Systematic control of protein interaction using a modular ER/K α-helix linker

Sivaraj Sivaramakrishnan, James A. Spudich

Research output: Contribution to journalArticlepeer-review

62 Scopus citations


Cellular functions of proteins are strongly influenced by their interactions with other proteins. The frequency of protein interactions is a function of the local concentration of two proteins and their affinity for one another.When two proteins are tethered together, the link between them influences their effective concentrations and therefore the frequency of their interaction. Currently no methods exist to systematically vary the effective concentration within this intramolecular interaction. Here we outline a modular, genetically encoded linker, namely, an ER/K [genetically encoded polypeptide motif based on alternating sequence of approximately four glutamic acid (E) followed by approximately four arginine (R) or lysine (K) residues] single α-helix that can be used to regulate the frequency of interaction between two proteins, or between a protein and a peptide, one at each end. We exploit the wide range of interaction affinities between calmodulin and its binding peptides, combined with FRET to determine the effect of the ER/K α-helix in regulating protein interactions. We find that increasing the length of the ER/K α-helix reduces the on rate of the intramolecular interaction without significantly affecting the off rate, regardless of the affinity of the bimolecular interaction.We outline a genetically encoded approach to determine the dissociation constant for both moderate (micromolar K d) and strong (nanomolar K d) protein interactions. Our studies demonstrate the use of the ER/K α-helix to systematically engineer FRET biosensors that detect changes in concentration or affinity of interacting proteins, and modulate enzyme autoinhibition. Our findings are consistent with the ER/K α-helix as a worm-like chain with rare, stochastic breaks in the helix backbone that may account for the behavior of myosin VI stepping along actin.

Original languageEnglish (US)
Pages (from-to)20467-20472
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number51
StatePublished - Dec 20 2011


  • Cell signaling
  • Modulation
  • Systematic protein affinity strength


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