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Probing Solution-Phase and Gas-Phase Structures of Trp-cage Cations by Chiral Substitution and Spectroscopic Techniques
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences.
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
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, Molecular biophysics.
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2006 (English)In: International Journal of Mass Spectrometry, ISSN 1387-3806, E-ISSN 1873-2798, Vol. 253, no 3, 263-273 p.Article in journal (Refereed) Published
Abstract [en]

The relevance of gas-phase protein structure to its solution structure is of the utmost importance in studying biomolecules by mass spectrometry. D-Amino acid substitutions within a minimal protein. Trp-cage. were used to correlate solution-phase properties as measured by circular dichroism with solution/gas-phase conformational features of protein cations probed via charge state distribution (CSD) in electrospray ionization. and gas-phase features revealed by tandem mass spectrometry (MS/MS). The gas-phase features were additionally supported by force-field molecular dynamics simulations. CD data showed that almost any single-residue D-substitution destroys the most prominent CD feature of the "native" all-L isomer, alpha-helicity. CSD was able to qualitatively assess the degree of compactness of solution-phase molecular structures. CSD results were consistent with the all-L form being the most compact in solution among all studied stereoisomers except for the D-Asn(1) isomer. D-substitutions of the aromatic Y(3), W(6) and Q(5) residues generated the largest deviations in CSD data among single amino acid substitutions. consistent with the critical role of these residues in Trp-cage stability. Electron capture dissociation of the stereoisomer dications gave an indication that some gas-phase structural features of Trp-cage are similar to those in solution. This result is supported by MDS data oil five of the studied stereoisomer dications in the gas-phase. The MDS-derived minimum-energy structures possessed more extensive hydrogen bonding than the solution-phase structure of the native form, deviating from the latter by 3-4 angstrom and were not 'inside-out' compared to native structures. MDS data could be correlated with CD data and even with ECD results. which aided in providing a long-range structural constraint for MDS. The overall conclusion is the general resemblance, despite the difference on the detailed level, of the preferred structures in both phases for the mini protein Trp-cage.

Place, publisher, year, edition, pages
2006. Vol. 253, no 3, 263-273 p.
Keyword [en]
solution structure; gas-phase structure; chirality; electron capture dissociation; charge state distribution
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
URN: urn:nbn:se:uu:diva-95865DOI: 10.1016/j.ijms.2006.04.012ISI: 000238967500014OAI: oai:DiVA.org:uu-95865DiVA: diva2:170227
Available from: 2007-05-07 Created: 2007-05-07 Last updated: 2016-08-24Bibliographically approved
In thesis
1. Secondary and Higher Order Structural Characterization of Peptides and Proteins by Mass Spectrometry
Open this publication in new window or tab >>Secondary and Higher Order Structural Characterization of Peptides and Proteins by Mass Spectrometry
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The work in this thesis has demonstrated the advantages and limitations of using MS based technologies in protein and peptide structural studies.

Tandem MS, specifically electron capture dissociation (ECD) have shown the ability to provide structural insights in molecules containing the slightest of all modifications (D-AA substitution). Additionally, it can be concluded that charge localization in molecular ions is best identified with ECD and to a lesser degree using CAD.

Fragment ion abundances are a quantifiable tool providing chiral recognition (RChiral). An analytical model demonstrating the detection and quantification of D-AAs within proteins and peptides has been achieved. ECD has demonstrated the ability to quantify stereoisomeric mixtures to as little as 1%. Chirality elucidation on a nano LC-MS/MS time scale has been shown.

The structures of various stereoisomers of the mini protein Trp Cage were explored, each providing unique ECD fragment ion abundances suggestive of gas phase structural differences. The uniqueness of these abundances combined with MDS data have been used in proposing a new mechanism in c and z fragment ion formation in ECD. This mechanism suggests initial electron capture on a backbone amide involved in (neutral) hydrogen bonding.

The wealth of solution phase (circular dichroism), transitition phase (charge state distribution, CSD) and gas phase (ECD) data for Trp Cage suggest that at low charge states (2+) the molecule has a high degree of structural similarity in solution- and gas- phases. Furthermore, quantitative information from CSD studies is garnered when using a “native” deuteriated form as part of the stereoisomeric mixture. It has also been shown that the stability of the reduced species after electron capture is indicative of the recombination energy release, which in turn is linked to the coulombic repulsion- a structural constraint that can be used for approximation of the inter-charge distance for various stereoisomers.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 58 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 304
Keyword
Engineering physics, Protein Structure, Mass Spectrometry, Electron Capture Dissociation, Tandem Mass Spectrometry, Teknisk fysik
Identifiers
urn:nbn:se:uu:diva-7861 (URN)978-91-554-6887-3 (ISBN)
Public defence
2007-05-28, B41, BMC, Husarg. 3, Uppsala, 10:15
Opponent
Supervisors
Available from: 2007-05-07 Created: 2007-05-07Bibliographically approved
2. 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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 360
Keyword
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
Identifiers
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
Opponent
Supervisors
Available from: 2007-11-09 Created: 2007-11-09 Last updated: 2016-08-24Bibliographically approved

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Patriksson, Alexandravan Der Spoel, David

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