Aliphatic semisynthetic variants of the amino-terminal residue of sperm whale myoglobin: enrichment with carbon-13 and determination and interpretation of terminal pK values

Abstract

The synthesis of a series of myoglobins substituted in the amino-terminal residue to provide variation in the aliphatic nature of the side chain and enrichment in 13C was accomplished by semisynthetic methods. The replacements for valine, the native first residue, included 13C-enriched glycine, alanine, valine, leucine, and isoleucine. The products were extensively characterized and found to be virtually indistinguishable by most physical methods. 13C NMR spectroscopy showed significant differences in the amino-terminal pK value, ranging from 7.72 for [Gly1]myoglobin to 7.15 for [Leu1]myoglobin. Consideration of the electrostatic effects of the charge matrix indicated a balance of interactions at this site not significantly altered by these variations in the side chain. By examination of the crystal structure, consideration of earlier work regarding the interactions of the side chain of Leu-2, and data regarding the motions of the terminal residue, it was concluded that the interaction of the side chain of the first residue with the hydrophobic cluster formed primarily by close contact of invariant residues Leu-2 and Leu-137 was the primary cause for the reduction in terminal pK values seen for the larger aliphatics. By restricting the freedom of the residue, this interaction limits the available hydration volume and consequently favors the unprotonated form of the amine. The concurrent observation of both functional elements in the series of alpha-amino-terminal residues brings out the interrelated consequences for the two categories of solvent interactions controlling structural and functional properties in a graded way.

Roles of the .beta.146 histidyl residue in the molecular basis of the Bohr effect of hemoglobin: a proton nuclear magnetic resonance study

“…The physical basis for these observations may involve the alteration of the electrostatic charge matrix of the protein molecule, as described by Matthew et al (1985), or alteration of other properties of the Hb molecule. As an example, substitution studies at the amino-terminal residue of myoglobin have demonstrated a 0.3 pH unit perturbation of the pK value of the terminal -amino group upon substitution of alanine for glycine at the amino terminus, without detectable conformational alteration of the protein molecule (Busch et al, 1985). The change of pX was attributed to an alteration of hydration in the vicinity of the amino acid residue, detected by a pH-dependent study of the dynamics of the amino-terminal residue.…”

Site-specific semisynthetic variant of human hemoglobin.

“…These pK differences for myoglobin appeared to, involve solvation effects rather than salt bridges (11).…”

“…Such comparisons have been shown to be sensitive indicators for the structural integrity of heme proteins (30). The agreements between the absorption ratios, particularly those involving the Soret band, indicate that the modified a chains renatured and positioned the heme properly (11,13,31).…”

“…We report here the production of two hemoglobin analogs by using chemical methods similar to those developed for semisynthetic studies on sperm whale myoglobin (10,11) and for reconstruction of hemoglobin tetramers (12)(13)(14) (17). Under these conditions the reaction was directed to the NH2 termini, reflecting the facility for anion binding at these loci (data not shown).…”

Carbamino group formation with peptides and proteins studied by mass spectrometry

“…Carbamino group formation has been studied with glycine [3] and other amino acids [4], bradykinin [2], and angiotensin [2], substance P [18], insulin [5], myoglobin (Mb) [3,19,20], and hemoglobin (Hb) [8 -15, 21, 22]. With proteins such as Mb and Hb, side-chain -amino groups can also form carbamino acids.…”