Fluorescent protein sensors for detection of analytes

• US 6,197,928 B1
• Filed: 03/14/1997
• Issued: 03/06/2001
• Est. Priority Date: 03/14/1997
• Status: Expired due to Term

• Current Assignee: National Institutes of Health (NIH) (Government of the United States of America)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Tsien, Roger Y., Miyawaki, Atsushi
• Primary Examiner(s): Scheiner, Laurie
• Assistant Examiner(s): Parkin, Jeffrey S.
• Application Number:
  US08/818,252
• Time in Patent Office: 1,453 Days
• Field of Search: 530/350, 530/402
• US Class Current: 530/350
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

Fluorescent indicators including a binding protein moiety, a donor fluorescent protein moiety, and an acceptor fluorescent protein moiety are described. The binding protein moiety has an analyte-binding region which binds an analyte and causes the indicator to change conformation upon exposure to the analyte. The donor moiety and the acceptor moiety change position relative to each other when the analyte binds to the analyte-binding region. The donor moiety and the acceptor moiety exhibit fluorescence resonance energy transfer when the donor moiety is excited and the distance between the donor moiety and the acceptor moiety is small. The indicators can be used to measure analyte concentrations in samples, such as calcium ion concentrations in cells.

Claims

1. A fluorescent indicator protein comprising:

a binding protein moiety having an analyte-binding domain which binds an analyte and causes the indicator to change conformation upon exposure to the analyte;

a donor fluorescent protein moiety covalently coupled to the binding protein moiety; and

an acceptor fluorescent protein moiety covalently coupled to the binding protein moiety,

wherein a conformational change is induced in the binding protein moiety upon analyte binding, which in turn induces conformational changes in the position or orientation of the donor fluorescent protein and acceptor fluorescent protein moieties with respect to one another, thereby altering the relative amounts of fluorescence from the two fluorescent protein moieties when the donor is excited by irradiation.

2. The indicator of claim 1, wherein the donor fluorescent protein moiety and the acceptor fluorescent protein moiety are Aequorea-related fluorescent protein moieties.

3. The indicator of claim 2, wherein the indicator fuirther includes a target peptide moiety, a linker moiety and the binding protein moiety further includes a peptide-binding domain for binding the target peptide moiety,

wherein the linker moiety covalently couples the binding protein moiety and the target peptide moiety together,

wherein the binding protein moiety and target peptide moiety are coupled to either an acceptor fluorescent protein moiety or a donor fluorescent protein moiety, and

wherein analyte binding to said analyte-binding domain induces conformational changes in the position or orientation of the target peptide moiety and the peptide-binding domain which in turn induces changes in the position or orientation of the donor fluorescent protein and acceptor fluorescent protein moieties with respect to one another, thereby altering the relative amounts of fluorescence from the two fluorescent protein moieties when the donor is excited by irradiation.

4. The indicator of claim 3, wherein the indicator further comprises a localization sequence.

5. The indicator of claim 4, wherein the localization sequence is a nuclear localization sequence, an endoplasrnic reticulum localization sequence, a peroxisome localization sequence, a mitochondrial localization sequence, a Golgi apparatus localization sequence, or a plasma membrane localization sequence.

6. The indicator of claim 3, wherein the donor fluorescent protein moiety is covalently coupled to the binding protein moiety and the acceptor fluorescent protein moiety is covalently coupled to the target peptide moiety.

7. The indicator of claim 3, wherein the linker moiety is a peptide moiety.

8. The indicator of claim 7, wherein the linker moiety includes between 1 amino acid residue and 30 amino acid residues.

9. The indicator of claim 7, wherein the indicator is a single polypeptide.

10. The indicator of claim 9, wherein one of the donor fluorescent protein moiety or the acceptor fluorescent protein moiety is covalently coupled to the carboxy terminus of the single polypeptide and the other of the donor fluorescent protein moiety or the acceptor fluorescent protein moiety is covalently coupled to the amino terminus of the single polypeptide.

