Structural and functional studies of fungal cellulose-binding domains by NMR spectroscopy

Dissertation

Maija-Liisa Mattinen

Research output: ThesisDissertationCollection of Articles

Abstract

Cellulose has a important environmental role in the preservation of the global carbon cycle and commercial significance as a raw material for industry. To understand the biodegradation of cellulose on the atomic level, it is important to be able to relate enzyme activities with the three-dimensional (3D) structures of cellulases and cellulose. The cellulolytic system of the fungus Trichoderma reesei is one of the best understood of all cellulolytic systems. Most cellulases from T. reesei have a cellulose-binding domain (CBD) specialised for binding to cellulose. As a means to understanding the interactions between cellulose and CBDs of cellobiohydrolase I (CBHI) and endoglucanase I (EGI) from T. reesei, 3D structures of these domains were studied by two-dimensional (2D) 1H NMR (nuclear magnetic resonance) techniques. Structural effects of mutations of conserved amino acids in CBDCBHI, Y5A, P16R, N29A, Y31A, Y32A and Q34A, were evaluated by comparing chemical shifts, coupling constants and NOEs of the backbone protons of the mutants and wild-type CBDCBHI. In general, the substitutions did not alter significantly the secondary structures of these engineered peptides. The adsorption experiments on cellulose showed that Y5A, Y31A and Y32A had lost nearly all their affinity to cellulose. For Y31A and Y32A the 3D structures revealed small local changes around the mutation on the flat face of CBD, which was expected to bind to cellulose. Therefore the structural roles of Tyr-31 and Tyr-32 must be minor, but their functional importance is clear since the mutants lacking these residues did not bind strongly to cellulose. In the case of Y5A the disruption of the structural framework at the N-terminus and the complete loss of affinity to cellulose implied that Tyr-5 has both structural and functional significance. The 3D structure of a synthetic CBDEGI was also determined by NMR spectroscopy. The structure was very similar to that of wild-type CBDCBHI. Within the precision of the structures, even the cellulose-binding face of CBDEGI was similar to that of CBDCBHI, apart from the place of attachment of the different side-chain. The determined NMR structure was also in agreement with an earlier modelled structure of CBDEGI. Finally, soluble cello-oligosaccharides were used as model compounds for cellobiose chains to investigate the interaction between CBD and cellulose by NMR spectroscopy. CBDs caused line broadening effects and decreasing T2 relaxation times for certain sugar resonances, whereas there were no effects in the presence of a mutant that bound weakly to cellulose. Experiments showed that the interactions between CBD and cellobiose units of sugars are specific, supporting the model presented for the CBD binding to crystalline cellulose. It remained uncertain, however, how well the cello-oligosaccharides mimicked the binding of CBD to cellulose.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • University of Helsinki
Supervisors/Advisors
  • Riekkola, Marja-Liisa, Supervisor, External person
Award date13 May 1998
Place of PublicationEspoo
Publisher
Print ISBNs951-38-5225-3
Electronic ISBNs951-38-5226-1
Publication statusPublished - 1998
MoE publication typeG5 Doctoral dissertation (article)

Fingerprint

nuclear magnetic resonance spectroscopy
cellulose
Trichoderma reesei
cellobiose
cellulases
mutants
manmade structures
cellulose 1,4-beta-cellobiosidase
sugars
mutation
carbohydrate binding
biodegradation
endo-1,4-beta-glucanase
oligosaccharides
protons

Keywords

  • cellulose
  • cellulase
  • cellobiohydrolase
  • endoglucanase
  • cellulose-binding domain
  • fungi
  • NMR spectroscopy
  • Trichoderma reesei
  • biopolymers

