Affinity electrophoresis: Principles and applications

Modular glycoside hydrolases that attack recalcitrant polymers generally contain noncatalytic carbohydratebinding modules (CBMs), which play a critical role in the action of these enzymes by localizing the appended catalytic domains onto the surface of insoluble polysaccharide substrates. Type B CBMs, which recognize single polysaccharide chains, display ligand specificities that are consistent with the substrates hydrolyzed by the associated catalytic domains. In enzymes that contain multiple catalytic domains with distinct substrate specificities, it is unclear how these different activities influence the evolution of the ligand recognition profile of the appended CBM. To address this issue, we have characterized the properties of a family 11 CBM (CtCBM11) in Clostridium thermocellum Lic26A-Cel5E, an enzyme that contains GH5 and GH26 catalytic domains that display ␤-1,4-and ␤-1,3-1,4-mixed linked endoglucanase activity, respectively. Here we show that CtCBM11 binds to both ␤-1,4-

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“…The nonbinding negative control was bovine serum albumin. Quantitative assessment of binding was carried out as described previously (21).…”

Section: Methodsmentioning

confidence: 99%

The Family 11 Carbohydrate-binding Module of Clostridium thermocellum Lic26A-Cel5E Accommodates β-1,4- and β-1,3–1,4-Mixed Linked Glucans at a Single Binding Site

Carvalho

Goyal²,

Prates³

et al. 2004

Journal of Biological Chemistry

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“…The method was carried out as described by Charnock et al (13) except that gels were subjected to electrophoresis for 3 h. AGE was also carried out in the presence of a range of oat spelt xylan concentrations (0.1 to 0.005 mg ml Ϫ1 ), and affinity constants for the interaction of native and mutant forms of CBM6 with ligand were determined using equations derived from Takeo (19). Crystallization-Crystals of CBM6 were grown by vapor phase diffusion using the hanging drop method.…”

Section: Methodsmentioning

confidence: 99%

The Location of the Ligand-binding Site of Carbohydrate-binding Modules That Have Evolved from a Common Sequence Is Not Conserved

Czjzek

Bolam

Mosbah

et al. 2001

Journal of Biological Chemistry

108

113

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Polysaccharide-degrading enzymes are generally modular proteins that contain non-catalytic carbohydrate-binding modules (CBMs), which potentiate the activity of the catalytic module. CBMs have been grouped into sequence-based families, and three-dimensional structural data are available for half of these families. Clostridium thermocellum xylanase 11A is a modular enzyme that contains a CBM from family 6 (CBM6), for which no structural data are available. We have determined the crystal structure of this module to a resolution of 2.1 Å. The protein is a ␤-sandwich that contains two potential ligand-binding clefts designated cleft A and B. The CBM interacts primarily with xylan, and NMR spectroscopy coupled with site-directed mutagenesis identified cleft A, containing Trp-92, Tyr-34, and Asn-120, as the ligand-binding site. The overall fold of CBM6 is similar to proteins in CBM families 4 and 22, although surprisingly the ligand-binding site in CBM4 and CBM22 is equivalent to cleft B in CBM6. These structural data define a superfamily of CBMs, comprising CBM4, CBM6, and CBM22, and demonstrate that, although CBMs have evolved from a relatively small number of ancestors, the structural elements involved in ligand recognition have been assembled at different locations on the ancestral scaffold.

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“…Bovine serum albumin was used as the non-specific binding fi negative control. Quantitative assessment of ligand binding characteristics of CtCBM11 and its mutants using affinity fi electrophoresis was carried out as described previously 19 .…”

Section: Construction Of Mutants Of Ctcbm11 By Site-directed Mutagenesismentioning

confidence: 99%

“…4). The affi nities of CBM11 and its mutants for ligands fi were quantifi ed and the association constants ( fi K ( ( a ) were determined by using an equation described elsewhere 19 . Fig.…”

Section: Carboxymethyl Cellulose +mentioning

confidence: 99%

“…1C shows a sample plot of 1/r versus lichenan concentration, where r is the relative migration distance of proteins in presence of affi nity ligand in the gel. The fi values of Ka were calculated by extrapolating the lines and taking inverse of value of intercept on x-axis of the plot as described by Takeo 19 . Table 3 lists the Ka values of CtCBM11 and mutants with soluble substrates used.…”

Section: Carboxymethyl Cellulose +mentioning

confidence: 99%

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Functional characterization and mutation analysis of family 11, Carbohydrate-Binding Module (CtCBM11) of cellulosomal bifunctional cellulase from Clostridium thermocellum

et al. 2007

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The non-catalytic, family 11 carbohydrate binding module (CtCBM11) belonging to a bifunctional cellulosomal cellulase from Clostridium thermocellum was hyperexpressed in E. coli and functionally characterized. Affi nity fi electrophoresis of CtCBM11 on nondenaturing PAGE containing cellulosic polysaccharides showed binding with β-glucan, lichenan, hydroxyethyl cellulose and carboxymethyl cellulose. In order to elucidate the involvement of conserved aromatic residues Tyr 22, Trp 65 and Tyr 129 in the polysaccharide binding, site-directed mutagenesis was carried out and the residues were changed to alanine. The results of affi nity electrophoresis and binding adsorption fi isotherms showed that of the three mutants Y22A, W65A and Y129A of CtCBM11, two mutants Y22A and Y129A showed no or reduced binding affi nity with polysaccharides. fi These results showed that tyrosine residue 22 and 129 are involved in the polysaccharide binding. These residues are present in the putative binding cleft and play a critical role in the recognition of all the ligands recognized by the protein.

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Affinity electrophoresis: Principles and applications

Cited by 76 publications

References 53 publications

The Family 11 Carbohydrate-binding Module of Clostridium thermocellum Lic26A-Cel5E Accommodates β-1,4- and β-1,3–1,4-Mixed Linked Glucans at a Single Binding Site

The Family 11 Carbohydrate-binding Module of Clostridium thermocellum Lic26A-Cel5E Accommodates β-1,4- and β-1,3–1,4-Mixed Linked Glucans at a Single Binding Site

The Location of the Ligand-binding Site of Carbohydrate-binding Modules That Have Evolved from a Common Sequence Is Not Conserved

Functional characterization and mutation analysis of family 11, Carbohydrate-Binding Module (CtCBM11) of cellulosomal bifunctional cellulase from Clostridium thermocellum

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