Site-directed mutagenesis within the central constriction site of ScrY (sucroseporin): Effect on ion transport and comparison of maltooligosaccharide binding to LamB of Escherichia coli

B. H. Kim, C. Andersen, Jens Kreth, C. Ulmke, K. Schmid, R. Benz

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

The 3-D structures of the maltooligosaccharide-specific LamB-channel of Escherichia coli (also called maltoporin) and sucrose-specific ScrY (sucroseporin) are known from X-ray crystallography. The central constriction of the channels formed by the external loop 3 is controlled by a number of different amino acids. The most prominent one of these, N192, D201 and F204, were replaced by site-directed mutagenesis into those of LamB, which, according to the 3-D model of both channels are localized at similar places. The ScrY single mutants ScrYN192R, ScrYD201Y and ScrYF204D and the ScrY triple mutant ScrY3113 (N192R + D201Y + F204D) were created together with the triple mutant ScrY3213, which lacks also amino acids 1 to 61 from the N-terminal end. The mutant proteins were purified to homogeneity and were reconstituted into lipid bilayer membranes. In these experiments, the single-channel conductance of the mutants in different salt solutions and the stability constants for binding of different maltooligosaccharides to the mutant channels was measured using titration experiments with carbohydrates. The carbohydrate-induced block of the channel function could also be used for the study of current noise through the different mutant ScrY-channels. The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants (k1 and k-1) of carbohydrate-binding to the binding site inside the channels. The results suggest that both on- and off-rate constants were affected by the mutations. Most of them showed a substantial effect on carbohydrate binding kinetics. Nevertheless, single-channel conductance and carbohydrate binding of ScrY3113 mutant were still different from that of LamB, suggesting that not only the amino acids of the central constriction but also the general architecture of both channels have a substantial influence on channel properties.

Original languageEnglish (US)
Pages (from-to)239-253
Number of pages15
JournalJournal of Membrane Biology
Volume187
Issue number3
DOIs
StatePublished - Jun 1 2002
Externally publishedYes

Fingerprint

Ion Transport
Site-Directed Mutagenesis
Constriction
Carbohydrates
Escherichia coli
Amino Acids
X Ray Crystallography
Lipid Bilayers
Mutant Proteins
Sucrose
Noise
Salts
Binding Sites
maltooligosaccharides
Mutation
Membranes

Keywords

  • Carbohydrate binding
  • LamB
  • Lipid bilayer membrane
  • Noise analysis
  • ScrY
  • Sucrose transport

ASJC Scopus subject areas

  • Biophysics
  • Physiology
  • Cell Biology

Cite this

Site-directed mutagenesis within the central constriction site of ScrY (sucroseporin) : Effect on ion transport and comparison of maltooligosaccharide binding to LamB of Escherichia coli. / Kim, B. H.; Andersen, C.; Kreth, Jens; Ulmke, C.; Schmid, K.; Benz, R.

In: Journal of Membrane Biology, Vol. 187, No. 3, 01.06.2002, p. 239-253.

Research output: Contribution to journalArticle

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abstract = "The 3-D structures of the maltooligosaccharide-specific LamB-channel of Escherichia coli (also called maltoporin) and sucrose-specific ScrY (sucroseporin) are known from X-ray crystallography. The central constriction of the channels formed by the external loop 3 is controlled by a number of different amino acids. The most prominent one of these, N192, D201 and F204, were replaced by site-directed mutagenesis into those of LamB, which, according to the 3-D model of both channels are localized at similar places. The ScrY single mutants ScrYN192R, ScrYD201Y and ScrYF204D and the ScrY triple mutant ScrY3113 (N192R + D201Y + F204D) were created together with the triple mutant ScrY3213, which lacks also amino acids 1 to 61 from the N-terminal end. The mutant proteins were purified to homogeneity and were reconstituted into lipid bilayer membranes. In these experiments, the single-channel conductance of the mutants in different salt solutions and the stability constants for binding of different maltooligosaccharides to the mutant channels was measured using titration experiments with carbohydrates. The carbohydrate-induced block of the channel function could also be used for the study of current noise through the different mutant ScrY-channels. The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants (k1 and k-1) of carbohydrate-binding to the binding site inside the channels. The results suggest that both on- and off-rate constants were affected by the mutations. Most of them showed a substantial effect on carbohydrate binding kinetics. Nevertheless, single-channel conductance and carbohydrate binding of ScrY3113 mutant were still different from that of LamB, suggesting that not only the amino acids of the central constriction but also the general architecture of both channels have a substantial influence on channel properties.",
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AU - Kim, B. H.

AU - Andersen, C.

AU - Kreth, Jens

AU - Ulmke, C.

AU - Schmid, K.

AU - Benz, R.

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AB - The 3-D structures of the maltooligosaccharide-specific LamB-channel of Escherichia coli (also called maltoporin) and sucrose-specific ScrY (sucroseporin) are known from X-ray crystallography. The central constriction of the channels formed by the external loop 3 is controlled by a number of different amino acids. The most prominent one of these, N192, D201 and F204, were replaced by site-directed mutagenesis into those of LamB, which, according to the 3-D model of both channels are localized at similar places. The ScrY single mutants ScrYN192R, ScrYD201Y and ScrYF204D and the ScrY triple mutant ScrY3113 (N192R + D201Y + F204D) were created together with the triple mutant ScrY3213, which lacks also amino acids 1 to 61 from the N-terminal end. The mutant proteins were purified to homogeneity and were reconstituted into lipid bilayer membranes. In these experiments, the single-channel conductance of the mutants in different salt solutions and the stability constants for binding of different maltooligosaccharides to the mutant channels was measured using titration experiments with carbohydrates. The carbohydrate-induced block of the channel function could also be used for the study of current noise through the different mutant ScrY-channels. The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants (k1 and k-1) of carbohydrate-binding to the binding site inside the channels. The results suggest that both on- and off-rate constants were affected by the mutations. Most of them showed a substantial effect on carbohydrate binding kinetics. Nevertheless, single-channel conductance and carbohydrate binding of ScrY3113 mutant were still different from that of LamB, suggesting that not only the amino acids of the central constriction but also the general architecture of both channels have a substantial influence on channel properties.

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