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 - 320 . (canceled)
321 . An isolated H-NOX protein comprising at least one mutation that alters the k off , k 1 , or k 2 for NO, the O 2 dissociation constant, the NO dissociation constant, or the NO reactivity compared to that of a corresponding wild type H-NOX protein, wherein the mutant H-NOX protein is not Thermoanaerobacter tengcongensis H-NOX Y140L, T. tengcongensis H-NOX W9F, T. tengcongensis H-NOX F78Y/Y140L, Legionella pneumophilia 2 H-NOX F142Y, Rattus norvegicus sGC β1 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 O 2 dissociation constant of the H-NOX protein is at least about 1 μM 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.
322 . The isolated H-NOX protein of claim 321 , 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 10 s −1 at 37° C., and wherein the O 2 dissociation constant of the H-NOX protein is at least about 1 μM at 37° C.
323 . The isolated H-NOX protein of claim 321 , wherein the NO dissociation constant of the mutant H-NOX protein is within 2 orders of magnitude of that of human hemoglobin alpha, and wherein the NO reactivity of the mutant H-NOX protein is at least 10-fold lower than that of human hemoglobin alpha.
324 . The isolated H-NOX protein of claim 321 , 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.
325 . The isolated H-NOX protein of claim 321 , wherein the NO reactivity of the mutant H-NOX protein is less than about 700 s −1 .
326 . The isolated H-NOX protein of claim 321 , wherein the NO reactivity of the mutant H-NOX protein is at least 100-fold lower than that of human hemoglobin alpha.
327 . The isolated H-NOX protein of claim 321 , wherein the rate of heme autoxidation of the H-NOX protein is less than about 1 h −1 at 37° C.
328 . The isolated H-NOX protein of claim 321 , wherein the H-NOX protein comprises at least one distal pocket mutation.
329 . The isolated H-NOX protein of claim 328 , wherein the distal pocket mutation is a mutation of a residue in alpha-helix A, D, E or G.
330 . The isolated H-NOX protein of claim 328 , wherein the distal pocket mutation corresponds to at least one of Thr4, Ile5, Thr8, Trp9, Trp67, Asn74, Ile75, Phe78, Phe82, Tyr140, and Leu144 of T. tengcongensis H-NOX.
331 . The isolated H-NOX protein of claim 328 , wherein the distal pocket mutation is a mutation in which a residue that corresponds to Tyr140 of T. tengcongensis H-NOX is replaced by any other amino acid or a residue that corresponds to Phe142 of L. pneumophilia 2 H-NOX is replaced by any other amino acid.
332 . The isolated protein of claim 328 , wherein the distal pocket mutation corresponds to an N74E mutation of T. tengcongensis , an N74A mutation of T. tengcongensis , or an N74H mutation of T. tengcongensis.
333 . The isolated protein of claim 328 , wherein the distal pocket mutations corresponds to an N74A/Y140H mutation of T. tengcongensis , or a W9F/N74A mutation of T. tengcongensis.
334 . The isolated protein of claim 332 , wherein the distal pocket mutation is an N74E mutation of T. tengcongensis , an N74A mutation of T. tengcongensis , or an N74H mutation of T. tengcongensis.
335 . The isolated protein of claim 334 , wherein the distal pocket mutations is an N74A/Y140H mutation of T. tengcongensis , or a W9F/N74A mutation of T. tengcongensis.
336 . The isolated H-NOX protein of claim 321 , wherein the H-NOX protein comprises at least one mutation that is not in the distal pocket
337 . The isolated H-NOX protein of claim 321 , wherein the corresponding wild-type H-NOX protein is a mammalian protein.
338 . The isolated H-NOX protein of claim 337 , wherein the corresponding wild-type H-NOX protein is a human protein.
339 . The isolated H-NOX protein of claim 338 , wherein the corresponding wild-type H-NOX protein is β1.
340 . The isolated H-NOX protein of claim 321 , wherein the corresponding wild-type H-NOX protein is a bacterial protein.
341 . The isolated H-NOX protein of claim 340 , wherein the corresponding wild-type H-NOX protein is a T. tengcongensis protein.
342 . The isolated H-NOX protein of claim 321 , wherein the H-NOX protein does not have a guanylyl cyclase catalytic domain.
343 . The isolated H-NOX protein of claim 321 , wherein the H-NOX protein is a fusion protein that includes an H-NOX domain and part or all of another protein.
344 . The isolated H-NOX protein of claims 321 , wherein the mutant H-NOX protein is covalently bound to another molecule or moiety or is part of a fusion protein.
345 . The isolated H-NOX protein of claim 344 , wherein the mutant H-NOX protein is covalently bound to polyethylene glycol.
346 . A recombinant nucleic acid encoding an H-NOX protein of claim 321 .
347 . A vector comprising a nucleic acid of claim 346 .
348 . A cell comprising a nucleic acid of claim 346 or the vector of claim 347 .
349 . A method of producing an H-NOX protein comprising culturing a cell comprising a nucleic acid encoding an H-NOX protein of any one of claim 321 under conditions suitable for production of the protein.
350 . The method of claim 349 , further comprising the step of purifying the H-NOX protein.