To probe sequence variation in a systematic manner, we drew on the observation that the temperature of the optimal enzyme activity correlates strongly with the temperature of the habitats of the originating organisms. Thus, thermophilic enzymes are slow compared to the mesophilic enzymes at ambient temperatures. We developed a bioinformatics approach to identify systematic amino acid changes that might be associated with temperature adaptation across bacterial glutamate transporters. We used this analysis as a guideline to introduce substitutions into GltPh with the aim to augment its transport activity at ambient temperatures without modifying the substrate-binding site. Gain-of-function was assessed by the transport of radiolabeled aspartate into artificial liposomes.
Over 30 single substitution variants were created. Top scorers, which increased transport up to five-fold, were further combined with each other, with functionally neutral substitutions and with previously documented gain-of-function GltPh-variants. This strategy resulted into GltPh-variants that increased the aspartate transport rate up to 25-fold.
A panel of GltPh-variants containing representative transporters across the catalytic range was selected for structure determination and measurement of elevator dynamics. So far, crystal structures at medium and high-resolution of several GltPh-variants did not reveal striking differences with structures of the wild-type protein. Elevator dynamics were measured by distance changes associated with the OFS-to-IFS reorientation of the substrate-binding domains using energy transfer between two site-specific fluorophores as a molecular ruler at the single molecule level by total internal reflection microscopy. These results showed that increased transport rates correlate well with increased elevator dynamics in single substitution variants. However, measurements on multi-substitution variants with the higher transport rates failed to confirm this correlation.
To determine the mechanism of accelerated aspartate transport in multi-substitution variants, we measured substrate affinities and release rates from the IFS. Although substitutions were selected to conserve the substrate binding site, aspartate affinities were lowered in some variants, but these changes did not appear to correlate linearly with increased transport rates, even though aspartate release by IFS-cross-linked GltPh-variants suggested that release was accelerated in multi-substitution variants.
Further details will be available from our publications:
1. Huysmans GHM*, Ciftci D, Wang X, Blanchard SC and Boudker O*. The high-energy transition state of a membrane transporter. *co-corresponding authors
2. Matin TR, Heath GR, Huysmans GHM, Boudker O and Scheuring S. Millisecond dynamics of unlabeled amino acid transporters.
3. Huang Y, Wang X, Lv G, Razavi A, Huysmans GH, Weinstein H, Bracken C, Elizier D and Boudker O. Monitoring Dynamics of Large Membrane Proteins by 19F Paramagnetic Longitudinal Relaxation: Domain Movement in a Glutamate Transporter Homolog.
4. Ciftci D, Huysmans GH, Wang X, He C, Terry D, Zhou Z, Fitzgerald G, Blanchard SC and Boudker O. Single-Molecule Transport Kinetics of a Glutamate Transporter Homologue Shows Static Disorder.
