Electrostatic effects in myoglobin. Application of the modified Tanford-Kirkwood theory to myoglobins from horse, California grey whale, harbor seal, and California sea lion.

The modified Tanford-Kirkwood electrostatic theory (Shire et al., 1974a) was applied to ferrimyoglobins from the following animal species: sperm whale (Physeter catodon), horse, California grey whale (Eschrichtius gibbosus), harbor seal (Phoca vitulina), and California sea lion (Zalophus californianus). Computations were made of the overall hydrogen ion titration curves of the proteins, and of pH and ionic strength variations of ionization equilibria for individual groups in the protein, with particular reference to the hemic acid ionization of the iron bound water molecule. Coordinates and static solvent accessibility were estimated in terms of the sperm whale myoglobin structure. Where possible, theoretical results and experimental data are compared. Some comparative features of charge and ionization properties among the various myoglobins are presented.     

On the difference in stability between horse and sperm whale myoglobins

The work in the literature on apomyoglobin is almost equally divided between horse and sperm whale myoglobins. The two proteins share high homology, show similar folding behavior, and it is often assumed that all folding phenomena found with one protein will also be found with the other. We report data at equilibrium showing that horse myoglobin was 2.1 kcal/mol less stable than sperm whale myoglobin at pH 5.0, and aggregated at high concentrations as measured by gel filtration and analytical ultracentrifugation experiments. The higher stability of sperm whale myoglobin was identified for both apo and holo forms, and was independent of pH from 5 to 8 and of the presence of sodium chloride. We also show that the substitution of sperm whale myoglobin residues Ala15 and Ala74 to Gly, the residues found at positions 15 and 74 in horse myoglobin, decreased the stability by 1.0 kcal/mol, indicating that helix propensity is an important component of the explanation for the difference in stability between the two proteins.     

A preliminary comparison of metmyoglobin molecules from seal and sperm whale

A preliminary residue by residue comparison of the conformations of the seal and sperm whale metmyoglobin molecules is reported. Data for the comparison were the atomic co-ordinates of all non-hydrogen atoms common to both structures 1184 equivalent pairs of atoms in all.The results of the comparison, though preliminary, indicate how the differences between the primary structures of the two molecules lead to the differences between their crystal structures and affect their interaction with ligands.     

Studies on cobalt myoglobins and hemoglobins. Interaction of sperm whale myoglobin and Glycera hemoglobin with molecular oxygen.

The pH dependence of the electron paramagnetic resonance (EPR) spectrum and oxygen affinity of cobaltous porphyrin-containing myoglobin (CoMb) have been examined. The hyperfine structures of the EPR spectrum of oxy-CoMb undergo small, reversible pH-dependent changes with pK values of 5.33, 5.55, and 5.25 +/- 0.05 for proto-, meso-, and deutero-CoMb's, respectively, whereas deoxy-CoMb does not exhibit any pH dependence of its EPR spectrum. The partial pressure of oxygen at half-saturation of proto-CoMb decreases from 26 to 42 Torr on lowering the pH from 7.0 to 4.8. For comparison, we have prepared cobaltous porphyrin-containing monomeric Glycera hemoglobin (CoHb (Glycera)), in which the distal histidyl group of myoglobin is replaced by a leucyl residue, and examined the equilibria and kinetics of its oxygenation and EPR spectrum. CoHb (Glycera) has exhibited a very low oxygen affinity (p50 = 7 X 10(2) Torr at 5 degrees) and a large dissociation rate constant (more than 8 X 10(4) S-1 at 5 degrees). The EPR spectrum of oxy-CoHb (Glycera) was affected by neither pH nor replacement of H2O with D2O. Low temperature photodissociation studies by EPR and spectrophotometry have shown that the photolyzed form of the ligated hemoglobin (Glycera) is similar to its deoxy form, in contrast to myoglobin which gives a new intermediate states as the photolyzed form. These differences between CoMb and CoHb (Glycera) are interpreted with relation to the possible role of the distal histidyl residue in CoMb.     

Titration behavior of individual tyrosine residues of myoglobins from sperm whale, horse, and red kangaroo.

