Folding of the β-Barrel Membrane Protein OmpA into Nanodiscs

Dee Ann K. Asamoto, Guipeun Kang, Judy E. Kim

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


Nanodiscs (NDs) are an excellent alternative to small unilamellar vesicles (SUVs) for studies of membrane protein structure, but it has not yet been shown that membrane proteins are able to spontaneously fold and insert into a solution of freely diffusing NDs. In this article, we present SDS-PAGE differential mobility studies combined with fluorescence, circular dichroism, and ultraviolet resonance Raman spectroscopy to confirm the spontaneous folding of outer membrane protein A (OmpA) into preformed NDs. Folded OmpA in NDs was incubated with Arg-C protease, resulting in the digestion of OmpA to membrane-protected fragments with an apparent molecular mass of ∼26 kDa (major component) and ∼24 kDa (minor component). The OmpA folding yields were greater than 88% in both NDs and SUVs. An OmpA adsorbed intermediate on NDs could be isolated at low temperature and induced to fold via an increase in temperature, analogous to the temperature-jump experiments on SUVs. The circular dichroism spectra of OmpA in NDs and SUVs were similar and indicated β-barrel secondary structure. Further evidence of OmpA folding into NDs was provided by ultraviolet resonance Raman spectroscopy, which revealed the intense 785 cm−1 structural marker for folded OmpA in NDs. The primary difference between folding in NDs and SUVs was the kinetics; the rate of folding was two- to threefold slower in NDs compared to in SUVs, and this decreased rate can tentatively be attributed to the properties of NDs. These data indicate that NDs may be an excellent alternative to SUVs for folding experiments and offer benefits of optical clarity, sample homogeneity, control of ND:protein ratios, and greater stability.

Original languageEnglish (US)
Pages (from-to)403-414
Number of pages12
JournalBiophysical journal
Issue number2
StatePublished - Jan 21 2020
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics


Dive into the research topics of 'Folding of the β-Barrel Membrane Protein OmpA into Nanodiscs'. Together they form a unique fingerprint.

Cite this