Fig. 3 shows one-dimensional ³¹P NMR spectra of peptide I (a), peptide I with 50% excess of mCrK₂₃ (b), and p-mCrK (c) in 50 mM MOPS buffer, pH 6.8, containing 1 mM sodium phosphate, 200 mM NaCl, 2 mM EDTA, 1 mM DTT, 2 mM benzamidine, and 0.02% NaN₃. The spectra were recorded at 35 °C with a 202 MHz spectrometer. The chemical shifts are referenced to external 85% phosphoric acid. The change in chemical shift of the phosphate peak (near 2 ppm) across the three spectra represents a difference of ~0.2 pH units. Fig. 4 shows that phosphorylated Crk is monomeric. The natural logarithm of the optical density (y) was plotted against the square of the distance moved in the centrifuge cell (r²). Data were recorded at 37 °C. Similar results were obtained at 20 °C. The methods involved hydrodynamic measurements in the same buffer used in NMR experiments. Analytical ultracentrifugation and data analysis are described in ref. 26. Initial sample concentration was determined from fringe count assuming an average refractive increment of 4.1 fringes mg⁻¹ml⁻¹ according to ref. 27. Molecular mass determination by light-scattering at 20 °C was essentially as described in ref. 26. A series of protein solutions at 1.06–2.73 mg ml⁻¹ were injected into a Dawn F multiangle laser light-scattering photometer. The common intercept on a Zimm plot of the extrapolations to zero angle and zero concentration yielded the reciprocal molecular mass; a value of 0.185 was assumed for dn/dc for the sample. However, p-mCrK appears to be able to bind these species only in the non-phosphorylated state. Thus, the intramolecular SH2-pTyr interaction may also inhibit intermolecular interactions involving the intervening N-terminal SH3 domain. Phosphorylation provides a mechanism for regulating the SH2/SH3 adaptor function of Crk. The direct identification of an intramolecular SH2-pTyr interaction in p-mCrK supports the idea that this is a general mechanism by which the activities of SH2-containing proteins, including Src-family kinases, may be regulated.Fig. 3 shows one-dimensional ³¹P NMR spectra of peptide I (a), peptide I with 50% excess of mCrK₂₃ (b), and p-mCrK (c) in 50 mM MOPS buffer, pH 6.8, containing 1 mM sodium phosphate, 200 mM NaCl, 2 mM EDTA, 1 mM DTT, 2 mM benzamidine, and 0.02% NaN₃. The spectra were recorded at 35 °C with a 202 MHz spectrometer. The chemical shifts are referenced to external 85% phosphoric acid. The change in chemical shift of the phosphate peak (near 2 ppm) across the three spectra represents a difference of ~0.2 pH units. Fig. 4 shows that phosphorylated Crk is monomeric. The natural logarithm of the optical density (y) was plotted against the square of the distance moved in the centrifuge cell (r²). Data were recorded at 37 °C. Similar results were obtained at 20 °C. The methods involved hydrodynamic measurements in the same buffer used in NMR experiments. Analytical ultracentrifugation and data analysis are described in ref. 26. Initial sample concentration was determined from fringe count assuming an average refractive increment of 4.1 fringes mg⁻¹ml⁻¹ according to ref. 27. Molecular mass determination by light-scattering at 20 °C was essentially as described in ref. 26. A series of protein solutions at 1.06–2.73 mg ml⁻¹ were injected into a Dawn F multiangle laser light-scattering photometer. The common intercept on a Zimm plot of the extrapolations to zero angle and zero concentration yielded the reciprocal molecular mass; a value of 0.185 was assumed for dn/dc for the sample. However, p-mCrK appears to be able to bind these species only in the non-phosphorylated state. Thus, the intramolecular SH2-pTyr interaction may also inhibit intermolecular interactions involving the intervening N-terminal SH3 domain. Phosphorylation provides a mechanism for regulating the SH2/SH3 adaptor function of Crk. The direct identification of an intramolecular SH2-pTyr interaction in p-mCrK supports the idea that this is a general mechanism by which the activities of SH2-containing proteins, including Src-family kinases, may be regulated.