Using flexible loop mimetics to extend Φ-value analysis to secondary structure interactions
| Autor: | Rudolf Volkmer-Engert, Neil M. Ferguson, Christopher M. Johnson, Alan R. Fersht, José R. Pires, Valerie Daggett, Jens Schneider-Mergener, Florian Toepert, Hartmut Oschkinat, Yong Ping Pan |
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| Rok vydání: | 2001 |
| Předmět: |
Circular dichroism
Molecular Sequence Data Protein Structure Secondary WW domain chemistry.chemical_compound Thioether Humans Amino Acid Sequence Loop modeling Nuclear Magnetic Resonance Biomolecular Protein secondary structure Adaptor Proteins Signal Transducing Multidisciplinary Sequence Homology Amino Acid biology Chemistry Circular Dichroism Molecular Mimicry YAP-Signaling Proteins Protein engineering Biological Sciences Phosphoproteins Folding (chemistry) Crystallography biology.protein Spectrophotometry Ultraviolet Carrier Proteins Linker Transcription Factors |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America, 98(23): 13008-13013 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.221467398 |
| Popis: | Chemical synthesis allows the incorporation of nonnatural amino acids into proteins that may provide previously untried probes of their folding pathway and thermodynamic stability. We have used a flexible thioether linker as a loop mimetic in the human yes kinase-associated protein (YAP 65) WW domain, a three-stranded, 44-residue, β-sheet protein. This linkage avoids problems of incorporating sequences that constrain loops to the extent that they significantly change the nature of the denatured state with concomitant effects on the folding kinetics. An NMR solution structure shows that the thioether linker had little effect on the global fold of the domain, although the loop is apparently more dynamic. The thioether variants are destabilized by up to 1.4 kcal/mol (1 cal = 4.18 J). Preliminary Φ-value analysis showed that the first loop is highly structured in the folding transition state, and the second loop is essentially unstructured. These data are consistent with results from simulated unfolding and detailed protein-engineering studies of structurally homologous WW domains. Previously, Φ-value analysis was limited to studying side-chain interactions. The linkers used here extend the protein engineering method directly to secondary-structure interactions. |
| Databáze: | OpenAIRE |
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