Global redesign of a native β-barrel scaffold

Wolfe, Aaron; Mohammad, Mohammad M.; Thakur, Avinash; Movileanu, Liviu

doi:10.1016/j.bbamem.2015.10.006

Cited by 15 publications

(34 citation statements)

References 62 publications

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“…This is because in general membrane proteins have multiple tryptophan residues exposed to their hydrophobic interface. 24 We judge that it is not very convenient to use intrinsic tryptophan fluorescence for the FP-based spectroscopy studies for a number of reasons. They include complex contributions of individual-residue tryptophan spectra to the overall FP spectrum of the protein as well as rapid tryptophan quenching, because the indole nucleus is prone to electron donation during the excitation state.…”

Section: Discussionmentioning

confidence: 99%

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Quantification of Membrane Protein-Detergent Complex Interactions

Wolfe

Zhang

et al. 2017

J. Phys. Chem. B

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Although fundamentally significant in structural, chemical, and membrane biology, the interfacial protein-detergent complex (PDC) interactions have been modestly examined because of the complicated behavior of both detergents and membrane proteins in aqueous phase. Membrane proteins are prone to unproductive aggregation resulting from poor detergent solvation, but the participating forces in this phenomenon remain ambiguous. Here, we show that using rational membrane protein design, targeted chemical modification, and steady-state fluorescence polarization spectroscopy, the detergent desolvation of membrane proteins can be quantitatively evaluated. We demonstrate that depleting the detergent in the sample well produced a two-state transition of membrane proteins between a fully detergent-solvated state and a detergent-desolvated state, the nature of which depended on the interfacial PDC interactions. Using a panel of six membrane proteins of varying hydrophobic topography, structural fingerprint, and charge distribution on the solvent-accessible surface, we provide direct experimental evidence for the contributions of the electrostatic and hydrophobic interactions to the protein solvation properties. Moreover, all-atom molecular dynamics simulations report the major contribution of the hydrophobic forces exerted at the PDC interface. This semi-quantitative approach might be extended in the future to include studies of the interfacial PDC interactions of other challenging membrane protein systems of unknown structure. This would have practical importance in protein extraction, solubilization, stabilization, and crystallization.

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Section: Discussionmentioning

confidence: 99%

“…23–24 fhua Δc/Δ5l_t7 was created by inverse PCR using pPR-IBA1- fhua Δc/ Δ 5l-6×His + plasmid as a template. The PCR product was self-ligated to create pPR-IBA1- fhua Δc/ Δ 5l_t7-6×His +.…”

Section: Methodsmentioning

confidence: 99%

Quantification of Membrane Protein-Detergent Complex Interactions

Wolfe

Zhang

et al. 2017

J. Phys. Chem. B

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“…Neither did deletion of the plug give similar significant increase in high conductance channels, nor did pruning the loop do the same without pruning strand 8 [37]. Ultimately for FhuA, the more extreme the loop-pruning, the higher the conductance [38,39]. However, as would be expected, it has also been shown that in some proteins deletion of the plug does in fact lead to higher conductance.…”

Section: Outer Membrane Protein Designmentioning

confidence: 99%

“…Replacing positive residues with negative residues at the constriction site of P. denitrificans porin was also effective in creating cation selectivity [47]. A similar approach was also attempted by neutralizing the negatively charged residues on the loops of FhuA [39]. …”

Section: Outer Membrane Protein Designmentioning

confidence: 99%

Outer membrane protein design

Slusky

2017

Current Opinion in Structural Biology

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Membrane proteins are the gateway to the cell. These proteins are also a control center of the cell as information from the outside is passed through membrane protein signaling networks to the cellular machinery. The design of membrane proteins seeks to harness the power of these gateways and signaling networks. This review will focus on the design of the membrane proteins that are in the outer membrane, a membrane which only exists for gram negative bacteria, mitochondria and chloroplasts. Unlike other membrane proteins, outer membrane proteins are uniquely shaped as β-barrels. Herein, I describe most known examples of outer-membrane, β-barrel design to date, focusing particularly on categorizing designs as 1) structural deconstruction, 2) structural changes, 3) chemical function design, and 4) the creation of new folds.

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“…We employed FhuA ΔC/Δ5L, an extensive truncation mutant (Fig. 1), 24 so that FhuA was converted from a non-ion conducting transmembrane protein 25 to a large-conductance nanopore. Therefore, this redesigned protein nanopore enabled parallel inspections of the anisotropy readout of detergent-refolded FhuA in solution and its ion transport activity, as judged by single-molecule electrophysiology in planar lipid membranes.…”

Section: Introductionmentioning

confidence: 99%

Interrogating Detergent Desolvation of Nanopore-Forming Proteins by Fluorescence Polarization Spectroscopy

et al. 2017

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Understanding how membrane proteins interact with detergents is of fundamental and practical significance in structural and chemical biology as well as in nanobiotechnology. Current methods for inspecting protein-detergent complex (PDC) interfaces require high concentrations of protein and are of low throughput. Here, we describe a scalable, spectroscopic approach that uses nanomolar protein concentrations in native solutions. This approach, which is based on steady-state fluorescence polarization (FP) spectroscopy, kinetically resolves the dissociation of detergents from membrane proteins and protein unfolding. For satisfactorily solubilizing detergents, at concentrations much greater than the critical micelle concentration (CMC), the fluorescence anisotropy was independent of detergent concentration. In contrast, at detergent concentrations comparable with or below the CMC, the anisotropy readout underwent a time-dependent decrease, showing a specific and sensitive protein unfolding signature. Functionally reconstituted membrane proteins into a bilayer membrane confirmed predictions made by these FP-based determinations with respect to varying refolding conditions. From a practical point of view, this 96-well analytical approach will facilitate a massively parallel assessment of the PDC interfacial interactions under a fairly broad range of micellar and environmental conditions. We expect that these studies will potentially accelerate research in membrane proteins pertaining to their extraction, solubilization, stabilization, and crystallization, as well as reconstitution into bilayer membranes.

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Global redesign of a native β-barrel scaffold

Cited by 15 publications

References 62 publications

Quantification of Membrane Protein-Detergent Complex Interactions

Quantification of Membrane Protein-Detergent Complex Interactions

Outer membrane protein design

Interrogating Detergent Desolvation of Nanopore-Forming Proteins by Fluorescence Polarization Spectroscopy

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