Differential Enzyme Flexibility Probed Using Solid-State Nanopores

doi:10.1021/acsnano.8b00734

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Differential Enzyme Flexibility Probed Using Solid-State Nanopores

2018-04-09

Eugene I. Shakhnovich

赵

赵清 (Qing Zhao)

Meni Wanunu (Meni Wanunu) *

DOI：10.1021/acsnano.8b00734

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Abstract

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Enzymes and motor proteins are dynamic macromolecules that coexist in a number of conformations of similar energies. Protein function is usually accompanied by a change in structure and flexibility, often induced upon binding to ligands. However, while measuring protein flexibility changes between active and resting states is of therapeutic significance, it remains a challenge. Recently, our group has demonstrated that breadth of signal amplitudes in measured electrical signatures as an ensemble of individual protein molecules is driven through solid-state nanopores and correlates with protein conformational dynamics. Here, we extend our study to resolve subtle flexibility variation in dihydrofolate reductase mutants from unlabeled single molecules in solution. We first demonstrate using a canonical protein system, adenylate kinase, that both size and flexibility changes can be observed upon binding to a substrate that locks the protein in a closed conformation. Next, we investigate the influence of voltage bias and pore geometry on the measured electrical pulse statistics during protein transport. Finally, using the optimal experimental conditions, we systematically study a series of wild-type and mutant dihydrofolate reductase proteins, finding a good correlation between nanopore-measured protein conformational dynamics and equilibrium bulk fluorescence probe measurements. Our results unequivocally demonstrate that nanopore-based measurements reliably probe conformational diversity in native protein ensembles.

Keywords:

protein flexibility

solid-state nanopore

bis-ANS

DHFR

adenylate kinase

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