11. The indicator of claim 2 wherein the binding protein moiety is calmodulin, cGMP-dependent protein kinase, a steroid hormone receptor, a ligand binding domain of a steroid hormone receptor, protein kinase C, inositol-1,4,5-triphosphate receptor, or recoverin.

12. The indicator of claim 3, wherein the binding protein moiety is calmodulin.

13. The indicator of claim 12, wherein the donor fluorescent protein moiety is the green fluorescent protein mutant P4-3, the green fluorescent protein mutant EBFP, or the green fluorescent protein mutant W1B, and the acceptor fluorescent protein moiety is the green fluorescent protein mutant S65T the green fluorescent protein mutant EGFP, or the green fluorescent protein mutant 10C.

14. The indicator of claim 12, wherein the target peptide moiety is a calnodulin-binding domain of skeletal muscle myosin light chain kinase (skMLCKp), smooth muscle myosin light chain kinase (smMLCK), calmodulin kinase II (CaMKII), Caldesmon, Calspermin, phosphofructokinase, calcineurin, phosphorylase kinase, Ca2+ATPase, 59 Kda phosphodiseterase (PDE), 60 Kda phosphodiseterase (PDE), nitric oxide synthase, type I adenylyl cyclase, Bordetella pertussis adenylyl cyclase, Neuromodulin, Spectrin, myristoylated alanine-rich C kinase substrate (MARCKS), MacMARCKS(F52), b-Adducin, heat shock protein HSP90a, human immunodeficiency virus envelop glycoprotin 160(HIV-1 gp160), brush-border myosin heavy chain-I (BBMHBI), Dilute myosin heavy chain (MHC), Mastoparan, Melittin, Glucagon, Secretin, vasoactive intestinal peptide (VIP), gastrin inhibitory peptide (GIP), or calmodulin binding peptide-2 (Model Peptide CBP2).

15. The indicator of claim 14, wherein the target peptide moiety is the calmodulin-binding domain of skeletal muscle myosin light chain kinase (skMLCK).

16. The indicator of claim 15, wherein the linker moiety is -Gly—

Gly-.

17. A fluorescent indicator protein further comprising:

a target peptide;

a binding protein moiety having an analyte-binding domain which binds an analyte and causes the indicator to change conformation upon exposure to the analyte, and a peptide-binding domain for binding the target peptide moiety;

a linker moiety that covalently couples the binding protein and the target peptide moiety and is a peptide moiety;

a donor fluorescent protein moiety covalently coupled to the binding protein moiety; and

an acceptor fluorescent protein moiety covalently coupled to the target peptide moiety,

wherein analyte binding to said analyte-binding domain induces conformational changes in the position or orientation of the target peptide moiety and the peptide-binding domain which in turn induces changes in the position or orientation of the donor fluorescent protein and acceptor fluorescent protein moieties with respect to one another, thereby altering the relative amounts of fluorescence from the two fluorescent protein moieties when the donor is excited by irradiation.

18. The indicator of claim 17, wherein the donor fluorescent protein moiety and the acceptor fluorescent protein moiety are Aequorea-related fluorescent protein moieties.

19. The indicator of claim 18, wherein the donor fluorescent protein moiety is the green fluorescent protein mutant, P4-3, the green fluorescent protein mutant, EBFP, or the green fluorescent protein mutant, W1B, and the acceptor fluorescent protein moiety is the green fluorescent protein mutant, S65T, the green fluorescent protein mutant, EGFP, or the green fluorescent protein mutant 10c.

20. The indicator of claim 19, wherein the binding protein moiety is calmodulin.

21. The indicator of claim 20, wherein the target peptide moiety is the calmodulin binding domain, (M13).

22. The indicator of claim 21, wherein one of the donor fluorescent protein moiety or the acceptor fluorescent protein moiety is located at the carboxy terminus of the single polypeptide and the other of the donor fluorescent protein moiety or the acceptor fluorescent protein moiety is located at the amino terminus of the single polypeptide.

