title: Thermodynamic and kinetic characterization of apoHmpH, a fast-folding bacterial globin.
abstract: Despite the widespread presence of the globin fold in most living organisms, only eukaryotic globins have been employed as model proteins in folding/stability studies so far. This work introduces the first thermodynamic and kinetic characterization of a prokaryotic globin, that is, the apo form of the heme-binding domain of flavohemoglobin (apoHmpH) from Escherichia coli. This bacterial globin has a widely different sequence but nearly identical structure to its eukaryotic analogues. We show that apoHmpH is a well-folded monomeric protein with moderate stability at room temperature [apparent Delta G degrees (UN(w))=-3.1+/-0.3 kcal mol(-1); m(UN)=-1.7 kcal mol(-1) M(-1)] and predominant alpha-helical structure. Remarkably, apoHmpH is the fastest-folding globin known to date, as it refolds about 4- to 16-fold more rapidly than its eukaryotic analogues (e.g., sperm whale apomyoglobin and soybean apoleghemoglobin), populating a compact kinetic intermediate (beta(I)=0.9+/-0.2) with significant helical content. Additionally, the single Trp120 (located in the native H helix) becomes locked into a fully native-like environment within 6 ms, suggesting that this residue and its closest spatial neighbors complete their folding at ultrafast (submillisecond) speed. In summary, apoHmpH is a bacterial globin that shares the general folding scheme (i.e., a rapid burst phase followed by slower rate-determining phases) of its eukaryotic analogues but displays an overall faster folding and a kinetic intermediate with some fully native-like traits. This study supports the view that the general folding features of bacterial and eukaryotic globins are preserved through evolution while kinetic details differ.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA.
authors.forename: Ye-Jin
authors.initials: YJ
authors.lastname: Eun
authors.forename: Neşe
authors.initials: N
authors.lastname: Kurt
authors.forename: Ashok
authors.initials: A
authors.lastname: Sekhar
authors.forename: Silvia
authors.initials: S
authors.lastname: Cavagnero
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004093:Dihydropteridine Reductase / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D019143:Evolution, Molecular; D005914:Globins / Q000737:chemistry* / Q000378:metabolism*; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D017510:Protein Folding; D013816:Thermodynamics
chemical_list: D029968:Escherichia coli Proteins; D006420:Hemeproteins; C585967:apoHmpH protein, E coli; D005914:Globins; D004093:Dihydropteridine Reductase; C076538:hmp protein, E coli; D009247:NADH, NADPH Oxidoreductases