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Sequential Equilibrium Unfolding of a Four-helix Bundle Peptide Shows that Urea and Guanidine have Different Effects on Tertiary and Secondary Structure. A CD and Time-resolved Fluorescence Spectroscopy Study
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Chemical Physics. (Kemisk fysik)
(Kemisk fysik)
(English)In: Biophysical Journal, ISSN 0006-3495Article in journal (Refereed) Submitted
Abstract [en]

In this paper we show, using time-resolved fluorescence and CD spectroscopy, that equilibrium unfolding of the homodimeric four-helix bundle peptide (KE2D15)2 occurs via different pathways depending on the denaturant. The initial effect of guanidine hydrochloride (GdHCl) and urea is similar insofar that the tertiary structure is slightly destabilized. This is accompanied by a slight increase in helicity, which we believe is due to stabilization of the backbone at the expense of hydrophobic core stability. With GdHCl we observe an almost immediate (≥2 M GdHCl) dissociation of the dimer into helical monomers, while the effect of urea is to stabilize the helices and induce a solvent- or urea-separated state that persists up to about 5 M urea. At high urea concentrations, the peptide exists in monomeric but helical form. The partial and full dissociation of the dimeric four-helix bundle is monitored through time-resolved fluorescence spectroscopy. Through the use of time-resolved fluorescence, we can assess the heterogeneity of the partly and fully denatured states even though the denaturation is carried out at equilibrium conditions. The width of the fluorescence lifetime distributions are analyzed in terms of conformational space of the peptide.

Keyword [en]
conformational dynamics, protein folding, chemical denaturants, time-resolved fluorescence spectroscopy, CD spectroscopy
URN: urn:nbn:se:uu:diva-109375OAI: oai:DiVA.org:uu-109375DiVA: diva2:272260
Available from: 2009-10-14 Created: 2009-10-14 Last updated: 2009-10-15Bibliographically 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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