The nature of protein dipole moments: experimental and calculated permanent dipole of .alpha.-chymotrypsin

Abstract

The electric dichroism of a-chymotrypsin has been measured in buffers of various pH values and ion compositions. The stationary dichroism obtained as a function of the electric field strength is not compatible with an induced dipole mechanism and clearly shows that a-chymotrypsin is associated with a substantial permanent dipole moment. After correction for the internal directing electric field according to a sphere model, the dipole moment is 1.6 X C m at p H 8.3 (corresponding to 480 D). This value decreases with decreasing pH (to 1.2 X C m at pH 4.2), but is almost independent of the monovalent salt concentration in the range from 2 to 12 m M and of Mg2+ addition up to 1 mM. The assignment of the permanent dipole moment is confirmed by analysis of the dichroism rise curves. The dichroism decay time constants of (31 f 1) ns at 2 OC can be represented by a spherical model with a radius of 25-26 A, which is consistent with the known X-ray structure. The limiting linear dichroism is slightly dependent on the buffer composition and demonstrates subtle variations of the protein structure. As a complement to the experimental results, electric and hydrodynamic parameters of a-chymotrypsin have been calculated according to the known X-ray structure. Bead model simulations provide the center of diffusion, which is used to calculate dipole moments according to the equilibrium charge distribution evaluated from standard pK values. In addition, dipole moments are also calculated according to the Tanford-Kirkwood model, which is modified by a more detailed consideration of solvation energies and evaluated from an equilibrium distribution of protein states generated by Monte Carlo techniques. The static dipole moment obtained by the first procedure is consistent with the experimental results over the whole pH range investigated, whereas the Monte Carlo results agree with the experimental data from pH 8.3 to 5.7. The fluctuating dipole moment evaluated by the second procedure gives rise to a minor contribution only, which is usually less than 20% of the static dipole moment. In summary, experimental and theoretical results are in satisfactory agreement and demonstrate the existence of a substantial "true" permanent dipole moment associated with a-chymotrypsin.%e assessment of electric properties for biological macromolecules proved to be particularly difficult-mainly due to the fact that these molecules usually bear a large number of charged residues. In most cases these residues attract counterions, which form a counterion atmosphere with a particularly high polarizability and thus cause large contributions to the measured electric properties. Although the polarization *Present address:

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