PREPARATION AND CHARACTERIZATION OF A BIFUNCTIONALLY SPIN-LABELED MUTANT OF MURINE EPIDERMAL GROWTH-FACTOR FOR SATURATION-TRANSFER ELECTRON-PARAMAGNETIC-RESONANCE STUDIES OF THE GROWTH-FACTOR RECEPTOR COMPLEX

In this report we describe the production of a [Lys3, Tyr22] murine ep idermal growth factor (mEGF) mutant for spin-labeling with bis(sulfo-N -succinimidyl)-[N-15, H-2(16)]doxyl-2-spiro-4'-pimelate ([N-15,H-2(16) ]BSSDP) in order to study the rotational dynamics of the EGF/EGF recep tor complex by saturation-transfer electron paramagnetic resonance (ST -EPR). Previous results [Faulkner-O'Brien et al. (1991) Biochemistry 3 0, 8976-8985] indicated that the reaction of [N-15, H-2(16)]BSSDP with wild-type mEGF did not yield a product useful for ST-EPR studies of t he EGF/EGF receptor complex because the major product, in which [N-15, H-2(16)]BSSDP was attached only at the amino terminus of mEGF, lacked rigid motional coupling of the spin probe to the protein, and the mor e tightly coupled bidentate product was unstable. Using oligonucleotid e-mediated site-directed mutagenesis of a synthetic gene for mEGF, we replaced Tyr3 with Lys and His22 with Tyr in wild-type mEGF to produce a mutant mEGF suitable for [N-15, H-2(16)]BSSDP labeling. The [Lys3,T yr22]mEGF was expressed in Escherichia coli HB101 transformed with a p IN-III-ompA3-[Lys3,Tyr22] mEGF plasmid and was purified from the bacte rial periplasm using a simple two step purification method. The [N-15, H-2(16)]BSSDP reacted with [Lys3,Tyr22]mEGF in high yield, and EPR an alysis of the major product revealed tight motional coupling between t he spin probe and the protein. Biological activity, as assessed by sti mulation of EGF receptor autophosphorylation and dimerization, was not affected by either the mutations or the addition of the spin label. T he [N-15, H-2(16)]BSSDP-modified [Lys3,Tyr22]mEGF was shown to be equi potent with mEGF in EGF receptor competition binding assays using A431 cells; in EPR studies, mEGF also was shown to specifically block [N-1 5, H-2(16)]BSSDP-modified [Lys3,Tyr22]mEGF binding to the EGF receptor in A431 membrane vesicles. Using the [N-15, H-2(16)]BSSDP-modified [L ys3,Tyr22]mEGF, we now report the first measurement of the rotational dynamics of the EGF/EGF receptor complex in A431 membrane vesicles by ST-EPR.