Using flexible loop mimetics to extend phi-value analysis to secondary structure interactions

Proc Natl Acad Sci U S A. 2001 Nov 6;98(23):13008-13. doi: 10.1073/pnas.221467398. Epub 2001 Oct 30.


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, beta-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 Phi-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, Phi-value analysis was limited to studying side-chain interactions. The linkers used here extend the protein engineering method directly to secondary-structure interactions.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adaptor Proteins, Signal Transducing*
  • Amino Acid Sequence
  • Carrier Proteins / chemistry*
  • Circular Dichroism
  • Humans
  • Molecular Mimicry*
  • Molecular Sequence Data
  • Nuclear Magnetic Resonance, Biomolecular
  • Phosphoproteins / chemistry*
  • Protein Structure, Secondary*
  • Sequence Homology, Amino Acid
  • Spectrophotometry, Ultraviolet
  • Transcription Factors
  • YAP-Signaling Proteins


  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • Phosphoproteins
  • Transcription Factors
  • YAP-Signaling Proteins
  • YAP1 protein, human

Associated data

  • PDB/1K5R