title: Folding of bovine pancreatic trypsin inhibitor (BPTI) variants in which almost half the residues are alanine.
abstract: Recent studies indicate that a fraction of the information contained in an amino acid sequence may be sufficient for specifying a native protein structure. An earlier alanine-scanning experiment conducted on bovine pancreatic trypsin inhibitor (BPTI; 58 residues) suggested that if cumulative mutations have additive effects on protein stability, a native protein structure could be built from BPTI sequences that contained many alanine residues distributed throughout the protein. To test this hypothesis, we designed and produced six BPTI mutants containing from 21 to 29 alanine residues. We found that the melting temperature of mutants containing up to 27 alanine residues (48 % of the total number of residues) could be predicted quite well by the sum of the change in melting temperature for the single mutations. Additionally, these same mutants folded into a native-like structure, as judged by their cooperative thermal denaturation curves and heteronuclear multiple quantum correlation (HMQC) NMR spectra. A BPTI mutant containing 22 alanine residues was further shown by 2D and 3D-NMR to fold into a structure very similar to that of native BPTI, and to be a functional trypsin inhibitor. These results provide insight into the extent to which native protein structure and function can be achieved with a highly simplified amino acid sequence.
authors.affiliation: Howard Hughes Medical Institute, Whitehead Institute for Biomedical Research Department of Biology, MIT, Cambridge, MA, 02142, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Kuroda
authors.forename: P S
authors.initials: PS
authors.lastname: Kim
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D007611:Aprotinin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D002942:Circular Dichroism; D014644:Genetic Variation / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics; D014357:Trypsin / Q000378:metabolism
chemical_list: D007611:Aprotinin; D014357:Trypsin; D000409:Alanine