Cite this

Mattinen, M-L. (1998). Structural and functional studies of fungal cellulose-binding domains by NMR spectroscopy: Dissertation. Espoo: VTT Technical Research Centre of Finland.
Mattinen, Maija-Liisa. / Structural and functional studies of fungal cellulose-binding domains by NMR spectroscopy : Dissertation. Espoo : VTT Technical Research Centre of Finland, 1998. 72 p.
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abstract = "Cellulose has a important environmental role in the preservation of the global carbon cycle and commercial significance as a raw material for industry. To understand the biodegradation of cellulose on the atomic level, it is important to be able to relate enzyme activities with the three-dimensional (3D) structures of cellulases and cellulose. The cellulolytic system of the fungus Trichoderma reesei is one of the best understood of all cellulolytic systems. Most cellulases from T. reesei have a cellulose-binding domain (CBD) specialised for binding to cellulose. As a means to understanding the interactions between cellulose and CBDs of cellobiohydrolase I (CBHI) and endoglucanase I (EGI) from T. reesei, 3D structures of these domains were studied by two-dimensional (2D) 1H NMR (nuclear magnetic resonance) techniques. Structural effects of mutations of conserved amino acids in CBDCBHI, Y5A, P16R, N29A, Y31A, Y32A and Q34A, were evaluated by comparing chemical shifts, coupling constants and NOEs of the backbone protons of the mutants and wild-type CBDCBHI. In general, the substitutions did not alter significantly the secondary structures of these engineered peptides. The adsorption experiments on cellulose showed that Y5A, Y31A and Y32A had lost nearly all their affinity to cellulose. For Y31A and Y32A the 3D structures revealed small local changes around the mutation on the flat face of CBD, which was expected to bind to cellulose. Therefore the structural roles of Tyr-31 and Tyr-32 must be minor, but their functional importance is clear since the mutants lacking these residues did not bind strongly to cellulose. In the case of Y5A the disruption of the structural framework at the N-terminus and the complete loss of affinity to cellulose implied that Tyr-5 has both structural and functional significance. The 3D structure of a synthetic CBDEGI was also determined by NMR spectroscopy. The structure was very similar to that of wild-type CBDCBHI. Within the precision of the structures, even the cellulose-binding face of CBDEGI was similar to that of CBDCBHI, apart from the place of attachment of the different side-chain. The determined NMR structure was also in agreement with an earlier modelled structure of CBDEGI. Finally, soluble cello-oligosaccharides were used as model compounds for cellobiose chains to investigate the interaction between CBD and cellulose by NMR spectroscopy. CBDs caused line broadening effects and decreasing T2 relaxation times for certain sugar resonances, whereas there were no effects in the presence of a mutant that bound weakly to cellulose. Experiments showed that the interactions between CBD and cellobiose units of sugars are specific, supporting the model presented for the CBD binding to crystalline cellulose. It remained uncertain, however, how well the cello-oligosaccharides mimicked the binding of CBD to cellulose.",
keywords = "cellulose, cellulase, cellobiohydrolase, endoglucanase, cellulose-binding domain, fungi, NMR spectroscopy, Trichoderma reesei, biopolymers",
author = "Maija-Liisa Mattinen",
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year = "1998",
language = "English",
isbn = "951-38-5225-3",
series = "VTT Publications",
publisher = "VTT Technical Research Centre of Finland",
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school = "University of Helsinki",

}

Mattinen, M-L 1998, 'Structural and functional studies of fungal cellulose-binding domains by NMR spectroscopy: Dissertation', Doctor Degree, University of Helsinki, Espoo.

Structural and functional studies of fungal cellulose-binding domains by NMR spectroscopy : Dissertation. / Mattinen, Maija-Liisa.

Espoo : VTT Technical Research Centre of Finland, 1998. 72 p.

Research output: ThesisDissertationCollection of Articles

TY - THES

T1 - Structural and functional studies of fungal cellulose-binding domains by NMR spectroscopy

