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Conformational Switching Between 310, α and ∏-helical States in a 12 Amino Acid Long Peptide Studied by Time-resolved Fluorescence and CD Spectroscopy
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Chemical Physics. (Kemisk fysik)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Chemical Physics. (Kemisk fysik)
(English)Article in journal (Refereed) In press
Abstract [en]

We have measured the end-to-end distance of a small peptide using time-resolved fluorescence energy transfer experiments and CD spectroscopy at various concentrations of TFE. The peptide comprises tryptophan as the donor and nitrotyrosine as the acceptor. The results show that the peptide is to a large degree helical even in the absence of TFE, and that addition of TFE to the solutions favors short, α-helical structures. Because of the nanosecond time resolution in the time-resolved fluorescence experiments, we are able to resolve four groups of donor–acceptor distances. The distances themselves do not change much with addition of TFE, however, the populations of the subgroups changes with TFE concentration. We assign the four resolvable distances to be, in decreasing length order, two forms of elongated, 310-helical structures, π-helical, and α-helical structures. The presence of multiple helical forms is supported by the fact that at least three components are needed to describe the change in CD upon addition of TFE. As we are observing the peptide under equilibrium conditions, the results tell that the peptide is at all TFE concentrations undergoing length changes, which are also accompanied by changes in hydration/solvent exposure. Addition of TFE does not appear to change the peptide structures, but changes the energy landscape in favor of short, α-helical structures.

Keyword [en]
Time-resolved fluorescence spectroscopy, CD spectroscopy, conformational dynamics
URN: urn:nbn:se:uu:diva-109392OAI: oai:DiVA.org:uu-109392DiVA: diva2:272259
Available from: 2009-10-14 Created: 2009-10-14 Last updated: 2010-01-14Bibliographically approved
In thesis
1. Structural Transitions in Helical Peptides: The Influence of Water – Implications for Molecular Recognition and Protein Folding
Open this publication in new window or tab >>Structural Transitions in Helical Peptides: The Influence of Water – Implications for Molecular Recognition and Protein Folding
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fluctuations in protein structure are vital to function. This contrasts the dominating structure-function paradigm, which connects the well-defined three-dimensional protein structure to its function. However, catalysis is observed in disordered enzymes, which lack a defined structure. Disordered proteins are involved in molecular recognition events as well. The aim of this Thesis is to describe the structural changes occuring in protein structure and to investigate the mechanism of molecular recognition.

Protein architecture is classified in a hierarchical manner, that is, it is categorized into primary, secondary, and tertiary levels. One of the major questions in biology today is how proteins fold into a defined three-dimensional structure. Some protein folding models, like the framework model, suggest that the secondary structure, like α-helices, is formed before the tertiary structure. This Thesis raises two questions: First, are structural fluctuations that occur in the protein related to the folding of the protein structure? Second, is the hierarchic classification of the protein architecture useful to describe said structural fluctuations?

Kinetic studies of protein folding show that important dynamical processes of the folding occur on the microsecond timescale, which is why time-resolved fluorescence spectroscopy was chosen as the principal method for studying structural fluctuations in the peptides. Time-resolved fluorescence spectroscopy offers a number of experimental advantages and is useful for characterizing typical structural elements of the peptides on the sub-microsecond timescale. By observing the fluorescence lifetime distribution of the fluorescent probe, which is a part of the hydrophobic core of a four-helix bundle, it is shown that the hydrophobic core changes hydration state, from a completely dehydrated to a partly hydrated hydrophobic core. These fluctuations are related to the tertiary structure of the four-helix bundle and constitute structural transitions between the completely folded four-helix bundle and the molten globule version. Equilibrium unfolding of the four-helix bundle, using chemical denaturants or increased temperature, shows that the tertiary structure unfolds before the secondary structure, via the molten globule state, which suggests a hierarchic folding mechanism of the four-helix bundle.

Fluctuations of a 12 amino acid long helical segment, without tertiary structure, involve a conformational search of different helical organizations of the backbone.

Binding and recognition of a helix-loop-helix to carbonic anhydrase occurs through a partly folded intermediate before the final tertiary and bimolecular structure is formed between the two biomolecules. This confirms the latest established theory of recognition that the binding and the folding processes are coupled for the binding molecules.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 90 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 683
protein dynamics, protein folding, molten globule, time-resolved fluorescence spectroscopy, CD spectroscopy, molecular recognition, structure-function paradigm
National Category
Atom and Molecular Physics and Optics
Research subject
Physical Chemistry
urn:nbn:se:uu:diva-109396 (URN)978-91-554-7637-3 (ISBN)
Public defence
2009-11-30, Häggsalen, Ångströmlaboratoriet, Lägerhyddsvägen 1 Polacksbacken, Uppsala, 10:30 (English)
Available from: 2009-11-09 Created: 2009-10-14 Last updated: 2010-12-16Bibliographically approved

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