Prediction and verification of glycosyltransferase activity by bioinformatics analysis and protein engineering

Dietlind L. Gerloff; Elena I. Ilina; Camille Cialini; Uxue Mata Salcedo; Michel Mittelbronn; Tanja Müller

doi:10.1016/j.xpro.2022.101905

Prediction and verification of glycosyltransferase activity by bioinformatics analysis and protein engineering

Dietlind L. Gerloff
, Elena I. Ilina
, Camille Cialini
, Uxue Mata Salcedo
, Michel Mittelbronn
, Tanja Müller^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Research › peer-review

3 Citations (Scopus)

Abstract

A significant number of proteins are annotated as functionally uncharacterized proteins. Within this protocol, we describe how to use protein family multiple sequence alignments and structural bioinformatics resources to design loss-of-function mutations of previously uncharacterized proteins within the glycosyltransferase family. We detail approaches to determine target protein active sites using three-dimensional modeling. We generate active site mutants and quantify any changes in enzymatic function by a glycosyltransferase assay. With modifications, this protocol could be applied to other metal-dependent enzymes. For complete details on the use and execution of this protocol, please refer to Ilina et al. (2022).¹

Original language	English
Article number	101905
Journal	STAR Protocols
Volume	4
Issue number	1
Early online date	16 Dec 2022
DOIs	https://doi.org/10.1016/j.xpro.2022.101905
Publication status	Published - 17 Mar 2023

Keywords

Bioinformatics
Molecular Biology
Protein Biochemistry
Protein expression and purification
Sequence analysis

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10.1016/j.xpro.2022.101905Licence: CC BY-NC-ND

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title = "Prediction and verification of glycosyltransferase activity by bioinformatics analysis and protein engineering",

abstract = "A significant number of proteins are annotated as functionally uncharacterized proteins. Within this protocol, we describe how to use protein family multiple sequence alignments and structural bioinformatics resources to design loss-of-function mutations of previously uncharacterized proteins within the glycosyltransferase family. We detail approaches to determine target protein active sites using three-dimensional modeling. We generate active site mutants and quantify any changes in enzymatic function by a glycosyltransferase assay. With modifications, this protocol could be applied to other metal-dependent enzymes. For complete details on the use and execution of this protocol, please refer to Ilina et al. (2022).1",

keywords = "Bioinformatics, Molecular Biology, Protein Biochemistry, Protein expression and purification, Sequence analysis",

author = "Gerloff, \{Dietlind L.\} and Ilina, \{Elena I.\} and Camille Cialini and \{Mata Salcedo\}, Uxue and Michel Mittelbronn and Tanja M{\"u}ller",

note = "Funding Information: The authors would like to thank the Luxembourg National Research Fund (FNR) for the support (FNR PEARL P16/BM/11192868 grant). The illustrations used in the graphical abstract and Figures 4 , 5 , and 6 were created and exported with publication licenses from Biorender.com ( https://biorender.com ). Additionally, the authors would like to thank the reviewers for their valuable comments and suggestions, which helped us to improve the quality of the manuscript. Publisher Copyright: {\textcopyright} 2022 The Author(s)",

year = "2023",

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doi = "10.1016/j.xpro.2022.101905",

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TY - JOUR

T1 - Prediction and verification of glycosyltransferase activity by bioinformatics analysis and protein engineering

AU - Gerloff, Dietlind L.

AU - Ilina, Elena I.

AU - Cialini, Camille

AU - Mata Salcedo, Uxue

AU - Mittelbronn, Michel

AU - Müller, Tanja

N1 - Funding Information: The authors would like to thank the Luxembourg National Research Fund (FNR) for the support (FNR PEARL P16/BM/11192868 grant). The illustrations used in the graphical abstract and Figures 4 , 5 , and 6 were created and exported with publication licenses from Biorender.com ( https://biorender.com ). Additionally, the authors would like to thank the reviewers for their valuable comments and suggestions, which helped us to improve the quality of the manuscript. Publisher Copyright: © 2022 The Author(s)

PY - 2023/3/17

Y1 - 2023/3/17

N2 - A significant number of proteins are annotated as functionally uncharacterized proteins. Within this protocol, we describe how to use protein family multiple sequence alignments and structural bioinformatics resources to design loss-of-function mutations of previously uncharacterized proteins within the glycosyltransferase family. We detail approaches to determine target protein active sites using three-dimensional modeling. We generate active site mutants and quantify any changes in enzymatic function by a glycosyltransferase assay. With modifications, this protocol could be applied to other metal-dependent enzymes. For complete details on the use and execution of this protocol, please refer to Ilina et al. (2022).1

AB - A significant number of proteins are annotated as functionally uncharacterized proteins. Within this protocol, we describe how to use protein family multiple sequence alignments and structural bioinformatics resources to design loss-of-function mutations of previously uncharacterized proteins within the glycosyltransferase family. We detail approaches to determine target protein active sites using three-dimensional modeling. We generate active site mutants and quantify any changes in enzymatic function by a glycosyltransferase assay. With modifications, this protocol could be applied to other metal-dependent enzymes. For complete details on the use and execution of this protocol, please refer to Ilina et al. (2022).1

KW - Bioinformatics

KW - Molecular Biology

KW - Protein Biochemistry

KW - Protein expression and purification

KW - Sequence analysis

UR - https://www.scopus.com/pages/publications/85144022186

UR - https://pubmed.ncbi.nlm.nih.gov/36528856

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DO - 10.1016/j.xpro.2022.101905

M3 - Article

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AN - SCOPUS:85144022186

SN - 2666-1667

VL - 4

JO - STAR Protocols

JF - STAR Protocols

IS - 1

M1 - 101905

ER -

Prediction and verification of glycosyltransferase activity by bioinformatics analysis and protein engineering

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