Journal article
Hydrophobic Modulation of Heme Properties in Heme Protein Maquettes
Biochemistry (Easton), Vol.40(35), pp.10550-10561
09/04/2001
DOI: 10.1021/bi002806h
PMID: 11523997
Abstract
We have investigated the properties of the two hemes bound to histidine in the H10 positions of the uniquely structured apo form of the heme binding four-helix bundle protein maquette [H10H24-L6I,L13F](2), here called [I(6)F(13)H(24)](2) for the amino acids at positions 6 (I), 13 (F) and 24 (H), respectively. The primary structure of each alpha-helix, alpha-SH, in [I(6)F(13)H(24)](2) is Ac-CGGGEI(6)WKL.H(10)EEF(13)LKK.FEELLKL.H(24)EERLKK.L-CONH(2). In our nomenclature, [I(6)F(13)H(24)] represents the disulfide-bridged di-alpha-helical homodimer of this sequence, i.e., (alpha-SS-alpha), and [I(6)F(13)H(24)](2) represents the dimeric four helix bundle composed of two di-alpha-helical subunits, i.e., (alpha-SS-alpha)(2). We replaced the histidines at positions H24 in [I(6)F(13)H(24)](2) with hydrophobic amino acids incompetent for heme ligation. These maquette variants, [I(6)F(13)I(24)](2), [I(6)F(13)A(24)](2), and [I(6)F(13)F(24)](2), are distinguished from the tetraheme binding parent peptide, [I(6)F(13)H(24)](2), by a reduction in the heme:four-helix bundle stoichiometry from 4:1 to 2:1. Iterative redesign has identified phenylalanine as the optimal amino acid replacement for H24 in the context of apo state conformational specificity. Furthermore, the novel second generation diheme [I(6)F(13)F(24)](2) maquette was related to the first generation diheme [H10A24](2) prototype, [L(6)L(13)A(24)](2) in the present nomenclature, via a sequential path in sequence space to evaluate the effects of conservative hydrophobic amino acid changes on heme properties. Each of the disulfide-linked dipeptides studied was highly helical (>77% as determined from circular dichroism spectroscopy), self-associates in solution to form a dimer (as determined by size exclusion chromatography), is thermodynamically stable (-DeltaG(H)2(O) >18 kcal/mol), and possesses conformational specificity that NMR data indicate can vary from multistructured to single structured. Each peptide binds one heme with a dissociation constant, K(d1) value, tighter than 65 nM forming a series of monoheme maquettes. Addition of a second equivalent of heme results in heme binding with a K(d2) in the range of 35-800 nM forming the diheme maquette state. Single conservative amino acid changes between peptide sequences are responsible for up to 10-fold changes in K(d) values. The equilibrium reduction midpoint potential (E(m7.5)) determined in the monoheme state ranges from -156 to -210 mV vs SHE and in the diheme state ranges from -144 to -288 mV. An observed heme-heme electrostatic interaction (>70 mV) in the diheme state indicates a syn global topology of the di-alpha-helical monomers. The heme affinity and electrochemistry of the three H24 variants studied identify the tight binding sites (K(d1) and K(d2) values <200 nM) having the lower reduction midpoint potentials (E(m7.5) values of -155 and -260 mV) with the H10 bound hemes in the parent tetraheme state of [H10H24-L6I,L13F](2), here called [I(6)F(13)H(24)](2). The results of this study illustrate that conservative hydrophobic amino acid changes near the heme binding site can modulate the E(m) by up to +/-50 mV and the K(d) by an order of magnitude. Furthermore, the effects of multiple single amino acid changes on E(m) and K(d) do not appear to be additive.
Details
- Title: Subtitle
- Hydrophobic Modulation of Heme Properties in Heme Protein Maquettes
- Creators
- Brian R Gibney - The Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine,University of Pennsylvania, Philadelphia, Pennsylvania 19104Steve S Huang - The Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine,University of Pennsylvania, Philadelphia, Pennsylvania 19104Jack J Skalicky - The Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine,University of Pennsylvania, Philadelphia, Pennsylvania 19104Ernesto J Fuentes - University of Iowa, Biochemistry and Molecular BiologyA. Joshua Wand - The Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine,University of Pennsylvania, Philadelphia, Pennsylvania 19104P. Leslie Dutton - The Johnson Research Foundation, Department of Biochemistry and Biophysics, School of Medicine,University of Pennsylvania, Philadelphia, Pennsylvania 19104
- Resource Type
- Journal article
- Publication Details
- Biochemistry (Easton), Vol.40(35), pp.10550-10561
- Publisher
- American Chemical Society
- DOI
- 10.1021/bi002806h
- PMID
- 11523997
- ISSN
- 0006-2960
- eISSN
- 1520-4995
- Language
- English
- Date published
- 09/04/2001
- Academic Unit
- Biochemistry and Molecular Biology
- Record Identifier
- 9984024408802771
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