Some examples of how NMR can provide information about biological systems

Some examples of how NMR can provide information about biological systems. Autoinhibited Multi-Domain Proteins are Critical in Many Signal Transduction Pathways. Numerous multi-domain proteins transmit signals from the T-cell receptor. Rosen lab.

Some examples of how NMR can provide information about biological systems Autoinhibited Multi-Domain Proteins are Critical in Many Signal Transduction Pathways

Numerous multi-domain

proteins transmit signals

from the T-cell receptor

Rosen lab Vav Signal Transduction Pathways proto-oncoprotein is a key GEF that regulates Rho family GTPases

A member of the Dbl family of guanine nucleotide exchange factors (GEF) for the Rho family of GTP binding proteins.

Important in hematopoiesis, playing a role in T-cell and B-cell development and activation.

DH domain is inhibited by contacts with the Acidic (Ac) region and is relieved by phosphorylation of the Ac region tyrosines

A Helix From the Ac Domain Binds in the DH Active Site: Autoinhibition by Occlusion

Y3 is buried in the interface

Aghazadeh, et al. Cell, 102: 625-633. Phosphorylation Disrupts Autoinhibitory Interactions Autoinhibition by Occlusion Aghazadeh, et al. Cell, 102: 625-633.

Amide resonances from N-terminal (Ac region) helix collapse to the center of

the 1H/15N HSQC spectra and become extremely intense

13Ca and 13Cb assignments indicate that the N-terminus is random coil

How is Y3 Accessed by Kinases? Autoinhibition by Occlusion A general problem in autoinhibition/allostery: activators must contact buried sites ?

Linearity of chemical Autoinhibition by Occlusion

shifts across multiple

perturbations indicates

a two-state equilibrium

Chemical Shift Can Report on Population Distribution closed 50:50 mixture open wobs = powo + (1-po)wc Mutants Sample a Range of Population Autoinhibition by OcclusionDistributions Open Closed

Linearity strongly suggests an

equilibrium between Y3-

bound and Y3-unbound states

wobs = powo + (1-po)wc Conformational equilibrium controls Vav activation by Src family kinases Vav Autoinhibition by Occlusion WASP/Cdc42 Science. 1998 Jan 23;279(5350):509-14. Nature. 2000 Mar 9;404(6774):151-8. Nature. 1999 May 27;399(6734):379-83. Rosen lab