De Novo Design and Molecular Assembly of a Transmembrane Diporphyrin-Binding Protein Complex

Abstract

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 secondshell 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.Significant progress has been achieved in the computational design of functional water-soluble proteins.1-6 However, the de novo design of membrane proteins remains difficult despite recent advances in understanding the factors that drive membrane protein folding and association. 7 Here, we present the de novo design of a membrane protein PRIME (PoRphyrins In MEmbrane) that utilizes a non-natural iron diphenylporphyrin (Fe III DPP) with the ultimate goal of facilitating electron transfer across a bilayer. Transmembrane (TM) electron transfer lies at the heart of photosynthesis and ATP production in a variety of organisms and thus is of great fundamental interest and, potentially, of practical importance.Considerable progress has been made in the design of watersoluble multiheme proteins [8][9][10][11] and amphiphilic maquettes [12][13][14][15] that position a single heme in the membrane phase. Also, in an elegant study, Cordova et al. 16 designed a TM peptide that binds a single heme between two helices whose geometry is defined by a GXXXG motif. To be generally useful for transmembrane electron transfer, it is important to also design systems that position multiple redox-active cofactors sufficiently close to provide a multicentered pathway for electrons to rapidly pass across the bilayer.Here we extend previous computational methods used for the design of water-soluble multiheme proteins to membrane targets. The design of PRIME is based on the backbone of a water-soluble multiporphyrinbinding peptide. 17,18 Its fold appeared to be particularly well suited for a membrane environment, because it has a tight interhelical "Ala-coil" motif, which is favored in both water and membrane-soluble proteins. 19Four helices were arranged in a D 2 -symmetrical bundle to bind two Fe(II/III) diphenylporphyrins in a bis-His geometry. In the design, the His ligands are stabilized by a bifurcated second-shell hydrogen bond with a main chain carbonyl and a Thr (T18) hydroxyl from a neighboring helix (Figure 1). 20Following the optimization of the coordination sphere of the iron, the backbone was repacked to produce a final sequence. Sid...

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