The titration behavior of individual tyrosine residues of myoglobins has been studied by observing the pH dependence of the chemical shifts of Czeta and Cgamma of these residues in natural abundance of 13C Fourier transform NMR spectra (at 15.18 MHz, in 20-mm sample tubes, at 37 degrees) of cyanoferrimyoglobins from sperm whale, horse, and red kangaroo. A comparison of the pH dependence of the spectra of the three proteins yielded specific assignments for the resonance of Tyr-151 (sperm whale) and Tyr-103 (sperm whale and horse). Selective proton decoupling yielded specific assignments for Czeta of Tyr-146 of the cyanoferrimyoglobins from horse and kangaroo, but not the corresponding assignment for sperm whale. The pH dependence of the chemical shifts indicated that only Tyr-151 and Tyr-103 are titratable tyrosine residues. Even at pH 12, Tyr-146 did not begin to titrate. The titration behavior of C zeta and Cgamma of Tyr-151 of sperm whale cyanoferrimyoglobin yielded a single pK value of 10.6. The pH dependence of the chemical shift of each of the resonances of Tyr-103 of the cyanoferrimyoglobins from horse and sperm whale could not be fitted with the use of a single pK value, but was consistent with two pK values (about 9.8 and 11.6). Furthermore, the resonances of Czeta and Cgamma of Tyr-103 broadened at high pH. The titration behavior of the tyrosines of sperm whale carbon monoxide myoglobin and horse ferrimyoglobin was also examined. A comparison of all the experimental results indicated that Tyr-151 is exposed to solvent, Tyr-146 is not exposed, and Tyr-103 exhibits intermediate behavior. These results for myoglobins in solution are consistent with expectations based on the crystal structure.     

1H-NMR comparative study of the active site in shark (Galeorhinus japonicus), horse, and sperm whale deoxy myoglobins.

1H-NMR spectra of deoxy myoglobins (Mbs) from shark (Galeorhinus japonicus), horse, and sperm whale have been studied to gain insights into their active site structure. It has been demonstrated for the first time that nuclear Overhauser effect (NOE) can be observed between heme peripheral side-chain proton resonances of these paramagnetic complexes. Val-E11 methyl and His-F8 C delta H proton resonances of these Mbs were also assigned from the characteristic shift and line width. The hyperfine shift of the former resonance was used to calculate the magnetic anisotropy of the protein. The shift analysis of the latter resonance, together with the previously assigned His-F8 N delta H proton resonance, revealed that the strain on the Fe-N epsilon bond is in the order horse Mb approximately whale Mb < shark Mb and that the hydrogen bond strength of the His-F8 N delta H proton to the main-chain carbonyl oxygen in the preceding turn of the F helix is in the order shark Mb < horse Mb < whale Mb. Weaker Feporphyrin interaction in shark Mb was manifested in a smaller shift of the heme methyl proton resonance and appears to result from distortion of the coordination geometry in this Mb. Larger strain on the Fe-N epsilon bond in shark Mb should be to some extent attributed to its lowered O2 affinity (P50 = 1.1 mmHg at 20 degrees C), compared to whale and horse Mbs.     

Electrostatic interactions in sperm whale myoglobin. Site specificity, roles in structural elements, and external electrostatic potential distributions

The electrostatic free energy contribution to the stability of sperm whale ferrimyoglobin was evaluated according to the static accessibility modified Tanford-Kirkwood model. The electrostatic free energy contribution of each distinct structural element was divided into one term arising from interactions between it and other elements (interelemental) and another from interactions within the particular element itself (intraelemental). At pH 7 the majority of the terms were found to be stabilizing. The interelemental terms are the dominant ones for most structural elements. The small interelemental terms of the C and D helices are compensated by large intraelemental interactions which stabilize these short helices. Perturbations in pH can be accommodated by the structural elements through a redistribution of stabilizing and destabilizing interactions. The electrostatic potentials calculated at the surface of the protein indicate that the internal compensation of local potentials achieved during folding results in a generally neutral protein-solvent interface save for two distinct areas of nonzero potential. The accessibility of each charged atom to solvent was analyzed in terms of the surface area lost to charged, polar and nonpolar atoms separately. The net solvent accessibility lost parallels closely that lost to nonpolar atoms alone, indicating a specific role for nonpolar atoms in defining dielectric shielding of charged atoms, aside from their participation in the well-known hydrophobic interactions.     

