BIOCHEMISTRY - L. Stryer - 1984

VOLUME 1

PART I. CONFORMATION AND DYNAMICS

CHAPTER 3. OXYGEN CARRIERS: MYOGLOBIN AND HEMOGLOBIN

3.8. Myoglobin Has Essentially the Same Structure in Dissolved and Crystalline States

High-resolution X-ray crystallography has yielded a remarkably detailed, albeit static, picture of the Myoglobin molecule. A crucial question arises: does dissolved myoglobin share the same Structure as crystalline myoglobin? Crystalline myoglobin differs from dissolved myoglobin in that it is subjected to a high Ionic strength environment [3 M (NH₄)₂SO₄] during crystallization, and intermolecular interactions are established between individual molecules. Could these factors alter The myoglobin structure to such an extent that the X-ray crystallographic picture fails to reflect its biological function? The answer to this question is a resounding no. A wealth of evidence demonstrates that The structure of myoglobin in solution and in the crystalline state is remarkably similar.

1. In the crystalline state, myoglobin is functionally active: it can bind oxygen, although the reaction proceeds less rapidly than in solution. Overall, the reactivity of myoglobin in the crystalline state is reduced.

2. The absorption spectrum characteristic of the heme group in myoglobin is identical in both dissolved and crystalline states. Absorption spectra serve as a highly sensitive indicator of the microenvironment surrounding the heme group.

3. The α-Helix content in the dissolved molecule is estimated via optical rotatory dispersion and circular dichroism. The amount of α-helices measured in this way agrees closely with the number of α-helices derived from the crystal electron density map. It is highly unlikely that major conformational changes would occur without a corresponding change in the number of helical turns in the Cell/13.html">Protein Structure.

4. X-ray crystallographic analysis of seal myoglobin has revealed that the Tertiary Structure of this protein closely resembles that of whale myoglobin. In contrast, the crystal lattices of these myoglobins are quite distinct, and consequently, the intermolecular interactions within these two crystals differ. Thus, it is highly unlikely that significant structural distortions are caused by the crystal lattice.

3.9. Nonpolar Interactions Play a Crucial Role in Stabilizing the Conformation of Myoglobin

X-ray crystallography reveals the structure of myoglobin, but it does not explain why the molecule adopts this particular structure rather than another. Indeed, a central objective of Protein Chemistry is to determine how the Amino Acid Sequence dictates the three-dimensional structure of a protein. Studies on myoglobin have shown that the interior of the molecule consists of densely packed nonpolar residues. Amino acid residues such as valine, leucine, isoleucine, Methionine, and phenylalanine are hydrophobic. Given a choice between Water and a nonpolar environment, they clearly favor the latter. Furthermore, due to dense packing, their side chains are brought into close proximity, resulting in Van der Waals attractive forces between them. Thus, a significant portion of the attractive force driving the folding of the protein molecule is related to the Hydrophobicity of these amino acid residues—that is, their tendency to exclude water. These hydrophobic side chains are thermodynamically more stable when clustered in the interior of the molecule rather than exposed to an aqueous environment. The increase in stability upon protein folding is driven by the gain in Entropy that occurs when water molecules are no longer constrained in an ordered arrangement around exposed hydrophobic groups (Section 6.26). Consequently, a polypeptide chain in an aqueous solution spontaneously folds such that its hydrophobic side chains are sequestered in the interior, while its polar, charged side chains reside On the surface of the molecule.

3.10. The Unfolded Myoglobin Molecule Spontaneously Adopts a Functionally Active Configuration

Does the heme group influence the Spatial Structure of myoglobin? This question was answered by studying apomyoglobin, which is myoglobin devoid of its heme group. Apomyoglobin is prepared from myoglobin by lowering the solution pH to 3.5. Because this acidification weakens the bond between the heme and the protein, an organic solvent extraction is used to separate the heme from the protein. The aqueous phase containing apomyoglobin is then neutralized. Optical rotatory dispersion measurements have shown that at neutral pH, the degree of α-helix formation in apomyoglobin is 60%, which is significantly lower than that in myoglobin (75%). Hydrodynamic studies indicate that apomyoglobin is less compact than myoglobin. Moreover, the stability of apomyoglobin is considerably lower than that of myoglobin. Therefore, the presence of the heme group has a marked effect on the structure of myoglobin.

Is the folding of apomyoglobin and the subsequent incorporation of heme a spontaneous process? If urea or guanidine is added to a neutral solution of apomyoglobin, the protein molecule unfolds (denatures). The α-helix content under these conditions (i.e., in an 8 M urea solution) drops close to zero. Upon removal of urea by dialysis, the degree of α-helix formation increases back to 60%, meaning that the apomyoglobin refolds. Subsequent addition of heme to this solution results in The formation of biologically active myoglobin (Fig. 3.22). Upon reduction to ferromyoglobin, this renatured protein is capable of reversibly binding oxygen with its original efficiency. Consequently, the intricate three-dimensional structure of myoglobin is inherently encoded within The amino acid sequence of apomyoglobin when associated with the heme prosthetic group. This finding corroborated the universality of a principle first discovered during the investigation of Ribonuclease renaturation—namely, that the amino acid sequence determines protein conformation.

Class="center">Fig. 3.22. Formation of myoglobin from denatured apomyoglobin