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Design and synthesis of multi‐haem proteins

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DeGrado WF, Dutton PL: Design and synthesis of multi-haem proteins.

The rate constants for both CO and histidine binding to the pentacoordinate state were determined using laser flash photolysis: a 1-ns pulse-width frequency doubled YAG laser at 532 nm excites the preformed carbonmonoxyferrous complex, causing the detachment of the ligand CO. This transiently forms an unliganded pentacoordinate heme protein and the rates of these binding processes were determined by analyzing the multi-exponential rebinding traces taken as a function of CO concentration using the method of Hargrove ():

Design and synthesis of multi-haem proteins

Designed proteins containing bound cofactors, both natural and synthetic, hold great promise as inexpensive biocompatible catalysts useful in medicine, energy production and green industrial catalysis (-). Heme proteins were the first bioinorganic-cofactor containing proteins created over fifteen years ago (, ), and since then they have proven instructive as to the underlying engineering requirements which drive natural heme protein evolution (-).

De novo design and synthesis of heme proteins.

The family of hexacoordinate hemoglobins are characterized by the property that they are bis-histidine-ligated in the oxidized state and exist in a mixed bis- and mono-histidine ligation state when reduced (). The transient pentacoordination of the heme cofactor allows for the ligation of molecular oxygen. We have recently reported the design, bacterial expression, and biochemical analysis of the completely artificial hexacoordinate oxygen transport protein HP7 (). This was the culmination of a series of design projects involving helical bundles which bind heme (-). The homodimeric protein HP7 is composed of two helix-loop-helix peptides in which the loops connect via a disulfide in a topology we have termed the ‘candelabra’ motif () (see ).

We report the mutational analysis of an artificial oxygen transport protein, HP-7, which operates via a mechanism akin to human neuroglobin and cytoglobin. This protein destabilizes one of two heme-ligating histidine residues by coupling histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Replacement of these glutamate residues with alanine, which is uncharged, increases the affinity of the distal histidine ligand by a factor of thirteen. Paradoxically, it also decreases heme binding affinity by a factor of five in the reduced state and sixty in the oxidized state. Application of a three-state binding model, in which an initial pentacoordinate binding event is followed by a protein conformational change to hexacoordinate, provides insight into the mechanism of this seemingly counterintuitive result: the initial pentacoordinate encounter complex is significantly destabilized by the loss of the glutamate side chains, and the increased affinity for the distal histidine only partially compensates. These results point to the importance of considering each oxidation and conformational state in the design of functional artificial proteins.

towards the de novo synthesis of functional heme proteins.

Comprehensive spectroscopic study using UVvis, ES-MS, MCD, multifrequency EPR, and XAS showed striking similarity between the engineered CuA center in azurin and other native and engineered CuA centers, demonstrating the close structural relationship between the two copper centers and the power of redesigning metal-binding sites in elucidating the structure and function of metalloproteins.

The CC9 gene was synthesized (Biomatik Corp., Cambridge, ON) with an N-terminal TEV cut site and inserted between the BamHI and XhoI restriction sites of pET32a (+) (Novagen) as previously described (). Mutagenesis to produce CC9-H7F and HP7-H7F was accomplished using the Quikchange method (Stratagene Inc., La Jolla, CA). Mutagenized plasmid DNA was transformed into chemically competent DH5α E. coli, the resulting clones verified by sequencing, and a verified plasmid was transformed into BL21(DE3) cells for expression. Labelled and unlabeled proteins were expressed and purified as we have published previously (). Apoprotein concentrations were determined optically using ε280 = 11.5 mM-1cm-1 on the basis of their tryptophan content ().

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  • Chemical Synthesis of Proteins | Annual Review of …

    24/11/2009 · Structural analysis of heme proteins: implications for design and prediction

  • Glossary | Linus Pauling Institute | Oregon State University

    Design and synthesis of multi…

  • Publications - University of Illinois at Urbana–Champaign

    07/11/2000 · Design and synthesis of multi-haem proteins

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319 | Peiwen Wu, Yang Yu, Claire E

This review summarizes the progress in the synthesis of close models of complex metalloproteins, followed by a description of recent advances in using the approach for making novel compounds that are unprecedented in either inorganic chemistry or biology.

Clontech Laboratories, Inc. - Life Science Tools and …

UV−vis, elemental analysis, and EPR studies of the cyanide-bound CuBMb indicate that a spin-coupled, CN--bridged CuB-heme center is formed in the designed model protein, as in the native HCOs.

ABCG2 Gene - GeneCards | ABCG2 Protein | ABCG2 …

In the process, it introduces the CuA center in four different systems---native protein systems, soluble protein truncates of native proteins, synthetic models using organic molecules, and biosynthetic models using proteins as ligands---with a greater emphasis on biosynthetic models of CuA, especially on new, deeper insights gained from their studies.

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Peptides were synthesized on a Millipore Model 9050Plus solid-phase peptide synthesizer using FMOC-protected amino acids on a PAL–PEG–PS resin. Peptides were cleaved by stirring the dry resin for 2 h under nitrogen at room temperature with 8.8 : 0.5 : 0.2 : 0.5 TFA : phenol : triisoporpylsilane : water followed by precipitation with cold diethylether. Crude peptides were purified by reversed-phase HPLC using a 2.5 × 25-cm preparative Vydac C18 column and a linear H2O/CH3CN (0.1% TFA) gradient at a flow rate of 7 mL/min. Purified peptides were then lyophilized and stored at 4°C. Formation of the disulfide linked di-α-helical peptides was accomplished by stirring monomer peptides overnight in a 250 mM Tris buffer solution at pH 8.5. The disulfide-linked peptides were purified by reversed-phase HPLC as described above. Electrospray/ionization mass spectrometry (PeptidoGenic Research) was used to confirm the identity of the peptides.

ABCB10 Gene - GeneCards | ABCBA Protein | ABCBA …

Holoprotein complexes, containing 1.0 equivalent per homodimer, were prepared as before () by five consecutive additions of 0.2 equivalents of hemin in DMSO with at least ten minutes between additions. Proteins were then purified from any unbound heme by passage through a PD10 desalting columns pre-equilibrated with 250mM Boric Acid, 100mM KCl pH 9.0. Holoprotein solution concentrations were determined using experimentally derived Soret ε414 = 129 mM-1cm-1 for HP7-H7F and 118 mM-1cm-1 for CC9-H7F. If necessary, both holo- and apoproteins were concentrated using Centricon YM-10 spin concentrators (Millipore, Inc., Billerica, MA).

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