The D-galactose-H+ symporter, Ga1P, of Escherichia coil was subjected to mutagenesis to elucidate the molecular mechanism of substrate and ligand recognition, to define the roles of individual amino acid residues and to stabilize the protein in a conformation favorable for crystallization. Twenty-five mutants were constructed during the course of this study using the following four criteria: 1) elimination of protease susceptible sites; 2) sequence similarities and differences to close homologues; 3) residues based on previous mutagenesis in GLUT1 that might lock the protein in a conformation suitable for crystallization; and 4) residues putatively involved in electrostatic interactions. Almost all mutant proteins were shown to be expressed and inserted into the membrane at levels equivalent to the WT-GaIP(His)6, as verified by SDS-PAGE and Western blot. A combination of different criteria such as ability to transport sugars and bind high affinity inhibitory ligands, cytochalasin B and forskolin, enabled comparison between the WT- and GalP(His)6 mutants. Analyses of these mutants allowed for the following important observations. (1) Peptide-mass fingerprinting indicated that the outer membrane protease cleaves between Arg455 and Lys456 in the C-terminal end of GalP(His)6. Neutral substitution of these residues did not eliminate the C-terminal truncation, indicating that the adjacent residues might also serve as substrates for the outer-membrane proteases. However, the purified proteins of R455T and K456A mutants were observed to migrate as a single species, with the C-terminal protein not co-purifying with the full-length protein. Additionally, initial crystallization trials of K456A-GalP(His)6 appears promising. (2) Neutral substitution of Asp312 (D3 12G) severely impaired sugar-transport while retaining the ability to bind ligands. Furthermore an additional occluded-ligand binding state was observed shifting the equilibrium of the mutant towards the inward-facing conformation in the absence of substrate. This inwardly locked mutant can serve as a good candidate for crystallization. (3) When the Tyr273 residue (N-terminal end of TMH7) was substituted with Ile, the ability to transport D-glucose and D-galactose was severely impaired. Moreover, cytochalasin B affinity was completely lost and the affinity for forskolin reduced, indicating that this residue might be directly involved in sugar and cytochalasin B recognition. The Pro368 residue substitution by Gln drastically reduced D-glucose recognition while maintaining the affinity to D-galactose, indicating that Pro368 might participate in the discrimination of the C-4 hydroxyl group on sugar substrates. (4) Mutation of G1u438, Thr439 and Lys440 of the 'VPETIC' motif at the end of TMH12 indicated that these residues are likely to be involved in maintaining the overall conformation of the transporter through electrostatic interactions rather than having a direct involvement in substrate and ligand binding. Additional mutants not mentioned here were found to have either no effect or minor perturbations in either the structure or helical packing of the transporter indicating that they were not essential for the overall transport process.