PhD project: The use of mass spectrometry to probe the metamorphic protein Lymphotactin

Project description

Lymphotactin (Ltn) is a unique human chemokine; small secreted signalling proteins, which during immune response direct the migration of leukocytes towards areas of inflammation. Lymphotactin is the sole member of the C sub class of this important family of proteins and contains only one disulfide bond as opposed to the two normally found in the other classes of chemokines.1 Additionally, it contains a unique extended C terminus sequence which forms an intrinsically disordered tail. Lymphotactin exists in equilibrium between two distinct conformations, each of which performs a separate role in vivo.2 Interconversion between these two folds involves a complete restructuring of the core residues.3

Since the development of soft ionisation techniques such as electrospray ionisation (ESI) or nano-ESI, native mass spectrometry is emerging as a valuable tool for the analysis of proteins, due to its high sensitivity, large mass range and ability to observe intact complexes.4-6 Additionally, soft ionisation techniques have been coupled with spectroscopic gas phase techniques, such as gas phase IR.7,8 Through the use of such techniques there has been significant evidence for the presence of a variety of structural motifs within proteins in gas phase including α-helices.9-11 However, there has been no direct evidence of the presence and preservation of charged β-sheets in intact proteins in the gas phase despite some evidence reported for the preservation of charged β-sheet structures, in very small model peptide sections.12,13. We have shown for another family of small antimicrobial, chemokine like peptides (β-defensins) that single point mutations can affect gas phase structure and that this can be related to biological function. 14-16

We have begun to collaborate with Brian Volkaman – the leading investigator of lymphotactin, and have exciting evidence from recent work using ion mobility mass spectrometry (IM-MS)  and also electron capture dissociation (ECD) (using FT-ICR mass spectrometry) that both active folds of WT lymphotactin can be preserved in the solvent free environment of a mass spectrometer. Preliminary work has indicated that these gas phase measurements will be able to pinpoint at an amino acid level, which regions of the sequence are responsible for stabilising each fold. This project will extend the preliminary investigations. It will continue to use IM-MS and ECD to investigate the fold stability of lymphotactin, employing a series of single and double point mutants. The aim will be to use the gas phase findings to predict which region of sequence are critical for structure and most crucial to the conversion from one fold to the other. We will chart the energetics of the transition from the GAG binding fold (stable in the WT at 0ºC and high salt) and the chemokine fold (stable at   40ºC and low salt). We will also employ molecular dynamics to examine the conformations of this protein and the mutants and using well-honed group techniques compare experimental findings with those from simulation.

The student will be based in the group of Dr. Barran in the School of Chemistry and will work closely with Professor Cait MacPhee in the School of Physics, Barran and MacPhee currently have 4 shared PhD. students. The work will be predominantly Mass Spectrometry based, but will also involve training in other biophysical characterisation techniques. Functional assays will be conducted as part of this project, in investigating GAG binding (analogous to our work on defensins17) and also by our collaborators. The student will be able to travel to Wisconsin to take part in the experiments thRE. ECD will allow us to map the binding site of GAG on the protein and we have already observed that GAG binding to lymphotactin, causes fibril formation, which will be further investigated. This biophysical project, will determine the intrinsic interactions needed for functional fold, and will develop new methods that will be applicable to other proteins.

References

(1) Kelner, G.; Kennedy, J.; Bacon, K.; Kleyensteuber, S.; Largaespada, D.; Jenkins, N.; Copeland, N.; Bazan, J.; Moore, K.; Schall, T.; et, a. Science 1994266, 1395.
(2) Tuinstra, R. L.; Peterson, F. C.; Kutlesa, S.; Elgin, E. S.; Kron, M. A.; Volkman, B. F. PNAS 2008105, 5057.
(3) Tyler, R. C.; Murray, N. J.; Peterson, F. C.; Volkman, B. F. Biochemistry 201150, 7077.
(4)  Heck, A. J. R. Nat. Meth. 20085, 927.
(5)  Fenn, J.; Mann, M.; Meng, C.; Wong, S.; Whitehouse, C. Science 1989246, 64.
(6) Wilm, M. S.; Mann, M. Int. J. Mass Spectrom. Ion Processes 1994136, 167.
(7) Stearns, J. A.; Boyarkin, O. V.; Rizzo, T. R. J. Am. Chem. Soc. 2007129, 13820.
(8) Boyarkin, O. V.; Mercier, S. R.; Kamariotis, A.; Rizzo, T. R. J. Am. Chem. Soc. 2006128, 2816.
(9) Jarrold, M. F. Phys. Chem. Chem. Phys. 20079, 1659.
(10) Stearns, J. A.; Seaiby, C.; Boyarkin, O. V.; Rizzo, T. R. Phys. Chem. Chem. Phys. 200911, 125.
(11) Pagel, K.; Kupser, P.; Bierau, F.; Polfer, N. C.; Steill, J. D.; Oomens, J.; Meijer, G.; Koksch, B.; von Helden, G. Int. J. Mass Spectrom. 2009283, 161.
(12) Ruotolo, B. T.; Tate, C. C.; Russell, D. H. J. Am. Soc. Mass Spectrom. 200415, 870.
(13) Li, A.; Fenselau, C.; Kaltashov, I. A. Proteins: Struct., Funct., Genet. 1998Suppl 2, 22.
(14) McCullough, B. J.; Kalapothakis, J.; Eastwood, H.; Kemper, P.; MacMillan, D.; Taylor, K.; Dorin, J.; Barran, P. E. AnalyticalChemistry 200880, 6336.
(15) De Cecco, M.; Seo, E. S.; Clarke, D. J.; McCullough, B. J.; Taylor, K.; Macmillan, D.; Dorin, J. R.; Campopiano, D. J.;Barran, P. E. J Phys Chem B 2010114, 2312.
(16) Taylor, K.; Clarke, D. J.; McCullough, B.; Chin, W.; Seo, E.; Yang, D.; Oppenheim, J.; Uhrin, D.; Govan, J. R. W.; Campopiano,D. J.; MacMillan, D.; Barran, P.; Dorin,J. R. J Biol Chem 2008283, 6631.
(17) Seo, E. S.; Blaum, B. S.; Vargues, T.; De Cecco, M.; Deakin, J. A.; Lyon, M.; Barran, P. E.; Campopiano, D. J.; Uhrin, D. Biochemistry-Us 201049, 10486.

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