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Forster resonance energy transfer as a probe of membrane protein folding.

ABSTRACT: The folding reaction of a ?-barrel membrane protein, outer membrane protein A (OmpA), is probed with Förster resonance energy transfer (FRET) experiments. Four mutants of OmpA were generated in which the donor fluorophore, tryptophan, and acceptor molecule, a naphthalene derivative, are placed in various locations on the protein to report the evolution of distances across the bilayer and across the protein pore during a folding event. Analysis of the FRET efficiencies reveals three timescales for tertiary structure changes associated with insertion and folding into a synthetic bilayer. A narrow pore forms during the initial stage of insertion, followed by bilayer traversal. Finally, a long-time component is attributed to equilibration and relaxation, and may involve global changes such as pore expansion and strand extension. These results augment the existing models that describe concerted insertion and folding events, and highlight the ability of FRET to provide insight into the complex mechanisms of membrane protein folding. This article is part of a Special Issue entitled: Membrane protein structure and function.

SUBMITTER: Kang G

PROVIDER: S-EPMC3253952 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Förster resonance energy transfer as a probe of membrane protein folding.

Kang Guipeun G López-Peña Ignacio I Oklejas Vanessa V Gary Cyril S CS Cao Weihan W Kim Judy E JE

Biochimica et biophysica acta 20110907 2

The folding reaction of a β-barrel membrane protein, outer membrane protein A (OmpA), is probed with Förster resonance energy transfer (FRET) experiments. Four mutants of OmpA were generated in which the donor fluorophore, tryptophan, and acceptor molecule, a naphthalene derivative, are placed in various locations on the protein to report the evolution of distances across the bilayer and across the protein pore during a folding event. Analysis of the FRET efficiencies reveals three timescales fo ...[more]

PMID: 21925139

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Project description:A protein-protein Förster resonance energy transfer (FRET) system, employing probes at multiple positions, was designed to specifically monitor the dissociation of the H2A-H2B dimer from the nucleosome core particle (NCP). Tryptophan donors and Cys-AEDANS acceptors were chosen because, compared to previous NCP FRET fluorophores, they: (1) are smaller and less hydrophobic, which should minimize perturbations of histone and NCP structure; and (2) have an R0 of 20 A, which is much less than the dimensions of the NCP (approximately 50 A width and approximately 100 A diameter). Equilibrium protein unfolding titrations indicate that the donor and acceptor moieties have minimal effects on the stability of the H2A-H2B dimer and (H3-H4)2 tetramer. NCPs containing the various FRET pairs were reconstituted with the 601 DNA positioning element. Equilibrium NaCl-induced dissociation of the modified NCPs showed that the 601 sequence stabilized the NCP to dimer dissociation relative to weaker positioning sequences. This finding implies a significant role for the H2A-H2B dimers in determining the DNA sequence dependence of NCP stability. The free energy of dissociation determined from reversible and well-defined sigmoidal transitions revealed two distinct phases reflecting the dissociation of individual H2A-H2B dimers, confirming cooperativity as suggested previously; these data allow quantitative description of the cooperativity. The FRET system was then used to study the effects of the histone variant H2A.Z on NCP stability; previous studies have reported both destabilizing and stabilizing effects. H2A.Z FRET NCP dissociation transitions suggest a slight increase in stability but a significant increase in cooperativity of the dimer dissociations. Thus, the utility of this protein-protein FRET system to monitor the effects of histone variants on NCP dynamics has been demonstrated, and the system appears equally well-suited for dissection of the kinetic processes of dimer association and dissociation from the NCP.

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Forster resonance energy transfer as a probe of membrane protein folding.

Publications

Förster resonance energy transfer as a probe of membrane protein folding.

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