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Published January 16, 1996 | Supplemental Material
Journal Article Open

Phosphotransfer and CheY-Binding Domains of the Histidine Autokinase CheA Are Joined by a Flexible Linker


Multidimensional heteronuclear NMR techniques were applied to study a protein fragment of the histidine autokinase CheA from Escherichia coli. This fragment (CheA_(1-233)) contains the phosphotransfer domain and the CheY-binding domain joined by a linker region. Comparison of chemical shift and NOE cross-peak patterns indicates that the structures of the two domains in CheA_(1-233) remain nearly the same as in the two individual domain fragments, CheA1-134 and CheA_(124-257). Relaxation properties of the backbone ^(15)N nuclei were measured to study the rotational correlations of the two domains and properties of the linker region. Dynamics data were analyzed both by an isotropic motional model and an anisotropic motional model. The experimental T_1 and T_2 values, the derived rotational correlation times, and motional anisotropy are significantly different for the two domains, indicating the two domains reorient independently and the linker region is highly flexible. Dynamics data of CheA_(1-233) were also compared with those of CheA_(1-134). Our studies show that flexible domain linkers and extended and flexible terminal polypeptide chains can have significant effects on the motional properties of the adjacent structured regions. These observations suggest a model for the graded regulation of CheA autophosphorylation activity. In this model, the various activity states of the receptor are generated by controlling the access of the mean position of the kinase domain to the phosphotransfer domain. This would then modulate the diffusional encounter rate of the domains and hence activity over a wide and graded range of values.

Additional Information

© 1996 American Chemical Society. Received August 19, 1995; Revised Manuscript Received November 13, 1995. Publication Date (Web): January 16, 1996. D.F.L. was supported by a Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation Fellowship DRG-1195. M.M.M. is supported by an NIH training grant. R.V.S. was supported by a National Research Service Award Fellowship GM14767 from NIH. This work was supported by research grants GM33677 (F.W.D.) and AI-19296 (M.I.S.).

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