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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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Related in: MedlinePlus

The design of an artificial oxygen transport protein (6) begins with an extremely simple three amino acid near heptad repeat sequence (1) and progresses through the design stages via a series of amino acid changes (red) and trial intermediates (2-5) that are tested to reveal functional properties and clarify the roles of individual amino acids. In 2-5, helical sequences shown are linked by cysteine disulfide loops and self assemble in 4 helix bundles, while in 6 a longer loop (red) unites two identical sequences, with the loops themselves now disulfide linked, as shown at right.
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Figure 1: The design of an artificial oxygen transport protein (6) begins with an extremely simple three amino acid near heptad repeat sequence (1) and progresses through the design stages via a series of amino acid changes (red) and trial intermediates (2-5) that are tested to reveal functional properties and clarify the roles of individual amino acids. In 2-5, helical sequences shown are linked by cysteine disulfide loops and self assemble in 4 helix bundles, while in 6 a longer loop (red) unites two identical sequences, with the loops themselves now disulfide linked, as shown at right.

Mentions: The engineering-based design of functional synthetic proteins progresses through 4 stages (Figure 1): 1) assemble a simple, robust generic protein framework, such as a helical bundle, of appropriate size to sustain eventual cofactor binding and catalytic function; 2) insert cofactor binding amino acids, keeping the number of amino acid changes few to control complexity; 3) adjust the sequence for improved structural resolution; 4) iteratively test, redesign and add engineering elements to refine function. This method of designing proteins follows that long used by artists and architects who develop maquettes, simple models that are progressively altered to test and determine the ultimate characteristics of their constructions.


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

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

The design of an artificial oxygen transport protein (6) begins with an extremely simple three amino acid near heptad repeat sequence (1) and progresses through the design stages via a series of amino acid changes (red) and trial intermediates (2-5) that are tested to reveal functional properties and clarify the roles of individual amino acids. In 2-5, helical sequences shown are linked by cysteine disulfide loops and self assemble in 4 helix bundles, while in 6 a longer loop (red) unites two identical sequences, with the loops themselves now disulfide linked, as shown at right.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: The design of an artificial oxygen transport protein (6) begins with an extremely simple three amino acid near heptad repeat sequence (1) and progresses through the design stages via a series of amino acid changes (red) and trial intermediates (2-5) that are tested to reveal functional properties and clarify the roles of individual amino acids. In 2-5, helical sequences shown are linked by cysteine disulfide loops and self assemble in 4 helix bundles, while in 6 a longer loop (red) unites two identical sequences, with the loops themselves now disulfide linked, as shown at right.
Mentions: The engineering-based design of functional synthetic proteins progresses through 4 stages (Figure 1): 1) assemble a simple, robust generic protein framework, such as a helical bundle, of appropriate size to sustain eventual cofactor binding and catalytic function; 2) insert cofactor binding amino acids, keeping the number of amino acid changes few to control complexity; 3) adjust the sequence for improved structural resolution; 4) iteratively test, redesign and add engineering elements to refine function. This method of designing proteins follows that long used by artists and architects who develop maquettes, simple models that are progressively altered to test and determine the ultimate characteristics of their constructions.

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