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Mechanisms of a secondary active transporter function (25) | Dr Bernadette Byrne

Transporters are designed to bind cargo on one side of the membrane, undergo a conformational change and release it on the other side of the membrane. A range of different transporter structures have revealed that there are a number of different molecular mechanisms by which transporters can move their cargo across the membrane. This dissertation will involve a detailed description of one or more of the mechanisms of transport, describing both the structural and biochemical evidence to support the particular mechanism.

Dr Bernadette Byrne


Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh.

Abstract: GltPh from Pyrococcus horikoshii is a homotrimeric Na(+)-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters that take up the neurotransmitter glutamate. Each protomer in GltPh consists of a trimerization domain involved in subunit interactions and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na(+) and aspartate binding to GltPh. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na(+) binds with low affinity to the apoprotein (Kd 120 mm), with a particularly low kon value (5.1 m(-1)s(-1)). At least two sodium ions bind before aspartate. The binding of Na(+) requires a very high activation energy (Ea 106.8 kJ mol(-1)) and consequently has a large Q10 value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na(+) concentration present. Binding of aspartate was not observed in the absence of Na(+), whereas in the presence of high Na(+) concentrations (above the Kd for Na(+)) the dissociation constants for aspartate were in the nanomolar range, and the aspartate binding was fast (kon of 1.4 × 10(5) m(-1)s(-1)), with low Ea and Q10 values (42.6 kJ mol(-1) and 1.8, respectively). We conclude that Na(+) binding is most likely the rate-limiting step for substrate binding.

Pub.: 30 Apr '15, Pinned: 25 Jan '17