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

Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Journal of the American Chemical Society (Impact Factor: 12.11). 10/2010; 132(44):15516-8. DOI: 10.1021/ja107487b
Source: PubMed


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.

Download full-text


Available from: Ivan V Korendovych
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Natural proteins are concatenated amino acids with definite handedness or chirality, with their spatial orientation being preferentially left handed or L-chiral. This paper discusses the biophysics of stereo-chemical perturbation to proteins using D-(α) amino acid and its utility as an additional design alphabet while scripting novel protein structures.
    Full-text · Article · Dec 2010 · Systems and Synthetic Biology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The active sites of enzymes are lined with side chains whose dynamic, geometric, and chemical properties have been finely tuned relative to the corresponding residues in water. For example, the carboxylates of glutamate and aspartate are weakly basic in water but become strongly basic when dehydrated in enzymatic sites. The dehydration of the carboxylate, although intrinsically thermodynamically unfavorable, is achieved by harnessing the free energy of folding and substrate binding to reach the required basicity. Allosterically regulated enzymes additionally rely on the free energy of ligand binding to stabilize the protein in a catalytically competent state. We demonstrate the interplay of protein folding energetics and functional group tuning to convert calmodulin (CaM), a regulatory binding protein, into AlleyCat, an allosterically controlled eliminase. Upon binding Ca(II), native CaM opens a hydrophobic pocket on each of its domains. We computationally identified a mutant that (i) accommodates carboxylate as a general base within these pockets, (ii) interacts productively in the Michaelis complex with the substrate, and (iii) stabilizes the transition state for the reaction. Remarkably, a single mutation of an apolar residue at the bottom of an otherwise hydrophobic cavity confers catalytic activity on calmodulin. AlleyCat showed the expected pH-rate profile, and it was inactivated by mutation of its active site Glu to Gln. A variety of control mutants demonstrated the specificity of the design. The activity of this minimal 75-residue allosterically regulated catalyst is similar to that obtained using more elaborate computational approaches to redesign complex enzymes to catalyze the Kemp elimination reaction.
    Full-text · Article · Apr 2011 · Proceedings of the National Academy of Sciences
  • [Show abstract] [Hide abstract]
    ABSTRACT: Computational and theoretical methods are advancing protein design as a means to create and investigate proteins. Such efforts further our capacity to control, design and understand biomolecular structure, sequence and function. Herein, the focus is on some recent applications that involve using theoretical and computational methods to guide the design of protein sequence ensembles, new enzymes, proteins with novel cofactors, and membrane proteins.
    No preview · Article · Jun 2011 · Current opinion in chemical biology
Show more