Cell-free synthesis of the Ras-binding domain of c-Raf-1: binding studies to fluorescently labelled H-ras.

It has previously been shown that the transient kinetics of the interaction between the Ras-binding domain of c-Raf-1 and the proto-oncoprotein Ras can be followed by stopped-flow measurements using the 2',3'-(N-methylanthraniloyl) fluorescence of 2',3'-(N-methylanthraniloyl) guanyl-5'-yl-imidodiphosphate-labelled Ras. In continuation of this work, we demonstrate that the His-tagged Ras-binding domain of c-Raf-1 can also be synthesized in a cell-free expression system. After purification by Ni2+ affinity chromatography, His-tagged Ras-binding domain of c-Raf-1 could be isolated in sufficient amounts for biochemical and biophysical investigations. The results obtained describe the first example of a cell-free synthesized protein which has been used for stopped-flow measurements to determine the transient kinetics of protein-protein interactions with an effector.

Design, total chemical synthesis, and binding properties of a [Leu-91-N1-methyl-7-azaTrp]Ras-binding domain of c-Raf-1.

The Ras-binding domain (RBD) of c-Raf-1 has been synthesized chemically, taking advantage of the chemical ligation of two peptide fragments of the protein. This procedure allowed incorporation of an unnatural amino acid (N1-methyl-7-azatryptophan) at position 91 of RBD, producing a protein with fluorescent properties distinct from and distinguishable from those of proteins containing the natural fluorophore tryptophan. The resulting protein was shown to interact with Ras in a manner that was almost indistinguishable from that of unmodified RBD based on transient kinetic monitoring of the binding event. Modified RBD containing the L-isomer of the unnatural amino acid or its racemic D,L mixture appeared to interact identically with Ras. The approach demonstrates a general procedure for the introduction of unnatural amino acids that can be used for monitoring protein-protein interactions and for the introduction of an unnatural backbone structure at strategic positions.

Transient kinetic studies on the interaction of Ras and the Ras-binding domain of c-Raf-1 reveal rapid equilibration of the complex.

Transient kinetic methods have been used to analyze the interaction between the Ras-binding domain (RBD) of c-Raf-1 and a complex of H-Ras and a GTP analogue. The results obtained show that the binding is a two-step process, with an initial rapid equilibrium step being followed by an isomerization reaction occurring at several hundred per second. The reversal of this step determines the rate constant for dissociation, which is on the order of 10 s-1. The lifetime of the complex is therefore on the order of 50-100 ms, which is much shorter than the lifetime of GTP at the active site of H-Ras as determined by the intrinsic GTPase reaction. This suggests that multiple interactions of a single activated Ras molecule and Raf can occur, the number being limited by the competing interaction with GAP. The GDP complex of H-Ras binds more than 2 orders of magnitude more weakly than the GTP-analogue complex, mainly due to a significant weakening of the initial binding equilibrium reaction in the GDP state, thereby avoiding even short-lived recruitment of Raf to the plasma membrane by the inactive Ras form.

The photophobic receptor from Natronobacterium pharaonis: temperature and pH dependencies of the photocycle of sensory rhodopsin II.

The photocycle of the photophobic receptor sensory rhodopsin II from N. pharaonis was analyzed by varying measuring wavelengths, temperature, and pH, and by exchanging H2O with D2O. The data can be satisfactorily modeled by eight exponents over the whole range of modified parameters. The kinetic data support a model similar to that of bacteriorhodopsin (BR) if a scheme of irreversible first-order reactions is assumed. Eight kinetically distinct protein states can then be identified. These states are formed from five spectrally distinct species. The chromophore states Si correspond in their spectral properties to those of the BR photocycle, namely pSRII510 (K), pSRII495 (L), pSRII400 (M), pSRII485 (N), and pSRII535 (O). In comparison to BR, pSRII400 is formed approximately 10 times faster than the M state; however, the back-reaction is almost 100 times slower. Comparison of the temperature dependence of the rate constants with those from the BR photocycle suggests that the differences are caused by changes of DeltaS. The rate constants of the pSRII photocycle are almost insensitive to the pH variation from 9.0 to 5.5, and show only a small H2O/D2O effect. This analysis supports the idea that the conformational dynamics of pSRII controls the kinetics of the photocycle of pSRII.