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Fluorescence probe of Trp-cage protein conformation in solution and in gas phase
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
2007 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 129, no 21, 6726-6735 p.Article in journal (Refereed) Published
Abstract [en]

Measurements of protein unfolding in the absence of solvent, when combined with unfolding studies in solution, offer a unique opportunity to measure the effects of solvent on protein structure and dynamics. The experiments presented here rely on the fluorescence of an attached dye to probe the local conformational dynamics through interactions with a Trp residue and fields originating on charge sites. We present fluorescence measurements of thermal fluctuations accompanying conformational change of a miniprotein, Trp-cage, in solution and in gas phase. Molecular dynamics (MD) simulations are performed as a function of temperature, charge state, and charge location to elucidate the dye-protein conformational dynamics leading to the changes in measured fluorescence. The results indicate that the stability of the unsolvated protein is dominated by hydrogen bonds. Substituting asparagine for aspartic acid at position 9 results in a dramatic alteration of the solution unfolding curve, indicating that the salt bridge involving Lys8, Asp9, and Arg16 (+ - +) is essential for Trp-cage stability in solution. In contrast, this substitution results in minor changes in the unfolding curve of the unsolvated protein, showing that hydrogen bonds are the major contributor to the stability of Trp-cage in gas phase. Consistent with this hypothesis, the decrease in the number of hydrogen bonds with increasing temperature indicated by MD simulations agrees reasonably well with the experimentally derived enthalpies of conformational change. The simulation results display relatively compact conformations compared with NMR structures that are generally consistent with experimental results. The measured unfolding curves of unsolvated Trp-cage ions are invariant with the acetonitrile content of the solution from which they are formed, possibly as a result of conformational relaxation during or after desolvation. This work demonstrates the power of combined solution and gas-phase studies and of single-point mutations to identify specific noncovalent interactions which contribute to protein-fold stability. The combination of experiment and simulation is particularly useful because these approaches yield complementary information which can be used to deduce the details of structural changes of proteins in the gas phase.

Place, publisher, year, edition, pages
2007. Vol. 129, no 21, 6726-6735 p.
National Category
Biological Sciences
URN: urn:nbn:se:uu:diva-96402DOI: 10.1021/ja065092sISI: 000246686700032PubMedID: 17487969OAI: oai:DiVA.org:uu-96402DiVA: diva2:170966
Available from: 2007-11-09 Created: 2007-11-09 Last updated: 2011-01-31Bibliographically approved
In thesis
1. From Solution into Vacuum - Structural Transitions in Proteins
Open this publication in new window or tab >>From Solution into Vacuum - Structural Transitions in Proteins
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Information about protein structures is important in many areas of life sciences, including structure-based drug design. Gas phase methods, like electrospray ionization and mass spectrometry are powerful tools for the analysis of molecular interactions and conformational changes which complement existing solution phase methods. Novel techniques such as single particle imaging with X-ray free electron lasers are emerging as well. A requirement for using gas phase methods is that we understand what happens to proteins when injected into vacuum, and what is the relationship between the vacuum structure and the solution structure.

Molecular dynamics simulations in combination with experiments show that protein structures in the gas phase can be similar to solution structures, and that hydrogen bonding networks and secondary structure elements can be retained. Structural changes near the surface of the protein happen quickly (ns-µs) during transition from solution into vacuum. The native solution structure results in a reasonably well defined gas phase structure, which has high structural similarity to the solution structure.

Native charge locations are in some cases also preserved, and structural changes, due to point mutations in solution, can also be observed in vacuo. Proteins do not refold in vacuo: when a denatured protein is injected into vacuum, the resulting gas phase structure is different from the native structure.

Native structures can be protected in the gas phase by adjusting electrospray conditions to avoid complete evaporation of water. A water layer with a thickness of less than two water molecules seems enough to preserve native conditions.

The results presented in this thesis give confidence in the continued use of gas phase methods for analysis of charge locations, conformational changes and non-covalent interactions, and provide a means to relate gas phase structures and solution structures.

Place, publisher, year, edition, pages
Uppsala: Universitetsbiblioteket, 2007. viii, 44 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 360
molecular dynamics, computer simulations, mass spectrometry, electrospray ionization, free-electron laser, vacuum structure of proteins, solvation, desolvation, single molecule imaging
National Category
Other Basic Medicine
urn:nbn:se:uu:diva-8300 (URN)978-91-554-7014-2 (ISBN)
Public defence
2007-12-01, B7:113a, BMC, Husargatan 3, Uppsala, 13:00
Available from: 2007-11-09 Created: 2007-11-09 Last updated: 2016-08-24Bibliographically approved

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