UNIVERSITY OF MASSACHUSETTS
The overall scope of the parent grant is to understand how ATP allosterically inhibits the transport activity of the human, blood brain barrier glucose transport protein, GLUT1 .The supplement seeks additional support to identify sites of interaction between the GLUT1 loop 6 and C-terminus by MS analysis of ATP-induced chemical x-links between these domains. We have shown: 1) that GLUT1 loop 6 and C-terminal domains interact in an ATP-dependent manner; 2) that these interactions promote occlusion of C-terminal residues 477-492 and loop 6 residues 245-256 and, 3) that ATP binding facilitates chemical x-linking of the GLUT1 C-terminal domain to a GLUT1 domain that is N-terminal to residue K300. We propose to identity the sites of contact between the GLUT1 C terminus and GLUT1 sequence N-terminal to K300 by MS analysis of x-linked peptides using variable and zero-length chemical x-linkers. This will provide a high-resolution map of ATP-dependent contacts within each GLUT1 molecule. This new understanding will accelerate our basic and clinical research into GLUT1 biology in health and disease by revealing protein surfaces that regulate GLUT1 function. This principle may be important in the regulation of the remaining 12 members of the GLUT family and the broader family of MFS proteins. This project necessitates the purchase of HPLC instrumentation and MS reagents to undertake this research and extensive engagement with the scientific staff of the UMMS proteomics facility. The net effect is that we will couple the advancement of science and health to stimulation of the economy through research-associated staff expansion, staff retention and the procurement and utilization of relevant commercial, biotechnology resources.