Nuclear magnetic resonance studies of sperm whale myoglobin specifically enriched with 13C in the methionine methyl groups.

The Cepsilon methyl group of the 2 methionine residues in sperm whale myoglobin was enriched with respect to 13C. This was accomplished by treatment of the apomyoglobin at pH 4 at room temperature with a 100-fold proportion of 13CH3I to form an intermediate containing enriched S-methylmethionine. Unselective demethylation to regain the apomyoglobin structure was accomplished by treatment at pH 10.5 with 0.5 M dithioerythritol at 37 degrees for 18 h. Reagents were removed at each stage by dialysis against dilute sodium azide solution. Hemin was reincorporated to form the holoprotein in a way that avoided the presence of an excess of the small molecule. After chromatographic purification the enriched myoglobin was obtained in a yield of between 29 and 60%. The composition, absorbance spectrum, circular dichroism spectrum, isoionic point, electrophoretic behavior, and oxygen-binding behavior following reduction were all indistinguishable from those of the virgin protein. NMR measurements were made at 15.1, 25.2, and 67.9 MHz at 27-30 degrees. The two enriched loci are represented by separate resonances that appear slightly downfield of the spectral position of the corresponding resonance in free methionine. The positions of these resonances are sensitive to pH and to the ligand bound at the heme group which is approximately 17 A distant from each methionine Cepsilon. On the basis of two separate types of experiment the downfield resonance was assigned to methionine 55 and the upfield resonance to methionine 131. Part of the observed variations in chemical shift could be treated as arising from pseudocontact interactions but part was ascribed to structural changes communicated to the environment of each methionine residue as a result of changes in heme ligand, pH, or temperature. The linewidths of the methionine Cepsilon resonances are narrowed by increasing temperature according to an Arrhenius energy of activation of nearly 3 kcal. The spin-lattice relaxation times, T1, of the two methionine Cepsilon resonances at the three spectrometer frequencies were interpreted to indicate the existence of rotational motions in each side chain in addition to that about the Sdelta-Cepsilon bond. The results as a whole show that the two methionine side chains undergo continuous variations in environment, and that these variations are controlled by events at a distance within the protein structure. It is suggested that the structural lability serves the function of facilitating conformational variations and adjustments within the heme pocket.     

A grammatical theory for the conformational changes of simple helix bundles.

Polymers, including biomolecules such as proteins, have two particularly important types of single-molecule transitions: a helix-coil transition, driven by interactions that are local in the chain, and a collapse transition, driven by nonlocal interactions. A long-standing challenge of polymer statistical mechanics has been to deal with both types of transition in a single theoretical framework. The simplest paradigmatic problem would be a theory of helix-bundle folding. Here, we show how the machinery of formal grammars, originally developed in the context of linguistic analysis and programming-language compilation, provides a simple and general way to combine the Zimm-Bragg model of alpha-helices with the model of Chen and Dill for nonlocal interactions in antiparallel polymeric systems. We use a well-known construction in the theory of formal grammars to give the statistical mechanical partition function for two-helix bundles. Predictions are shown to be quite good in comparison to exact enumerations within a lattice model.     

Identification of the amino acid functional groups responsible for 30-S ribosome recognition of messenger RNA.

About 30 protein-selective chemical reagents have been tested for their ability to inhibit the mRNA binding activity of the 30-S ribosome. A number of reagents were investigated which have been shown by other workers to be capable of modifying free epsilon-amino groups of lysine and all were found to inactivate 30-S ribosomes completely for natural mRNA binding activity. Several reagents selective for histidine, tyrosine, and tryptophan were also found to inactivate. We suggest that the epsilon-amino groups of lysine play an important role in mRNA binding to the 30-S ribosome.