User:Nathan H. Kipniss/Notebook/20.109 Final Project
Elucidating the calcium binding and cooperative mechanisms of D24H Inverse Pericam mutant
In module 2 of 20.109, we created the D24H mutant of inverse pericam. The experimental results from the calcium binding assay was surprising as calcium affinity decreased (Kd increase), yet cooperativtiy increased. For a final research idea, I would like to propose the set of experiments that would elucidate how the addition of a histidine into the first binding loop of inverse pericam can make these changes.
-working under the assumption that the SDM did indeed work (sequencing with BLAST, discontinuous mega-blast suggests it did indeed work)
- consider looking at calmodulin only.
- Pharmaceutical companies often need to know how a drug is interacting with a target. These same techniques could be applied to calmodulin and its target, M13. I currently have a request with MIT libraries to purchase a critical paper (see below).
Papers and Summaries
Junker, JP et al. Single-molecule force spectroscopy distinguishes target binding modes of calmodulin. Proceedings of the National Academy of Sciences of the United States of America 106.34 (2009): 14361-6.
- Cooperativtiy in CaM and target proteins is target protein dependent.
- Multiple transitions rates exist in CaM and target protein binding (again, protein dependent).
- for skMLCK, one cooperative transition; no intermediates could be found with this approach.
- potential issues: this paper uses worm like chain models to interpret data. How valid is that approximation/assumption?
- this method slows kinetics to actually observe structural transitions
- skMLCK demands that CaM is completely folded when binding.
Waltersson, Y et al. Mutational effects on the cooperativity of Ca2+ binding in calmodulin.â Biochemistry 32.31 (1993): 7866-71.
-this paper is a bit dated
- Asp24 is seemingly understudied (in +Z position, either Asp or Asn).
- Paper addresses Asp22, since Asp in +Y position is conserved across all four binding loops.
- May not be a simple explanation to AA sequence and calcium binding.
- Only one Oxygen in the anionic amino acids coordinate calcium. The other may serve to "recruit" calcium ions.
- It may the distribution of charges about a binding site that has a cooperative role.
Papers Currently being read
Grossman,M et al. Achieving broad molecular insights into dynamic protein interactions by integrated structural-kinetic approaches. Current opinion in structural biology 21.5 (2011): 678-85. Web. 1 Mar. 2012.
Scapin, G. Structural Biology and Drug Discovery. Current Pharmaceutical Design. 2006
- trying to get access to!