23. The indicator of claim 17, wherein the indicator further comprises a nuclear localization signal, an endoplasmic reticulum localization signal, a peroxisome localization signal, a mitochondrial localization signal, a Golgi apparatus localization sequence, or a plasma membrane localization sequence.

24. A method for determining the concentration of an analyte in a sample comprising:

contacting the sample with a fluorescent indicator protein comprising a binding protein moiety having an analyte-binding domain which binds an analyte, a donor fluorescent protein moiety covalently coupled to the binding protein moiety, and an acceptor fluorescent protein moiety covalently coupled to the binding protein moiety, wherein analyte binding to the analyte binding domain causes a conformational change in the analyte binding domain which in turn induces conformational changes in the position or orientation of the donor fluorescent protein and acceptor fluorescent protein moieties with respect to one another, thereby altering the relative amounts of fluorescence from the two fluorescent protein moieties when the donor is excited by irradiation;

exciting the donor moiety; and

determining the degree of fluorescence resonance energy transfer in the sample corresponding to the concentration of the analyte in the sample.

25. The method of claim 24, wherein the step of determining the degree of fluorescence resonance energy transfer in the sample comprises measuring light emitted the acceptor fluorescent protein moiety.

26. The method of claim 24, wherein determining the degree of fluorescence resonance energy transfer in the sample comprises measuring light emitted from the donor fluorescent protein moiety, measuring light emitted from the acceptor fluorescent protein moiety, and calculating a ratio of the light emitted from the donor fluorescent protein moiety and the light emitted from the acceptor fluorescent protein moiety.

27. The method of claim 24, wherein the step of determining the degree of fluorescence resonance energy transfer in the sample comprises measuring the excited state lifetime of the donor moiety.

28. The method of claim 26, further comprising the steps of determining the concentration of the analyte at a first time after contacting the sample with the fluorescence indicator, determining the concentration of the analyte at a second time after contacting the sample with the fluorescence indicator, and calculating the difference in the concentration of the analyte at the first time and the second time, whereby the difference in the concentration of the analyte in the sample reflects a change in concentration of the analyte present in the sample.

29. The method of claim 28, further comprising the step of contacting the sample with a compound between the first time and the second time, whereby a difference in the concentration of the analyte in the sample between the first time and the second time indicates that the compound alters the presence of the analyte.

30. The method of claim 24, wherein the sample comprises an intact cell and the contacting step comprises incorporating the fluorescent indicator into the cell.

31. The method of claim 30, wherein step of incorporating the fluorescent indicator into the cell includes transfecting the cell with an expression vector comprising expression control sequences operably linked to a nucleic acid sequence coding for the expression of the fluorescent indicator.

32. The method of claim 24, wherein the analyte is calcium.

33. The method of claim 24, wherein the donor fluorescent protein moiety and the acceptor fluorescent protein moiety are Aequorea-related fluorescent protein moieties.

34. The method of claim 33, wherein the indicator further includes a target peptide moiety and a linker moiety that covalently couples the binding protein and the target peptide moiety and is a peptide moiety, and the binding protein moiety further includes a peptide-binding region for binding a target peptide moiety.

35. The method of claim 34, wherein the indicator is a single polypeptide.

36. The method of claim 34, wherein the binding protein moiety is calmodulin, the donor fluorescent protein moiety is the green fluorescent protein mutant, P4-3, the green fluorescent protein mutant, EBFP, or the green fluorescent protein mutant, W1B, the acceptor fluorescent protein moiety is the green fluorescent protein mutant, S65T, the green fluorescent protein mutant, EGFP, or the green fluorescent protein mutant, 10c, and the target peptide moiety is the calmodulin binding domain, M13.

37. The method of claim 36, wherein the indicator further comprises a localization sequence.