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Design and engineering of an O(2) transport protein.

Koder RL, Anderson JL, Solomon LA, Reddy KS, Moser CC, Dutton PL - Nature (2009)

Bottom Line: Here we introduce this method with the design of an oxygen transport protein, akin to human neuroglobin.For stable oxygen binding without haem oxidation, water is excluded by simple packing of the protein interior and loops that reduce helical-interface mobility.O(2) affinities and exchange timescales match natural globins with distal histidines, with the remarkable exception that O(2) binds tighter than CO.

View Article: PubMed Central - PubMed

Affiliation: The Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
The principles of natural protein engineering are obscured by overlapping functions and complexity accumulated through natural selection and evolution. Completely artificial proteins offer a clean slate on which to define and test these protein engineering principles, while recreating and extending natural functions. Here we introduce this method with the design of an oxygen transport protein, akin to human neuroglobin. Beginning with a simple and unnatural helix-forming sequence with just three different amino acids, we assembled a four-helix bundle, positioned histidines to bis-histidine ligate haems, and exploited helical rotation and glutamate burial on haem binding to introduce distal histidine strain and facilitate O(2) binding. For stable oxygen binding without haem oxidation, water is excluded by simple packing of the protein interior and loops that reduce helical-interface mobility. O(2) affinities and exchange timescales match natural globins with distal histidines, with the remarkable exception that O(2) binds tighter than CO.

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Left: the spectra of the oxidized (green), reduced (blue), carboxy-ferrous (black) or oxy-ferrous (red) artificial oxygen transport protein 6 with either heme B (A) or heme A as the cofactor (B). These spectra are obtained at -15C where these spectra are stable for more than an hour. Right: stopped-flow spectral changes for mixing the reduced heme B proteins with oxygen at 15C. The fully designed oxygen transport protein 6 (C), shows the transformation of the reduced heme (blue) to the oxy-ferrous state (red) which eventually becomes oxidized (green), while the early intermediate 2 (D) proceeds directly and rapidly to the oxidized form.
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Figure 2: Left: the spectra of the oxidized (green), reduced (blue), carboxy-ferrous (black) or oxy-ferrous (red) artificial oxygen transport protein 6 with either heme B (A) or heme A as the cofactor (B). These spectra are obtained at -15C where these spectra are stable for more than an hour. Right: stopped-flow spectral changes for mixing the reduced heme B proteins with oxygen at 15C. The fully designed oxygen transport protein 6 (C), shows the transformation of the reduced heme (blue) to the oxy-ferrous state (red) which eventually becomes oxidized (green), while the early intermediate 2 (D) proceeds directly and rapidly to the oxidized form.

Mentions: Each maquette 2 - 6 displays ferric and ferrous visible spectra indicative of six-coordinate bis-histidine ligated heme B, characteristic of cytochromes b, deoxy-neuroglobin and cytoglobin, and quite distinct from the five-coordinate myoglobin and hemoglobin. Because the <1 nM KD for binding of the first heme is much tighter than 50 nM KD for the second heme 22, we simplify spectral analysis by binding one heme per bundle (Figure 2A). NMR assignments unambiguously identify the first heme B to bind at H7 positions at the open end of the candelabra structure of 6. Only ferrous heme 6 shows rapid and complete conversion of the ferrous heme into the oxyferrous heme with a halftime of ~50 milliseconds measured by stopped-flow spectroscopy. This oxyferrous spectrum is remarkably similar to native neuroglobin (supplementary figure S6). The oxyferrous state is stable for tens of seconds before single electron transfer from ferrous heme to O2 appears to generate superoxide.


Design and engineering of an O(2) transport protein.

Koder RL, Anderson JL, Solomon LA, Reddy KS, Moser CC, Dutton PL - Nature (2009)

Left: the spectra of the oxidized (green), reduced (blue), carboxy-ferrous (black) or oxy-ferrous (red) artificial oxygen transport protein 6 with either heme B (A) or heme A as the cofactor (B). These spectra are obtained at -15C where these spectra are stable for more than an hour. Right: stopped-flow spectral changes for mixing the reduced heme B proteins with oxygen at 15C. The fully designed oxygen transport protein 6 (C), shows the transformation of the reduced heme (blue) to the oxy-ferrous state (red) which eventually becomes oxidized (green), while the early intermediate 2 (D) proceeds directly and rapidly to the oxidized form.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539743&req=5

Figure 2: Left: the spectra of the oxidized (green), reduced (blue), carboxy-ferrous (black) or oxy-ferrous (red) artificial oxygen transport protein 6 with either heme B (A) or heme A as the cofactor (B). These spectra are obtained at -15C where these spectra are stable for more than an hour. Right: stopped-flow spectral changes for mixing the reduced heme B proteins with oxygen at 15C. The fully designed oxygen transport protein 6 (C), shows the transformation of the reduced heme (blue) to the oxy-ferrous state (red) which eventually becomes oxidized (green), while the early intermediate 2 (D) proceeds directly and rapidly to the oxidized form.
Mentions: Each maquette 2 - 6 displays ferric and ferrous visible spectra indicative of six-coordinate bis-histidine ligated heme B, characteristic of cytochromes b, deoxy-neuroglobin and cytoglobin, and quite distinct from the five-coordinate myoglobin and hemoglobin. Because the <1 nM KD for binding of the first heme is much tighter than 50 nM KD for the second heme 22, we simplify spectral analysis by binding one heme per bundle (Figure 2A). NMR assignments unambiguously identify the first heme B to bind at H7 positions at the open end of the candelabra structure of 6. Only ferrous heme 6 shows rapid and complete conversion of the ferrous heme into the oxyferrous heme with a halftime of ~50 milliseconds measured by stopped-flow spectroscopy. This oxyferrous spectrum is remarkably similar to native neuroglobin (supplementary figure S6). The oxyferrous state is stable for tens of seconds before single electron transfer from ferrous heme to O2 appears to generate superoxide.

Bottom Line: Here we introduce this method with the design of an oxygen transport protein, akin to human neuroglobin.For stable oxygen binding without haem oxidation, water is excluded by simple packing of the protein interior and loops that reduce helical-interface mobility.O(2) affinities and exchange timescales match natural globins with distal histidines, with the remarkable exception that O(2) binds tighter than CO.

View Article: PubMed Central - PubMed

Affiliation: The Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
The principles of natural protein engineering are obscured by overlapping functions and complexity accumulated through natural selection and evolution. Completely artificial proteins offer a clean slate on which to define and test these protein engineering principles, while recreating and extending natural functions. Here we introduce this method with the design of an oxygen transport protein, akin to human neuroglobin. Beginning with a simple and unnatural helix-forming sequence with just three different amino acids, we assembled a four-helix bundle, positioned histidines to bis-histidine ligate haems, and exploited helical rotation and glutamate burial on haem binding to introduce distal histidine strain and facilitate O(2) binding. For stable oxygen binding without haem oxidation, water is excluded by simple packing of the protein interior and loops that reduce helical-interface mobility. O(2) affinities and exchange timescales match natural globins with distal histidines, with the remarkable exception that O(2) binds tighter than CO.

Show MeSH
Related in: MedlinePlus