De novo design and molecular assembly of a transmembrane diporphyrin-binding protein complex

Abstract

The de novo design of membrane proteins remains difficult despite recent advances in understanding the factors that drive membrane protein folding and association. We have designed a membrane protein PRIME (PoRphyrins In MEmbrane) that positions two non-natural iron diphenylporphyrins (Fe(III)DPP's) sufficiently close to provide a multicentered pathway for transmembrane electron transfer. Computational methods previously used for the design of multiporphyrin water-soluble helical proteins were extended to this membrane target. Four helices were arranged in a D(2)-symmetrical bundle to bind two Fe(II/III) diphenylporphyrins in a bis-His geometry further stabilized by second-shell hydrogen bonds. UV-vis absorbance, CD spectroscopy, analytical ultracentrifugation, redox potentiometry, and EPR demonstrate that PRIME binds the cofactor with high affinity and specificity in the expected geometry.

Figure 1

a) Evolution of the four-helix porphyrin binding bundles: from water-soluble proteins through amphiphilic maquettes to PRIME. Hydrophilic residues are shown in blue, hydrophobic residues - in green, metal cofactor – in brown. b) The design of the iron coordination site in PRIME. c) General approach to the design of PRIME. The final repacked model of PRIME in a bilayer (yellow) viewed along the directions parallel (d) and normal (e) to the membrane. The sequence of PRIME is Ac-AIYGILAHSL ASILALLTGF LTIW-CONH₂.

Figure 2. PRIME’s specificity

Titration of different cofactors (12 μM) with PRIME peptides. Red circles – Fe^IIIDPP with PRIME (solid circles – slow equilibration method, 2 mM DPC; empty circles – 20 mM DPC); black triangles – hemin with PRIME in DPC (20 mM); green squares – Fe^IIIDPP with PRIME T18A in DPC (20 mM); blue circles – Fe^IIIDPP with PRIME A15I G19I in DPC (20 mM). Cofactor concentration was 12 μM in all cases. Unless explicitly noted, all samples were reconstituted using fast equilibration method in 10 mM phosphate buffer (pH 7.4) as described in the Supporting Information.

Figure 3

CD spectra of PRIME in the absence (blue) and in the presence of Fe^IIIDPP. Experimental details are given in the Supporting Information. In the case of PRIME-Fe^IIIDPP, MRE values in the 360-460 nm region are normalized to the number of cofactor molecules.

Figure 4

EPR spectrum of Fe^IIIDPP (1.5 mM) bound to PRIME (3.1 mM) in DPC micelles (185 mM) in frozen glass containing 30% glycerol. The peak at g = 6.0 represents high-spin iron(III) diphenylporphyrin not bound to two histidine residues; the peak at g = 4.3 represents a small amount of high-spin non-heme Fe(III).