uu.seUppsala University Publications
Change search
ReferencesLink to record
Permanent link

Direct link
Recognition and binding of a helix-loop-helix peptide to carbonic anhydrase occurs via partly folded intermediate structures
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)
2010 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 98, no 3, 425-433 p.Article in journal (Refereed) Published
Abstract [en]

We have studied the association of a fluorescently labeled helix–loop–helix peptide scaffold carrying a benzensulfonamide ligand to carbonic anhydrase using steady state and time-resolved fluorescence spectroscopy. The helix–loop–helix peptide, developed for biosensing applications, is labeled with the fluorescent probe dansyl, which serves as a polarity-sensitive reporter of the binding event. Using maximum entropy analysis of the fluorescence lifetime of the dansyl at 1:1 stoichiometry reveals three characteristic fluorescence lifetime groups, which are interpreted as differently interacting peptide–protein structures. We characterize these as mostly bound but unfolded, bound and partly folded, and strongly bound and folded peptide–protein complexes. Furthermore, analysis of the fluorescence anisotropy decay resulted in three different dansyl rotational correlation times, namely 0.18, 1.2, and 23 ns. Using their amplitudes, we can correlate the lifetime groups with the corresponding fluorescence lifetime group. The 23 ns rotational correlation time, which appears with the same amplitude as a 17 ns fluorescence lifetime, shows that the dansyl fluophorophore follows the rotational diffusion of carbonic anhydrase when it is a part of the folded peptide–protein complex. A partly folded and partly hydrated interfacial structure is manifested by a 8 ns dansyl fluorescence lifetime and a 1.2 ns rotational correlation time. This structure, we believe, is similar to a molten-globule-like interfacial structure which allows faster segmental movements and a higher degree of solvent exposure of dansyl. Excitation of dansyl on the helix–loop–helix peptide through Förster energy transfer from one or several tryptophans in the carbonic anhydrase, shows that the helix–loop–helix scaffold binds to a tryptophan-rich domain of the carbonic anhydrase. We conclude that the binding of the peptide to carbonic anhydrase involves a transition from a disordered to ordered structure of the helix–loop–helix scaffold.

Place, publisher, year, edition, pages
2010. Vol. 98, no 3, 425-433 p.
Keyword [en]
molecular recognition, protein dynamics, time-resolved fluorescence spectroscopy, disordered proteins, disordered to ordered structural transition
National Category
Chemical Sciences
URN: urn:nbn:se:uu:diva-109373DOI: 10.1016/j.bpj.2009.10.038ISI: 000274313200010PubMedID: 20141756OAI: oai:DiVA.org:uu-109373DiVA: diva2:272258
Available from: 2009-10-14 Created: 2009-10-14 Last updated: 2010-12-16Bibliographically 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

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed
By organisation
Chemical Physics
In the same journal
Biophysical Journal
Chemical Sciences

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 418 hits
ReferencesLink to record
Permanent link

Direct link