Compositions and methods for the delivery of nitric oxide
H-NOX proteins are mutated to exhibit improved or optimal kinetic and thermodynamic properties for blood gas NO delivery. The engineered H-NOX proteins comprise mutations that impart altered NO or O 2 ligand-binding relative to the corresponding wild-type H-NOX domain, and are operative as physiologically compatible mammalian blood NO gas carriers. The invention also provides pharmaceutical compositions, kits, and methods that use wild-type or mutant H-NOX proteins for the treatment of any condition for which delivery of NO is beneficial.
1. An isolated H-NOX protein comprising at least one distal pocket mutation that alters the k off , k 1 , or k 2 for NO, the 0 2 dissociation constant, the NO dissociation constant, or the NO reactivity compared to that of a corresponding wild type H-NOX protein, wherein the H-NOX protein does not comprise a guanylyl cyclase catalytic domain, wherein the distal pocket mutation comprises a substitution at a residue that corresponds to at least one of Thr4, Ile5, Thr9, Trp9, Trp67, Asn74, Ile75, Phe78, Phe82, Tyr140, and Leu144 of a T. tengcongensis H-NOX of SEQ ID NO:54, wherein the mutant H-NOX protein is not Thermoanaerobacter tengcongensis H-NOX Y140L, T. tengcongensis H-NOX W9F, T. tengcongensis H-NOX W9F/Y140L, T. tengcongensis H-NOX F78Y/Y140L, Legionella pneumophilia 2 H-NOX F142Y, Rattus norvegicus sGC 1 H-NOX (1-385) I145Y; and wherein either (a) the k off , k 1 , or k 2 for NO of the H-NOX protein is between about 1×10 −4 s −1 and about 10 s −1 at 37° C., and the 0 2 dissociation constant of the H-NOX protein is at least about 1 M at 37° C., or (b) the NO dissociation constant of the H-NOX protein is within 2 orders of magnitude of that of human hemoglobin alpha, and the NO reactivity of the H-NOX protein is at least 10-fold lower than that of human hemoglobin alpha.
2. The isolated H-NOX protein of claim 1 , wherein the k off , k 1 , or k 2 for NO of the mutant H-NOX protein is between about 1×10 −4 s −1 and about 0.012 s −1 at 37° C.
3. The isolated H-NOX protein of claim 1 , wherein the NO reactivity of the mutant H-NOX protein is less than about 700 s −1 .
4. The isolated H-NOX protein of claim 1 , wherein the NO reactivity of the mutant H-NOX protein is at least 100-fold lower than that of human hemoglobin alpha.
5. The isolated H-NOX protein of claim 1 , wherein the rate of heme autoxidation of the H-NOX protein is less than about 1 h−1 at 37° C.
6. The isolated H-NOX protein of claim 1 , wherein the H-NOX protein comprises at least one mutation that is not in the distal pocket.
7. The isolated H-NOX protein of claim 1 , wherein the corresponding wild-type H-NOX protein is a human protein.
8. The isolated H-NOX protein of claim 1 , wherein the corresponding wild-type H-NOX protein is a T. tengcongensis protein.
9. The isolated H-NOX protein of claim 1 , wherein the H-NOX protein is a fusion protein that includes an H-NOX domain and part or all of another protein.
10. The isolated H-NOX protein of claim 1 , wherein the mutant H-NOX protein is covalently bound to another molecule or moiety or is part of a fusion protein.
11. The isolated H-NOX protein of claim 10 , wherein the mutant H-NOX protein is covalently bound to polyethylene glycol.
12. A recombinant nucleic acid encoding an H-NOX protein of claim 1 .
13. A vector comprising a nucleic acid of claim 12 .
14. A cell comprising a nucleic acid of claim 12 or the vector of claim 13 .
15. A method of producing an H-NOX protein comprising culturing a cell comprising a nucleic acid encoding an H-NOX protein of claim 1 under conditions suitable for production of the protein.
16. The method of claim 15 , further comprising the step of purifying the H-NOX protein.
17. A method of delivering NO to an individual comprising administering to the individual in need thereof the H-NOX protein of claim 1 in an amount sufficient to deliver an effective amount of NO to an individual, wherein the NO dissociation constant of the H-NOX protein is within 2 orders of magnitude of that of human hemoglobin alpha, and the NO reactivity of the H-NOX protein is at least 10-fold lower than that of human hemoglobin alpha.