Position-dependent interactions between cysteine residues and the helix dipole.

Each peptide bond of the protein backbone contains a dipole moment; the alignment of several dipoles upon formation of an α-helix generates electrostatic potential at the helix termini and results in the helix dipole effect (Wada 1976; Hol et al. 1978; Hol 1985). The amino and carboxy termini of each helix effectively carry a partial positive and negative charge, respectively. Experiments and theoretical calculations suggest that this electrostatic field is due primarily to the dipole moments of peptide bonds in the first two turns of the helix (Hol et al. 1978; Aqvist et al. 1991; Lockhart and Kim 1992, 1993; Sitkoff et al. 1994). Because of the presence of this field, charged residues are often found at helix termini (Richardson and Richardson 1988). Such helix dipole interactions stabilize helical conformations (Shoemaker et al. 1987) and native protein structures (Nicholson et al. 1991). Due to the high frequencies of naturally occurring aspartate, glutamate, and histidine residues at helix termini, these residues have received most of the attention in helix dipole studies. The need to understand interactions involving cysteine residues, however, has emerged partly due to interest in the biochemical properties of the thioredoxin family of enzymes. Proteins of the thioredoxin family share the same fold and interact with cysteine-containing substrates (Martin 1995). Human thioredoxin is an isomerase that reduces disulfide bonds through a mechanism involving an initial nucleophilic attack by a charged thiolate. The pKa of the catalytic cysteine is 6.3 (Forman-Kay et al. 1992), significantly below the intrinsic thiol pKa of 8.5 to 9.0. The NMR and crystal structures of reduced thioredoxin show no positively charged residues nearby that can stabilize the thiolate (Qin et al. 1994; Weichsel et al. 1996), but the cysteine residue of interest is located at the N-terminus of an α-helix. The structural basis of the altered pKa, however, is not clear. Hydrogen bond formation (Dillet et al. 1998) and proton sharing (Jeng et al. 1995) between cysteine thiols have also been proposed as possible explanations. In light of these multiple interpretations, a quantitative measurement of helix dipole interactions with cysteine thiolates would contribute to understanding the structural basis of catalysis. Two rodent hemoglobins with reactive cysteine residues have been identified and are thought to play roles in responses to oxidants or xenobiotics. Cysteine 125β in rat hemoglobin has an altered pKa of 6.9 (Rossi et al. 1998). In this case, a possible intramolecular hydrogen bond between the side chain of serine 123β and the thiolate was used to explain the low pKa. The homologous cysteine residue in guinea pig hemoglobin was shown to have a similar pKa of 7.4 (Miranda 2000). Although a threonine residue is found in place of serine 123β in the guinea pig protein, the helix dipole effect was proposed as an alternate explanation of the pKa shift. Hemoglobin could, therefore, serve as a model for the controlled study of helix dipole interactions with thiolate anions, but the multimeric nature of the protein would unnecessarily complicate analysis because the cysteine residue of interest is close to a subunit interface. Instead, sperm whale myoglobin was studied because the protein is monomeric and the crystal structure has been solved (Phillips et al. 1990). Cysteine residues were introduced via site-directed mutagenesis into the Ncap, N1, and N2 positions of the myoglobin helix homologous to the hemoglobin helix in which the highly reactive cysteine residue was found. The helix dipole effect at each position was quantitatively determined and found to be significantly stronger at the Ncap compared to the N1 and N2 positions. Molecular modeling of each mutant suggests a possible explanation for the position-dependent nature of the helix dipole effect. The results are also discussed with regard to the reactivity of cysteine residues in rodent hemoglobins and the thioredoxin family.