The de novo design of molecular switches: Manipulation of secondary structure to modulate conformational stability

The de novo design of molecular switching peptides is of increasing interest because it tests and extends our fundamental understanding of this process while laying the groundwork for the creation of new chemical and biological reagents. Addressing this problem through protein design requires conditional stabilization of the target fold with concomitant destabilization of alternative folds. In the following work, I have exploited properties of the α-helix to design two different classes of helical molecular switching peptides. These peptides undergo structural stabilization upon exposure to the appropriate chemical signal, phosphorylation or metal-binding. Once conformationally stabilized, they can then participate in further biologically relevant activities, oligomerization or membrane lysis. Both sets of switches exhibited successful design of inducible activity. Studying both systems has also contributed to our understanding of determinants of protein structure and structure-based activation

The de novo design of molecular switches: Manipulation of secondary structure to modulate conformational stability

The de novo design of molecular switching peptides is of increasing interest because it tests and extends our fundamental understanding of this process while laying the groundwork for the creation of new chemical and biological reagents. Addressing this problem through protein design requires conditional stabilization of the target fold with concomitant destabilization of alternative folds. In the following work, I have exploited properties of the α-helix to design two different classes of helical molecular switching peptides. These peptides undergo structural stabilization upon exposure to the appropriate chemical signal, phosphorylation or metal-binding. Once conformationally stabilized, they can then participate in further biologically relevant activities, oligomerization or membrane lysis. Both sets of switches exhibited successful design of inducible activity. Studying both systems has also contributed to our understanding of determinants of protein structure and structure-based activation

RGS/Gi2α interactions modulate platelet accumulation and thrombus formation at sites of vascular injury

Although much is known about extrinsic regulators of platelet function such as nitric oxide and prostaglandin I2 (PGI2), considerably less is known about intrinsic mechanisms that prevent overly robust platelet activation after vascular injury. Here we provide the first evidence that regulators of G-protein signaling (RGS) proteins serve this role in platelets, using mice with a G184S substitution in Gi2α that blocks RGS/Gi2 interactions to examine the consequences of lifting constraints on Gi2-dependent signaling without altering receptor:effector coupling. The results show that the Gi2α(G184S) allele enhances platelet aggregation in vitro and increases platelet accumulation after vascular injury when expressed either as a global knock-in or limited to hematopoietic cells. Biochemical studies show that these changes occur in concert with an attenuated rise in cyclic adenosine monophosphate levels in response to prostacyclin and a substantial increase in basal Akt activation. In contrast, basal cyclic adenosine monophosphate (cAMP) levels, agonist-stimulated increases in [Ca++]i, Rap1 activation, and α-granule secretion were unaffected. Collectively, these observations (1) demonstrate an active role for RGS proteins in regulating platelet responsiveness, (2) show that this occurs in a pathway-selective manner, and (3) suggest that RGS proteins help to prevent unwarranted platelet activation as well as limiting the magnitude of the normal hemostatic response