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Thermostability investigation of Fatty Acid Binding Protein from Cataglyphis fortis by fluorescence spectroscopy using genetically introduced tryptophan residues
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
2011 (English)Independent thesis Basic level (degree of Bachelor), 10,5 credits / 16 HE creditsStudent thesis
Abstract [en]

The desert ant Cataglyphis fortis is one of the hyperthermophilic species of Cataglyphis. It lives in the Sahara desert and forages during the hottest hours of the day when it can get up to 70˚C in the sand. The body temperature of the ant during the foraging runs can reach a maximum of 55˚C. Since C.fortis is one of few eukaryotic hyperthermophilic species, its proteins probably have a high thermostability. Investigating the thermostability can give valuable information about the principles of protein folding and stability in hyperthermophiles.Fatty acid binding proteins (FABPs) have an important role in the cell taking up and transporting fatty acids and regulating metabolic and inflammatory pathways. FABPs have been extensively studied and structures from several species have been determined. The determined structures of all FABPs are very similar why thermostability studies of FABP from C.fortis are highly relevant.Fluorescence spectroscopy is an easy and fast method to measure intrinsic protein fluorescence. Tryptophans were genetically introduced into three different positions in FABP to be used as environmental sensitive probes. Complementing the measurement results with a model of the 3D structure of FABP from C.fortis gave additional information about the ligand binding.The (local) thermostability of the mutants can be detected by shift in wavelength maximum during temperature ramping experiments. All mutants are stabilised in the presence of fatty acids. The mutant with tryptophan positioned closest to the supposed ligand binding residues (Y11W) is most affected. The mutant with tryptophan situated farthest from the supposed binding residues (Y52W) shows a stabilisation of Tm less evident than for Y11W. Thus, the structural changes following fatty acid binding are more obvious in the environment close to the binding site.However, the third mutant C87W shows no significant stabilisation although positioned closer to the fatty acid binding site than Y52. This is probably due to the size difference between the original and introduced amino acid in the mutation. Since the high value of the starting λmax for C87W implies that C87W is quite exposed to the aqueous solvent, the residue is likely to not have subsumed in the protein tertiary structure.Further, the myristic acid stabilise the melting temperature of all the mutants while octanoic acid only has a local effect of Y11W increasing the cooperativity. This implies different binding properties and that myristic acid stabilise the entire protein while octanoic acid only has a local stabilisation effect around the ligand binding site.

Place, publisher, year, edition, pages
2011. , 30 p.
Keyword [en]
FABP, Fatty acid binding protein, fluorescence, Cataglyphis fortis, thermostability, mutant
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-74238ISRN: LITH-IFM-G-EX--11/2463--SEOAI: oai:DiVA.org:liu-74238DiVA: diva2:481666
Subject / course
Chemistry
Uppsok
Physics, Chemistry, Mathematics
Supervisors
Examiners
Available from: 2012-01-23 Created: 2012-01-21 Last updated: 2012-01-23Bibliographically approved

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CiteExportLink to record
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