T2 - Dissertation

AU - Mattinen, Maija-Liisa

N1 - Project code: KET94137

PY - 1998

Y1 - 1998

N2 - Cellulose has a important environmental role in the preservation of the global carbon cycle and commercial significance as a raw material for industry. To understand the biodegradation of cellulose on the atomic level, it is important to be able to relate enzyme activities with the three-dimensional (3D) structures of cellulases and cellulose. The cellulolytic system of the fungus Trichoderma reesei is one of the best understood of all cellulolytic systems. Most cellulases from T. reesei have a cellulose-binding domain (CBD) specialised for binding to cellulose. As a means to understanding the interactions between cellulose and CBDs of cellobiohydrolase I (CBHI) and endoglucanase I (EGI) from T. reesei, 3D structures of these domains were studied by two-dimensional (2D) 1H NMR (nuclear magnetic resonance) techniques. Structural effects of mutations of conserved amino acids in CBDCBHI, Y5A, P16R, N29A, Y31A, Y32A and Q34A, were evaluated by comparing chemical shifts, coupling constants and NOEs of the backbone protons of the mutants and wild-type CBDCBHI. In general, the substitutions did not alter significantly the secondary structures of these engineered peptides. The adsorption experiments on cellulose showed that Y5A, Y31A and Y32A had lost nearly all their affinity to cellulose. For Y31A and Y32A the 3D structures revealed small local changes around the mutation on the flat face of CBD, which was expected to bind to cellulose. Therefore the structural roles of Tyr-31 and Tyr-32 must be minor, but their functional importance is clear since the mutants lacking these residues did not bind strongly to cellulose. In the case of Y5A the disruption of the structural framework at the N-terminus and the complete loss of affinity to cellulose implied that Tyr-5 has both structural and functional significance. The 3D structure of a synthetic CBDEGI was also determined by NMR spectroscopy. The structure was very similar to that of wild-type CBDCBHI. Within the precision of the structures, even the cellulose-binding face of CBDEGI was similar to that of CBDCBHI, apart from the place of attachment of the different side-chain. The determined NMR structure was also in agreement with an earlier modelled structure of CBDEGI. Finally, soluble cello-oligosaccharides were used as model compounds for cellobiose chains to investigate the interaction between CBD and cellulose by NMR spectroscopy. CBDs caused line broadening effects and decreasing T2 relaxation times for certain sugar resonances, whereas there were no effects in the presence of a mutant that bound weakly to cellulose. Experiments showed that the interactions between CBD and cellobiose units of sugars are specific, supporting the model presented for the CBD binding to crystalline cellulose. It remained uncertain, however, how well the cello-oligosaccharides mimicked the binding of CBD to cellulose.

AB - Cellulose has a important environmental role in the preservation of the global carbon cycle and commercial significance as a raw material for industry. To understand the biodegradation of cellulose on the atomic level, it is important to be able to relate enzyme activities with the three-dimensional (3D) structures of cellulases and cellulose. The cellulolytic system of the fungus Trichoderma reesei is one of the best understood of all cellulolytic systems. Most cellulases from T. reesei have a cellulose-binding domain (CBD) specialised for binding to cellulose. As a means to understanding the interactions between cellulose and CBDs of cellobiohydrolase I (CBHI) and endoglucanase I (EGI) from T. reesei, 3D structures of these domains were studied by two-dimensional (2D) 1H NMR (nuclear magnetic resonance) techniques. Structural effects of mutations of conserved amino acids in CBDCBHI, Y5A, P16R, N29A, Y31A, Y32A and Q34A, were evaluated by comparing chemical shifts, coupling constants and NOEs of the backbone protons of the mutants and wild-type CBDCBHI. In general, the substitutions did not alter significantly the secondary structures of these engineered peptides. The adsorption experiments on cellulose showed that Y5A, Y31A and Y32A had lost nearly all their affinity to cellulose. For Y31A and Y32A the 3D structures revealed small local changes around the mutation on the flat face of CBD, which was expected to bind to cellulose. Therefore the structural roles of Tyr-31 and Tyr-32 must be minor, but their functional importance is clear since the mutants lacking these residues did not bind strongly to cellulose. In the case of Y5A the disruption of the structural framework at the N-terminus and the complete loss of affinity to cellulose implied that Tyr-5 has both structural and functional significance. The 3D structure of a synthetic CBDEGI was also determined by NMR spectroscopy. The structure was very similar to that of wild-type CBDCBHI. Within the precision of the structures, even the cellulose-binding face of CBDEGI was similar to that of CBDCBHI, apart from the place of attachment of the different side-chain. The determined NMR structure was also in agreement with an earlier modelled structure of CBDEGI. Finally, soluble cello-oligosaccharides were used as model compounds for cellobiose chains to investigate the interaction between CBD and cellulose by NMR spectroscopy. CBDs caused line broadening effects and decreasing T2 relaxation times for certain sugar resonances, whereas there were no effects in the presence of a mutant that bound weakly to cellulose. Experiments showed that the interactions between CBD and cellobiose units of sugars are specific, supporting the model presented for the CBD binding to crystalline cellulose. It remained uncertain, however, how well the cello-oligosaccharides mimicked the binding of CBD to cellulose.

KW - cellulose

KW - cellulase

KW - cellobiohydrolase

KW - endoglucanase

KW - cellulose-binding domain

KW - fungi

KW - NMR spectroscopy

KW - Trichoderma reesei

KW - biopolymers

M3 - Dissertation

SN - 951-38-5225-3

T3 - VTT Publications

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -

Mattinen M-L. Structural and functional studies of fungal cellulose-binding domains by NMR spectroscopy: Dissertation. Espoo: VTT Technical Research Centre of Finland, 1998. 72 p.