PEDS Advance Access originally published online on January 18, 2008
Protein Engineering Design and Selection 2008 21(3):171-185; doi:10.1093/protein/gzm082
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The Trp-cage: optimizing the stability of a globular miniprotein
Department of Chemistry, University of Washington, Seattle, WA 98195, USA
1 To whom correspondence should be addressed. E-mail: andersen{at}chem.washington.edu
The Trp-cage, as the smallest miniprotein, remains the subject of numerous computational and experimental studies of protein folding dynamics and pathways. The original Trp-cage (NLYIQWLKDGGPSSGRPPPS, Tm = 42°C) can be significantly stabilized by mutations; melting points as high as 64°C are reported. In helical portions of the structure, each allowed replacement of Leu, Ile, Lys or Ser residues by Ala results in a 1.5 (±0.35) kJ/mol fold stabilization. No changes in structure or fluxionality of the core results upon stabilization. Contrary to the initial hypothesis, specific Pro/Trp interactions are not essential for core formation. The entropic advantage of Pro versus Ala (
SU = 11 ± 2 J/mol K) was measured at the solvent-exposed P17 site. Pro–Ala mutations at two of the three prolines (P12 and P18) that encage the indole ring result in less fold destabilization (2.3–3.4 kJ/mol). However, a P19A mutation reduces fold stability by 16 kJ/mol reflecting a favorable Y3/P19 interaction as well as Trp burial. The Y3/P19 hydrophobic staple interaction defines the folding motif as an 18-residue unit. Other stabilizing features that have been identified include a solvent-exposed Arg/Asp salt bridge (3.4–6 kJ/mol) and a buried H-bonded Ser side chain (
10 kJ/mol).
Keywords: buried polar sidechain/fast-folding miniprotein/hydrophobic core formation/motif minimization/proline-tryptophan interactions
Received November 15, 2007; revised November 27, 2007; accepted November 28, 2007.