St Andrews Research Repository

St Andrews University Home
View Item 
  •   St Andrews Research Repository
  • Biology (School of)
  • Biology
  • Biology Theses
  • View Item
  •   St Andrews Research Repository
  • Biology (School of)
  • Biology
  • Biology Theses
  • View Item
  •   St Andrews Research Repository
  • Biology (School of)
  • Biology
  • Biology Theses
  • View Item
  • Login
JavaScript is disabled for your browser. Some features of this site may not work without it.

Structural and functional study of the nitrate transporter, NrtA

Thumbnail
View/Open
IngridRodriguesNunesdaSilvaMPhilThesis.pdf (3.906Mb)
IngridRodriguesNunesdaSilvaMPhilThesis_APPENDIX 1.pdf (588.9Kb)
IngridRodriguesNunesdaSilvaMPhilThesis_APPENDIX 2.pdf (26.78Kb)
IngridRodriguesNunesdaSilvaMPhilThesis_APPENDIX 3.pdf (130.4Kb)
IngridRodriguesNunesdaSilvaMPhilThesis_APPENDIX 4.pdf (2.373Mb)
IngridRodriguesNunesdaSilvaMPhilThesis_APPENDIX 5.pdf (170.8Kb)
Date
2013
Author
Da Silva, Ingrid Rodrigues Nunes
Supervisor
Ruxton, Graeme D.
Metadata
Show full item record
Altmetrics Handle Statistics
Abstract
Membrane proteins play a crucial role in most cellular processes. Transporter integral proteins carry a whole variety of solute molecules across biological membranes. While the structure of thousands of soluble proteins has been determined, very few integral membrane proteins have been solved. The structures of several major facilitator superfamily (MFS) proteins that have been determined revealed structural similarities but these proteins exhibit strict substrate specificity and the structural and functional basis of this specificity is poorly understood. In this study, a membrane protein from the saprotrophic ascomycete Aspergillus nidulans NrtA, which is involved in the nitrate transport was investigated. This high-affinity nitrate transporter is a member of the MFS and shares similar structural features to other MFS proteins that have been fully characterised. In order to reach a better understanding of the function and the structure of NrtA certain amino acids - that could play a critical role in nitrate transport - were changed using oligonucleotide mediated site-directed mutagenesis. NrtA homologue sequences facilitated the identification of certain conserved and highly conserved amino acids for mutational studies. Alanine scanning mutagenesis was used to analyse highly conserved glycine residues in the conserved NRT2 motif, which were found to play important roles but not essential as mutants were able to uptake nitrate. Three other residues located adjacent to this highly conserved motif were also analysed, revealing that a phenylalanine at position 457 probably play an important, albeit not crucial, structural role, an asparagine at position 459 is critical for NrtA function, whereas a leucine at position 460 is not essential. A comparative study of NrtA residues that have been replaced by their residue-equivalent in NrtB showed that a slight change in the hydrophobicity around TM2 highly conserved arginine at position 87 and also around TM8 highly conserved arginine at position 368 can potentially be the cause of differences between NrtA and NrtB 6-fold rate of transport and 10-fold affinity to nitrate. A variety of conserved and highly conserved aromatic residues were analysed revealing a variety of roles. A residue located at the proposed nitrate binding site was also analysed and substitutions to other residues were not tolerated and no alteration to enzyme kinetics in mutant N364Q was found when using the radioactive tracer ¹³NO₃⁻. Understanding the role of certain amino acids on nitrate uptake should in turn enable development of improved methods for the prediction of NrtA’s structure.
Type
Thesis, MPhil Master of Philosophy
Collections
  • Biology Theses
URI
http://hdl.handle.net/10023/3740

Items in the St Andrews Research Repository are protected by copyright, with all rights reserved, unless otherwise indicated.

Advanced Search

Browse

All of RepositoryCommunities & CollectionsBy Issue DateNamesTitlesSubjectsClassificationTypeFunderThis CollectionBy Issue DateNamesTitlesSubjectsClassificationTypeFunder

My Account

Login

Open Access

To find out how you can benefit from open access to research, see our library web pages and Open Access blog. For open access help contact: openaccess@st-andrews.ac.uk.

Accessibility

Read our Accessibility statement.

How to submit research papers

The full text of research papers can be submitted to the repository via Pure, the University's research information system. For help see our guide: How to deposit in Pure.

Electronic thesis deposit

Help with deposit.

Repository help

For repository help contact: Digital-Repository@st-andrews.ac.uk.

Give Feedback

Cookie policy

This site may use cookies. Please see Terms and Conditions.

Usage statistics

COUNTER-compliant statistics on downloads from the repository are available from the IRUS-UK Service. Contact us for information.

© University of St Andrews Library

University of St Andrews is a charity registered in Scotland, No SC013532.

  • Facebook
  • Twitter