Chapter 7 Semisynthesis of K+ Channels

Komarov, Alexander G.; Linn, Kellie M.; Devereaux, Jordan; Valiyaveetil, Francis I.

doi:10.1016/s0076-6879(09)62007-3

Cited by 5 publications

(10 citation statements)

References 33 publications

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“…We used the previously described semisynthesis of the KcsA channel to introduce amide-to-ester substitutions into the selectivity filter (29,30). The semisynthesis provides a truncated but functional form of the KcsA channel.…”

Section: Resultsmentioning

confidence: 99%

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Using protein backbone mutagenesis to dissect the link between ion occupancy and C-type inactivation in K ⁺ channels

Matulef

Komarov

Costantino

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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K+ channels distinguish K + from Na + in the selectivity filter, which consists of four ion-binding sites (S1-S4, extracellular to intracellular) that are built mainly using the carbonyl oxygens from the protein backbone. In addition to ionic discrimination, the selectivity filter regulates the flow of ions across the membrane in a gating process referred to as C-type inactivation. A characteristic of Ctype inactivation is a dependence on the permeant ion, but the mechanism by which permeant ions modulate C-type inactivation is not known. To investigate, we used amide-to-ester substitutions in the protein backbone of the selectivity filter to alter ion binding at specific sites and determined the effects on inactivation. The amide-to-ester substitutions in the protein backbone were introduced using protein semisynthesis or in vivo nonsense suppression approaches. We show that an ester substitution at the S1 site in the KcsA channel does not affect inactivation whereas ester substitutions at the S2 and S3 sites dramatically reduce inactivation. We determined the structure of the KcsA S2 ester mutant and found that the ester substitution eliminates K + binding at the S2 site. We also show that an ester substitution at the S2 site in the K v AP channel has a similar effect of slowing inactivation. Our results link C-type inactivation to ion occupancy at the S2 site. Furthermore, they suggest that the differences in inactivation of K + channels in K + compared with Rb + are due to different ion occupancies at the S2 site.

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Section: Resultsmentioning

confidence: 99%

“…The ester mutants of the KcsA and K v AP channels were generated either by semisynthesis or by the in vivo nonsense suppression approach (29,30,32,33). Crystals of the KcsA Y78-ester were grown as a complex with a Fab fragment as described (5).…”

Section: Methodsmentioning

confidence: 99%

Using protein backbone mutagenesis to dissect the link between ion occupancy and C-type inactivation in K ⁺ channels

Matulef

Komarov

Costantino

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Akin to molecules, ground-state soliton molecules have low energies, consisting of bound solitons with the shortest separation, while excited-state soliton molecules possess higher energies, corresponding to bound solitons with larger separations. [24] Experimentally, ground-and excited-soliton molecules were observed under different settings of the polarization controllers in the laser. Firstly, we obtained a ground-state soliton molecule consisting of double solitons separated by 1.8 ps that was the shortest separation obtained in the experiments and then measured its formation dynamics.…”

Section: Ground-state Soliton Moleculementioning

confidence: 99%

Build‐Up of Dissipative Optical Soliton Molecules via Diverse Soliton Interactions

Peng

Zeng

2018

Laser & Photonics Reviews

149

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Solitons can form bound states that are frequently referred to as soliton molecules as they exhibit molecule‐like dynamics. The build‐up phase of the optical soliton molecule remains elusive. Here, by means of a time‐stretch technique that enables real‐time access to the spectral and temporal dynamics, rich nonlinear processes involved in the build‐up of soliton molecules are revealed in an ultrafast fibre laser. Specifically, the formation of closely‐ and well‐separated bound solitons are resolved. In both cases, the build‐up phases consist of three nonlinear stages including mode locking, soliton splitting, and soliton interactions. For closely‐separated bound solitons, soliton interactions display wide diversities in repeated measurements, including soliton attraction, repelling, collision, vibration, and annihilation. For well‐separated bound solitons, repulsive interactions dominate the soliton interactions. Numerical simulations corroborate these experimental observations. Furthermore, a conceptually different soliton molecule, the intermittent‐vibration soliton molecule, is discovered and characterized. It is the intermediate state between the vibrational and stationary soliton molecules. The author's findings could assist in the understanding of the build‐up phase of localized structures in different dissipative systems.

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“…A sandwich fusion strategy was used for expression of the Glt Ph 1–384 (N-peptide) thioester . The fusion protein consisted of Glt Ph residues 1–384 sandwiched between glutathione S -transferase (GST) at the N-terminus and the gyrA intein–chitin binding domain at the C-terminus.…”

Section: Experimental Proceduresmentioning

confidence: 99%

Engineering the Glutamate Transporter Homologue Glt_Ph Using Protein Semisynthesis

2015

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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 GltPh, an archaeal homolog of glutamate transporters. Semisynthesis enables the precise engineering of GltPh through the incorporation of unnatural amino acids and peptide backbone modifications. In the semisynthesis, the GltPh 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 GltPh. Biochemical characterization of the semisynthetic GltPh 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 analog, citrulline. Our studies demonstrate that Arg397 is required for high affinity substrate binding and based on our results we propose that Arg397 is involved in a Na+- 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 GltPh. Further, the approach developed in this study should also be applicable to other membrane transport proteins.

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Chapter 7 Semisynthesis of K+ Channels

Cited by 5 publications

References 33 publications

Using protein backbone mutagenesis to dissect the link between ion occupancy and C-type inactivation in K ⁺ channels

Using protein backbone mutagenesis to dissect the link between ion occupancy and C-type inactivation in K ⁺ channels

Build‐Up of Dissipative Optical Soliton Molecules via Diverse Soliton Interactions

Engineering the Glutamate Transporter Homologue Glt_Ph Using Protein Semisynthesis

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Chapter 7 Semisynthesis of K+ Channels

Cited by 5 publications

References 33 publications

Using protein backbone mutagenesis to dissect the link between ion occupancy and C-type inactivation in K + channels

Using protein backbone mutagenesis to dissect the link between ion occupancy and C-type inactivation in K + channels

Build‐Up of Dissipative Optical Soliton Molecules via Diverse Soliton Interactions

Engineering the Glutamate Transporter Homologue GltPh Using Protein Semisynthesis

Contact Info

Product

Resources

About

Using protein backbone mutagenesis to dissect the link between ion occupancy and C-type inactivation in K ⁺ channels

Using protein backbone mutagenesis to dissect the link between ion occupancy and C-type inactivation in K ⁺ channels

Engineering the Glutamate Transporter Homologue Glt_Ph Using Protein Semisynthesis