SOME PROTEIN PROPERTIES IMPORTANT FOR ENZYME CATALYSIS - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 150

polarization of the ligand folate and, therefore, affects the catalytic process. The
experimental data on site-specific titration curves for 12 histidine residues in carbon
monoxy sperm whale myoglobin by the 2D double quantum NMR technique were found
to agree with theoretical predictions obtained with using a numerical Poisson-Boltzmann
model and a Monte Carlo treatment (Bashford et al., 1993).
That asymmetry in packing the peptide amide dipole results in larger positive than
negative regions in proteins of all folding motifs was theoretically demonstrated (Gunner
et al., 2000). The following conclusion have been made: 1) the average side chain
potential in 305 proteins is 109 30 mV; 2) the larger oxygen at the negative end and

smaller proton at the positive end of the amide dipole yield positive potential potentials;
3) twice as many amides have their oxygens exposed than their amine protons; 4) 30%
of the Asp, Glu, Lys, and Arg are buried, while 60% of buried residues are acids and
only 40% bases; and 5) the positive backbone potential stabilizes ionization of 20% of
the acids by >3 pH units (-4.1 kcal/mol).
It was shown that optimization of conformational relaxation, specific ion-binding,
local hydrogen bonding networks, desolvatation and taking into consideration the
flipping of side chains of asparagine, histidine and glutamine around their 2, 2 and 3
torsion angles can improve results of calculations. (Alexov and Gunner, 1997;
Gunner and Alexev, 2000). These optimizations are applied to some well characterized
proteins: BPTI, hen egg white lysozyme and superoxide dismutase. The significance of
multi-conformational structure and hydroxyl group motion for the local dielectric
constant and electrostatic potential was demonstrated as a result of calculating
electrostatic, Lennard-Jones potentials, and torsion angle energies at each proton position
of hen egg lysozyme (Alexov and Gunner, 1997). Detailed analysis of effects of
functional group charges and dipoles and their distribution over protein globules on the
electrostatic potential in proteins has revealed some general features of the systems
under consideration.

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4.2.2. EXPERIMENTAL APPROACHES

Methods of investigation of electrostatic potential around charged molecules
Two types of experimental methods for the investigation of local electrostatic fields in
the vicinity of specific parts of biological molecules were proposed. The first group of
methods is based upon electrostatic measurements utilizing static local parameters, such
as the pK of a chosen protein or polypeptide functional group or the spectral
characteristics of a chromophore attached to a biopolymer, i.e. the Stark effect (Lockhart
and Kim, 1991, 1992; Sitkoff et al ., 1994 and references therein). For example, the
electric field at the backbone amide groups and amino terminus of an alpha helix in
water has been determined by measuring the Stark effect in the absorption band for a
covalently attached, neutral probe molecule. It was shown that the field at the interface
between the helix and the solvent is an order of magnitude stronger than expected from
the dielectric properties of bulk water. The dielectric screening effects are an order of
magnitude greater for the backbone-charge interactions than for the backbone-dipole
interactions. The results obtained by these various methods agree with the theoretically
predicted values in most cases. Nevertheless, it is necessary to bear in mind that