Techniques

Authors mutated the NA2 binding site (T354V, S355A) and perturbed the intracellular gate (inward gate) (Y268A) to yield the LeuTk variant. [8, 9, 10]. Mutation of Thr to Val, Ser and Tyr to Ala are used due to high size similarity, in which the mutation stabilises an inward-open state of LeuT. Polymerase chain reaction (PCR) is used for the procedure of site-directed mutagenesis. The mutants are subcloned into a pET16b plasmid containing a thrombin-cleavage site and a C-terminal octa-histidine tag. To verify the correct mutant sequences, all mutants were verified by focussed DNA sequencing.

The site-directed mutants LeuTK(Y108F), LeuTK(TS) and LeuTK(TSY) were produced by polymerase chain reaction (PCR) and subcloned into a pET16b plasmid containing a thrombin-cleavage

site and a C-terminal octa-histidine tag. All mutants were verified by DNA sequencing. The LeuT mutants were expressed and purified by purified by Ni-affinity chromatography. The mutant transporters were kept sodium-free by washing three times in sodium-free buffer to ensure that no endogenously bound

leucine was carried along.

Monoclonal conformationally-sensitive Fab fragments are used in order to obtain the crystals. For the outward-open structure, the substrate binding is weakened by mutating a tyrosine residue, which coordinates the substrate, to phenylalanine.