Dynamic Spin Label Study of the Barstar-Barnase Complex

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V. P. Timofeev^1*, T. G. Balandin², Ya. V. Tkachev¹, V. V. Novikov¹, V. A. Lapuk³, and S. M. Deev²

¹Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, 119991 Moscow, Russia; fax: (495) 135-1405; E-mail: tim@eimb.ru

²Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia; E-mail: deyev@ibch.ru

³Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky pr. 47, 117913 Moscow, Russia; E-mail: lapuk@ioc.ac.ru

^* To whom correspondence should be addressed.

Received January 30, 2007; Revision received April 13, 2007

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The dynamic spin label method was used to study protein-protein interactions in the model complex of the enzyme barnase (Bn) with its inhibitor barstar. The C40A mutant of barstar (Bs) containing a single cysteine residue was modified with two different spin labels varying in length and structure of a flexible linker. Each spin label was selectively bound to the Cys82 residue, located near the Bn-Bs contact site. The formation of the stable protein complex between Bn and spin labeled Bs was accompanied by a substantial restriction of spin label mobility, indicated by remarkable changes in the registered EPR spectra. Order parameter, S, as an estimate of rapid reorientation of spin label relative to protein molecule, was sharply increasing approaching 1. However, the rotational correlation time tau for spin-labeled Bs and its complex with Bn in solution corresponded precisely to their molecular weights. These data indicate that both Bs and its complex with Bn are rigid protein entities. Spin labels attached to Bs in close proximity to an interface of interaction with Bn, regardless of its structure, undergo significant restriction of mobility by the environment of the contact site of the two proteins. The results show that this approach can be used to investigate fusion proteins containing Bn or Bs.

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KEY WORDS: mutant spin-labeled barstar, barnase, spin label, EPR spectra of protein complex, order parameter, correlation time

DOI: 10.1134/S0006297907090118

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