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Hydrated and Dehydrated Tertiary Interactions - Opening and Closing - of a Four-helix Bundle Peptide
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 Biochemistry and Organic Chemistry. (Lars Baltzer)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Chemical Physics. (Kemisk fysik)
2009 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 97, no 2, 572-580 p.Article in journal (Refereed) Published
Abstract [en]

The structural heterogeneity and thermal denaturation of a dansyl-labeled four-helix bundle homodimeric peptide has been studied with steady state and time-resolved fluorescence spectroscopy and with circular dichroism. At room temperature the fluorescence decay of the polarity-sensitive dansyl, located in the hydrophobic core region, can be described by a broad distribution of fluorescence lifetimes, reflecting the heterogeneous microenvironment. However, the lifetime distribution is nearly bimodal, which we ascribe to the presence of two major conformational subgroups. Since the fluorescence lifetime reflects the water content of the four-helix bundle conformations we can use the lifetime analysis to monitor the change of hydration state of the hydrophobic core of the four-helix bundle. Increasing the temperature from 9 °C to 23 °C leads to an increased population of molten-globule-like conformations with a less ordered helical backbone structure. The fluorescence emission maximum remains constant in this temperature interval, and the hydrophobic core is not strongly affected. Above 30 °C the structural dynamics involve transient openings of the four-helix bundle structure as evidenced by the emergence of a water-quenched component and less negative CD. Above 60 °C the homodimer starts to dissociate, as shown by the increasing loss of CD and narrow, short-lived fluorescence lifetime distributions.

Place, publisher, year, edition, pages
Elsevier , 2009. Vol. 97, no 2, 572-580 p.
Keyword [en]
time-resolved fluorescence spectroscopy, protein dynamics, CD-spectroscopy
National Category
Other Basic Medicine
URN: urn:nbn:se:uu:diva-109369DOI: 10.1016/j.bpj.2009.04.055ISI: 000268428700019OAI: oai:DiVA.org:uu-109369DiVA: diva2:272256
Available from: 2009-10-14 Created: 2009-10-14 Last updated: 2012-08-03Bibliographically 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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