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.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-109396 |
| Date | January 2009 |
| Creators | Lignell, Martin |
| Publisher | Uppsala universitet, Kemisk fysik, Uppsala : Acta Universitatis Upsaliensis |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, info:eu-repo/semantics/doctoralThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, 1651-6214 ; 683 |
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