User:Nathan H. Kipniss/Notebook/20.109 Final Project

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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.


Fitted Matlab binding curves


-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. €œSingle-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.” 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. œAchieving 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!