7 research outputs found
Investigating the Bacterial Glutamate Transporter Homolog GltPh with Unnatural Amino Acids
Recommended from our members
Individual Ion Binding Sites in the K(+) Channel Play Distinct Roles in C-type Inactivation and in Recovery from Inactivation.
The selectivity filter of K(+) channels contains four ion binding sites (S1-S4) and serves dual functions of discriminating K(+) from Na(+) and acting as a gate during C-type inactivation. C-type inactivation is modulated by ion binding to the selectivity filter sites, but the underlying mechanism is not known. Here we evaluate how the ion binding sites in the selectivity filter of the KcsA channel participate in C-type inactivation and in recovery from inactivation. We use unnatural amide-to-ester substitutions in the protein backbone to manipulate the S1-S3 sites and a side-chain substitution to perturb the S4 site. We develop an improved semisynthetic approach for generating these amide-to-ester substitutions in the selectivity filter. Our combined electrophysiological and X-ray crystallographic analysis of the selectivity filter mutants show that the ion binding sites play specific roles during inactivation and provide insights into the structural changes at the selectivity filter during C-type inactivation
Individual Ion Binding Sites in the K(+) Channel Play Distinct Roles in C-type Inactivation and in Recovery from Inactivation.
The selectivity filter of K(+) channels contains four ion binding sites (S1-S4) and serves dual functions of discriminating K(+) from Na(+) and acting as a gate during C-type inactivation. C-type inactivation is modulated by ion binding to the selectivity filter sites, but the underlying mechanism is not known. Here we evaluate how the ion binding sites in the selectivity filter of the KcsA channel participate in C-type inactivation and in recovery from inactivation. We use unnatural amide-to-ester substitutions in the protein backbone to manipulate the S1-S3 sites and a side-chain substitution to perturb the S4 site. We develop an improved semisynthetic approach for generating these amide-to-ester substitutions in the selectivity filter. Our combined electrophysiological and X-ray crystallographic analysis of the selectivity filter mutants show that the ion binding sites play specific roles during inactivation and provide insights into the structural changes at the selectivity filter during C-type inactivation
Engineering the Glutamate Transporter Homologue Glt<sub>Ph</sub> Using Protein Semisynthesis
Glutamate transporters catalyze the
concentrative uptake of glutamate
from synapses and are essential for normal synaptic function. Despite
extensive investigations of glutamate transporters, the mechanisms
underlying substrate recognition, ion selectivity, and the coupling
of substrate and ion transport are not well-understood. Deciphering
these mechanisms requires the ability to precisely engineer the transporter.
In this study, we describe the semisynthesis of Glt<sub>Ph</sub>,
an archaeal homologue of glutamate transporters. Semisynthesis allows
the precise engineering of Glt<sub>Ph</sub> through the incorporation
of unnatural amino acids and peptide backbone modifications. In the
semisynthesis, the Glt<sub>Ph</sub> polypeptide is initially assembled
from a recombinantly expressed thioester peptide and a chemically
synthesized peptide using the native chemical ligation reaction followed
by in vitro folding to the native state. We have developed a robust
procedure for the in vitro folding of Glt<sub>Ph</sub>. Biochemical
characterization of the semisynthetic Glt<sub>Ph</sub> indicates that
it is similar to the native transporter. We used semisynthesis to
substitute Arg397, a highly conserved residue in the substrate binding
site, with the unnatural analogue, citrulline. Our studies demonstrate
that Arg397 is required for high-affinity substrate binding, and on
the basis of our results, we propose that Arg397 is involved in a
Na<sup>+</sup>-dependent remodeling of the substrate binding site
required for high-affinity Asp binding. We anticipate that the semisynthetic
approach developed in this study will be extremely useful in investigating
functional mechanisms in Glt<sub>Ph</sub>. Further, the approach developed
in this study should also be applicable to other membrane transport
proteins