title: Probing the folding pathway of a beta-clam protein with single-tryptophan constructs.
abstract: BACKGROUND
  Cellular retinoic acid binding protein I (CRABPI) is a small, predominantly beta-sheet protein with a simple architecture and no disulfides or cofactors. Folding of mutants containing only one of the three native tryptophans has been examined using stopped-flow fluorescence and circular dichroism at multiple wavelengths.
  
  
  RESULTS
  Within 10 ms, the tryptophan fluorescence of all three mutants shows a blue shift, and stopped-flow circular dichroism shows significant secondary structure content. The local environment of Trp7, a completely buried residue located near the intersection of the N and C termini, develops on a 100 ms time scale. Spectral signatures of the other two tryptophan residues (87 and 109) become native-like in a 1 s kinetic phase.
  
  
  CONCLUSIONS
  Formation of the native beta structure of CRABPI is initiated by rapid hydrophobic collapse, during which local segments of chain adopt significant secondary structure. Subsequently, transient yet specific interactions of amino acid residues restrict the arrangement of the chain topology and initiate long-range associations such as the docking of the N and C termini. The development of native tertiary environments, including the specific packing of the beta-sheet sidechains, occurs in a final, highly cooperative step simultaneous with stable interstrand hydrogen bonding.
authors.affiliation: Department of Chemistry, University of Massachusetts, Amherst, MA 01003, USA.
authors.forename: P L
authors.initials: PL
authors.lastname: Clark
authors.forename: B F
authors.initials: BF
authors.lastname: Weston
authors.forename: L M
authors.initials: LM
authors.lastname: Gierasch
mesh_terms: D002942:Circular Dichroism; D005453:Fluorescence; D007700:Kinetics; D015335:Molecular Probes; D017433:Protein Structure, Secondary; D018168:Receptors, Retinoic Acid / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D015335:Molecular Probes; D018168:Receptors, Retinoic Acid; C082927:retinoic acid binding protein I, cellular; D014364:Tryptophan