Transporters, a major class of membrane proteins, mediate substrate translocations that are implicated in a variety of disease-related physiology, including aberrant cellular deposition of nutrients, ab- normal cell death or proliferation, and efflux of drug. So, understanding the transport mechanism is theo- retically significant, as well as fundamental to addressing relevant questions, such as disease etiology at the molecular level and drug design. Despite the importance, efforts to gain structural understanding of transport mechanism have long been hampered by the experimental difficulty associated with the complex nature of the bilayer environment where these proteins reside. Very recently, an idea of common transport mechanism has been highlighted by the breakthroughs in structural investigations, which revealed that functionally diverse transporters share a surprisingly similar topology composed of dual structural subunits with inverted symmetry. Here, I plan to pursue basic biomedical quest on establishing a unified transport mechanism by investigating metal- and sugar-transportation as model systems, using methods of experimental, computational and struc- tural biophysics. In implementing the work, my expertise in membrane protein structure and thermodynamic folding will be broadened by the leading research of my sponsor, William DeGrado, in diverse fields of protein structure-function-dynamics relationship, molecular biophysics of membrane proteins and de novo protein design.
Specific Aims : Following specific aims will be pursued:
Aim 1. To test the importance of symmetric dual topo- logy in transport function, I will use de novo protein design approach to generate zinc transport function in a minimalist model transporter composed of a symmetrically designed helix bundle.
Aim 2. To test the impor- tance of conformational dynamics in function, I will examine whether the activity by galactose transporter vSGLT, as well as de novo zinc transporter peptides, reflect the expected conformational change with respect to kinetic behaviors.
Aim 3. To test the corollary of the importance of structural dynamics in transportation, I will use both protein design and genetic screening approaches to generate a transmembrane peptide that allosterically locks the vSGLT or model transporter in one conformational state by binding the target trans- porter, and test the transportation inhibition effect of the de novo allosteric binder peptide. Reemphasis of the proposal's innovation: Abated zinc translocation is related to the lack of zinc-induced apoptosis in prostate cancer, while specific sugar transporters are up regulated in other tumor cells to meet the increased demand for nutrient. So, understanding the metal- and sugar-transportation function using the minimalist zinc transporters and vSGLT as model systems is extensively relevant. Completion of the proposed project will not only provide an excellent training opportunity, but also present conceptual mechanistic theories for transport function, which is potentially important in therapeutics development.
Transporters are proteins that reside in the biological membrane and control the movement of variety of molecules in and out of cells, as well as cellular organelles. As maintaining the chemical balance is critical for cell survival, understanding how transporters function is central to exploring physiologically central questions, such as how transport function is generated at the molecular level, how impaired transport function is related to diseases, such as cancer or heart disease, and drug design. This proposal is aimed at providing the conceptual understanding for transporter mechanism, along with novel ways of selectively regulating the transport function, achieving of which will be theoretically significant, as well as potentially fundamental to therapeutics development.
|Joh, Nathan H; Wang, Tuo; Bhate, Manasi P et al. (2014) De novo design of a transmembrane Zn²?-transporting four-helix bundle. Science 346:1520-4|