Fourier transform infrared analysis of purified lactose permease: A monodisperse lactose permease preparation is stably folded, α-helical, and highly accessible to deuterium exchange

Jason S. Patzlaff, Jeffrey A. Moeller, Bridgette A. Barry, Robert J Brooker

Research output: Contribution to journalArticle

60 Citations (Scopus)

Abstract

The lactose permease, encoded by the lacY gene of Escherichia coli, is an integral membrane protein that functions as a proton and lactose symporter. In this study, we have characterized a novel monodisperse, purified preparation of lactose permease, as well as functionally reconstituted lactose permease, using spectroscopic techniques. The purification of monodisperse lactose permease has been aided by the development of a lacY gene product containing an amino-terminal six histidine affinity tag. In the novel purification method described here, lactose permease is purified from β-dodecyl maltoside-solubilized membrane vesicles using three sequential column steps: hydroxyapatite, nickel-nitriloacetic acid (Ni-NTA) affinity, and cation-exchange chromatography. The hydroxyapatite step was shown to be essential in reducing aggregation of the final purified protein. Amino acid composition analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis support the conclusion that the protein has been purified to greater than 90% homogeneity. The protein has been successfully reconstituted and has been shown to be active for lactose transport. Fourier transform infrared (FT-IR) spectroscopy has been performed on monodisperse lactose permease and on proteoliposomes containing functional lactose permease. FT-IR spectroscopy supports the conclusion that the monodisperse lactose permease preparation is 80% α-helical and stably folded at 20 °C; thermal denaturation is first detected at 70 °C. Because the purified protein is also readily susceptible to 2H exchange, these results suggest that the protein is conformationally flexible and that 2H exchange is facilitated as the result of conformational fluctuations from the folded state.

Original languageEnglish (US)
Pages (from-to)15363-15375
Number of pages13
JournalBiochemistry
Volume37
Issue number44
DOIs
StatePublished - Nov 3 1998

Fingerprint

Deuterium
Fourier Analysis
Fourier transforms
Infrared radiation
Fourier Transform Infrared Spectroscopy
Durapatite
Lactose
Proteins
Purification
Genes
Symporters
Denaturation
lactose permease
Active Biological Transport
Chromatography
Nickel
Electrophoresis
Histidine
Sodium Dodecyl Sulfate
Escherichia coli

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Fourier transform infrared analysis of purified lactose permease : A monodisperse lactose permease preparation is stably folded, α-helical, and highly accessible to deuterium exchange. / Patzlaff, Jason S.; Moeller, Jeffrey A.; Barry, Bridgette A.; Brooker, Robert J.

In: Biochemistry, Vol. 37, No. 44, 03.11.1998, p. 15363-15375.

Research output: Contribution to journalArticle

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abstract = "The lactose permease, encoded by the lacY gene of Escherichia coli, is an integral membrane protein that functions as a proton and lactose symporter. In this study, we have characterized a novel monodisperse, purified preparation of lactose permease, as well as functionally reconstituted lactose permease, using spectroscopic techniques. The purification of monodisperse lactose permease has been aided by the development of a lacY gene product containing an amino-terminal six histidine affinity tag. In the novel purification method described here, lactose permease is purified from β-dodecyl maltoside-solubilized membrane vesicles using three sequential column steps: hydroxyapatite, nickel-nitriloacetic acid (Ni-NTA) affinity, and cation-exchange chromatography. The hydroxyapatite step was shown to be essential in reducing aggregation of the final purified protein. Amino acid composition analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis support the conclusion that the protein has been purified to greater than 90{\%} homogeneity. The protein has been successfully reconstituted and has been shown to be active for lactose transport. Fourier transform infrared (FT-IR) spectroscopy has been performed on monodisperse lactose permease and on proteoliposomes containing functional lactose permease. FT-IR spectroscopy supports the conclusion that the monodisperse lactose permease preparation is 80{\%} α-helical and stably folded at 20 °C; thermal denaturation is first detected at 70 °C. Because the purified protein is also readily susceptible to 2H exchange, these results suggest that the protein is conformationally flexible and that 2H exchange is facilitated as the result of conformational fluctuations from the folded state.",
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