict how a given protein sequence will fold and orient itself in a given phospholipid environment. The ability to regulate lipid composition temporally along with substituted cysteine accessibility method (SCAM TM ) (Bogdanov et al., Methods, 2005, 36, 148-171.) provides a powerful means to investigate molecular details of dynamic topogenesis and observe dynamic changes in protein topology as a function of membrane lipid composition.
Experimental questions we focus on:
• Whether folding and insertion are coupled or separable events?
• Is lipid and structurally specific folding of extramembrane domains related to overall transmembrane topogenesis?
• Where and how membrane proteins make their topological "decision"?
• Are topogenic signals recognized and “decoded” by given lipid profile or “interpreted” by insertion machinery?
• Can phospholipid composition of the membrane alone affect the topology and therefore the function of membrane protein?
• Transmembrane protein topology: static or dynamic? Is lipid bilayer really non-flipping zone for integral membrane protein?
• How to derive highly dynamic membrane protein topogenesis from relatively static experimental data such as endpoints topologies of membrane proteins?
• Can lipids manipulate the single membrane protein substrate to achieve multiple topological forms?
• Can polytopic membrane protein adopt two stable alternative transmembrane topological isoforms within one biological membrane?
• Can one transmembrane protein topology fluctuate within one membrane with time?
• Can a topological “mistake” (a domain trapped on the “wrong” site of the membrane) be corrected?
• What “flip-flopping” membrane proteins would need to have?
• Is “positive-inside” rule absolute? Are positively charged residues still more potent topological determinants than negatively charged ones?
• How to overcome energy costing flip-flop movement of large hydrophilic domain across the membrane?
• How transmembrane protein topological inversions governed and controlled: thermodynamically or kinetically?
SCAM TM is utilized to monitor lipid-dependent transmembrane topological switches and end-point topologies in vivo (intact cells) or in vitro (liposomes).