Bio-engineered hyper-functional “super” helicases
View Patent ↗Conformationally-constrained helicases having improved activity and strength are provided. Methods of making conformationally-constrained helicases having improved activity and strength are provided. Methods of using conformationally-constrained helicases having improved activity and strength are provided.
1. A method of catalyzing an unwinding reaction of a double-stranded DNA, comprising contacting the double-stranded DNA with a conformationally-constrained helicase
wherein the conformationally-constrained helicase is selected from the group consisting of a Rep helicase from E. coli , a UvrD helicase from E. coli , a PcrA helicase from B. stearothermophilus , or a homolog thereof;
wherein the conformationally-constrained helicase comprises a first subdomain comprising a 1A or a 1B subdomain having a first amino acid and a second subdomain comprising a 2B subdomain having a second amino acid,
wherein the first amino acid corresponds to any one of positions 84-116 or 178-196 of the helicase amino acid sequence, relative to SEQ ID NO:32;
wherein the second amino acid corresponds to any one of positions 388-411, 422-444, and 518-540 of the helicase amino acid sequence, relative to SEQ ID NO:32;
wherein a side chain of the first amino acid is covalently crosslinked to a side chain of the second amino acid with a linker to form an active, conformationally-constrained helicase;
wherein said first amino acid is less than about 20 Å from said second amino acid when the helicase is in an active conformation, and
wherein the conformationally-constrained helicase comprises at least one degree of freedom less than a helicase that is not constrained as such.
2. The method of claim 1 , wherein the conformationally-constrained helicase is chemically crosslinked.
3. The method of claim 1 , wherein the conformationally-constrained helicase comprises SEQ ID NO:4 or SEQ ID NO:12.
4. The method of claim 1 , wherein the linker comprises an alkyl having a length in the range from C7 to C23.
5. The method of claim 1 , wherein the first amino acid is covalently crosslinked to the second amino acid by a disulfide bond or a chemical crosslinker.
6. The method of claim 5 , wherein the chemical crosslinker is a bis-maleimide crosslinker.
7. The method of claim 5 , wherein the chemical crosslinker has a length of from about 6 Å to about 25 Å.
8. The method of claim 1 , wherein the chemical crosslinker is selected from the group consisting of