Biocurious

/ a biophysics blog

Hot off the Press: Allosteric Control through Mechanical Tension

Posted 13 August 2005 by PhilipJ under

As reported in yesterday’s Physical Review Letters (and subsequent Focus article), scientists at UCLA have inserted a “molecular spring” on the enzyme guanylate kinase (GK), allowing them to control enzymatic activity by applying or releasing tension in the spring.

Through site-directed mutagenesis, the wild-type GK molecule was given cystein DNA handles at either lobe (shown in magenta in the picture). A 60mer of single-stranded DNA (ssDNA) was then attached via cross-linkers to the cystein mutations, resulting in a “u” shaped handle attached to the enzyme. With the attached ssDNA handle, no obvious affect on enzymatic activity was observed.

Upon introduction of a complementary piece of ssDNA, the ssDNA handle becomes a double-stranded DNA (dsDNA) handle. dsDNA is significantly more rigid than ssDNA, having a persistence length some 50 times longer, and must bend in order to maintain the end-to-end distance imposed by the attachment points on the protein. In doing so, mechanical stress is exerted on the enzyme, resulting in some change in the enzyme’s structure. It was estimated that up to 10 pN of force might be exerted on the molecule by the dsDNA handle.

GK’s enzymatic activity is to bind ATP and GMP near its “jaw”, and transfer Pi between them. With a dsDNA handle attached, however, the enzyme’s affinity for GMP dropped by a factor of 10, while its affinity for ATP dropped by a factor of 2. It is inferred then, that the GMP binding site is disrupted fairly significantly by the dsDNA handle, while the ATP binding site is modified in a smaller way.

This is a cool example of mechanical control of enzymatic activity in an entirely different way than has been common in the past (for e.g., optical tweezers have been used extensively to study kinesin, DNA and RNA polymerases, etc).

Here is the abstract, while the full article requires a subscription.

  

  1. Youngseon Choi    4 May 07    #

    cool!


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