title: A minimal sequence code for switching protein structure and function.
abstract: We present here a structural and mechanistic description of how a protein changes its fold and function, mutation by mutation. Our approach was to create 2 proteins that (i) are stably folded into 2 different folds, (ii) have 2 different functions, and (iii) are very similar in sequence. In this simplified sequence space we explore the mutational path from one fold to another. We show that an IgG-binding, 4beta+alpha fold can be transformed into an albumin-binding, 3-alpha fold via a mutational pathway in which neither function nor native structure is completely lost. The stabilities of all mutants along the pathway are evaluated, key high-resolution structures are determined by NMR, and an explanation of the switching mechanism is provided. We show that the conformational switch from 4beta+alpha to 3-alpha structure can occur via a single amino acid substitution. On one side of the switch point, the 4beta+alpha fold is >90% populated (pH 7.2, 20 degrees C). A single mutation switches the conformation to the 3-alpha fold, which is >90% populated (pH 7.2, 20 degrees C). We further show that a bifunctional protein exists at the switch point with affinity for both IgG and albumin.
authors.affiliation: Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, MD 20850, USA.
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Alexander
authors.forename: Yanan
authors.initials: Y
authors.lastname: He
authors.forename: Yihong
authors.initials: Y
authors.lastname: Chen
authors.forename: John
authors.initials: J
authors.lastname: Orban
authors.forename: Philip N
authors.initials: PN
authors.lastname: Bryan
mesh_terms: D006801:Humans; D007074:Immunoglobulin G; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D012709:Serum Albumin; D020860:Subtilisin / Q000235:genetics*
chemical_list: D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D020860:Subtilisin