User:Steven J. Koch/His-tag lifetime
Skoch3 18:13, 17 July 2007 (EDT):This is a document I wrote in December 2003 for people who were considering using Ni-NTA / hexahistidine tags as a tethering method. I think the lifetime of this attachment is too short for long-term attachment, if using only a single his-tag.
Steve’s best guess conclusions'[1]':
- The Ni-NTA/hexahistidine bond has a zero-force lifetime (inverse off-rate) of ~100 seconds[2-4].
- Thus a single his-tag is inappropriate for long-term cargo transport and / or storage
- However, in the case of the DARPA tagging module, this lifetime is probably long enough to keep the QDs from diffusing around the module too much (i.e., they will detach every once in a while, but very soon find another post to bind.
- Further indication of a relatively short lifetime is the fact that Ni-NTA chromatography works: i.e., the lifetime is short enough for imidazole to compete for binding sites.
- For Ni-NTA/hexahistidine, the characteristic force for decreasing the lifetime appears to be about 20 pN[2].
- This is thus a complicated situation, since kinesin typically doesn’t produce force in the 20 pN range. Thus, passing off would be more like a “pause and wait ~100s” situation, rather than a “rip-off the his-tag” situation.
- Depending on the needs and device construction, this may or may not be OK. For example, in DARPA tagging module, if you keep running into more QDs, it will keep slowing down the module.
- For a particular anti-his antibody[5], there is possible indication that the characteristic force appeared to be ~ 3 pN. (You would expect there to be little correlation between NTA/his and anti-his/his.)
- This is thus a complicated situation, since kinesin typically doesn’t produce force in the 20 pN range. Thus, passing off would be more like a “pause and wait ~100s” situation, rather than a “rip-off the his-tag” situation.
- Multiple hexahistidine tags can dramatically increase the lifetime[3, 6, 7]
- However, if the characteristic force is really >=20 pN (see above), then multiple his-tags may in fact preclude passing off of cargo[7].
List of important literature on this subject
- Hinterdorfer AFM studies of Ni-NTA/hexahistidine[2]
- Gaub AFM studies of Metal2+-NTA/hexahistidine[4] (Information on Co2+, Cu2+, Ni2+, Zn2+)
- Pluckthun “zero force” studies of Ni-NTA/hexahistidine lifetime using the BIACORE apparatus[3].
- Block his-tagged lamda exonuclease study, using antibody against polyhistidine[5].
- Bachand and Montemagno, multiply his-tagged ATPase and microspheres.[6, 7]
1. It is easier to explain these data analyses in person, and the differences between lifetimes and "strength"
2. Hinterdorfer, P., et al., Surface attachment of ligands and receptors for molecular recognition force microscopy. COLLOIDS AND SURFACES B-BIOINTERFACES, 2002. 23(2-3): p. 115-123.
3. Nieba, L., et al., BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip. Anal Biochem, 1997. 252(2): p. 217-28.
4. Schmitt, L., et al., A metal-chelating microscopy tip as a new toolbox for single-molecule experiments by atomic force microscopy. Biophys J, 2000. 78(6): p. 3275-85.
5. Perkins, T.T., et al., Sequence-dependent pausing of single lambda exonuclease molecules. Science, 2003. 301(5641): p. 1914-8.
6. Soong, R.K., et al., Powering an inorganic nanodevice with a biomolecular motor. Science, 2000. 290(5496): p. 1555-8.
7. Soong, R.K., et al., Evaluating Adhesion Strength of Biological Molecules to Nanofabricated Structures. Technical Proceedings of the 1999 International Conference on Modeling and Simulation of Microsystems, 1999: p. 95-98.
