NANOSCALE PROTEIN DYNAMICS AND LONG- RANGE ALLOSTERY IN CELL SIGNALING Zimei Bu 1 and David J. E. Callaway 1,2 1 City College of New York and 2 New York University School of Medicine

Multiple domains in proteins give rise to a great deal of flexibility and mobility, leading to protein domain dynamics. Nanoscale domain motions can only be directly observed using spectra measured by neutron spin echo spectroscopy (our new frontier!). They are essential for: nanoscale allostery catalysis regulatory activity transport of metabolites formation of protein assemblies cellular locomotion

90 O NHERF1 is an elongated protein with multiple modular domains PDZ1 PDZ2 CT PDZ1 PDZ2 CT 57.1 Å 45.8 Å NHERF1 from SAXS PDZ domains

Structural changes in NHERF1 upon binding to ezrin 110 Å Ezrin PDZ2 PDZ1 NHERF1 Ezrin-induces long-range interdomain allostery in NHERF1

Applying neutron spin echo spectroscopy to study long-range coupled protein domain motion Ferenc Mezei Nanosecond to microsecond time scales Å: nano length scales

Shape fluctuations in a protein that ONLY NSE can see!

Protein motion—low Reynolds number Overdamped creeping motions--(badminton at bottom of molasses pool, not a cruise ship crossing the Atlantic!) Effective diffusion constant D eff (Q)

Mobility tensor H defines dynamics— our new technique! The Q dependence of the decay rates of the NSE measured correlation functions is defined by the mobility tensor

Mobility tensor v = H F H is the mobility tensor, and yields the velocity of a domain given the force applied on it or another subunit. NSE yields H, given structure. (Bu et al, PNAS, 2005)

PDZ1 PDZ2 CT NHERF1 The dynamics of (unbound) NHERF1 alone is well described by a rigid-body model Only inputs to calculations are diffusion constant from PFG NMR and SANS coordinates—no need to fit NSE data or use MD! Farago et al Biophys J 2010

PDZ1 PDZ2 FERM CT Binding to ezrin activates inter- domain motions in NHERF1 more than 100 Å away!! Rigid body Farago et al Biophys J 2010

Binding to FERM activates inter-domain motions in NHERF1 - A simple four-point model describes all

110 Å Ezrin PDZ2 PDZ1 NHERF1 Binding to ezrin activates nanoscale inter-domain motions in NHERF1

Neutron spin echo spectroscopy allows us to see coupled interdomain motion in proteins for the very first time Our analyses show that these motions can be revealed by utilizing nonequilibrium statistical mechanics (mobility tensor)—no need for mnolecular dynamics or multiparameter fits

Acknowledgements NIH ILL, NIST, and ORNL