Globins

Perhaps the most important complex of iron(II) is heme (or haeme). Haemoglobin, the iron-containing constituent of the blood, consists essentially of a protein, globin, attached through a nitrogen atom at one coordination position of an octahedral complex of iron(II). Of the other five coordination positions, four (in a plane) are occupied by nitrogen atoms, each of which is part of an organic... 

Having built a hidden Markov model for a particular family of proteins, it can then b< used to search a database. A score is computed for each sequence in the database anc those sequences that score significantly more than other sequences of a similar length ar( identified. This was demonstrated for two key families of proteins, globins and kinases ii the original paper [Krogh et al. 1994]. For the kinases, 296 sequences with a Z score abov<... 

An article entitled Memo globin Its Occurrence Structure and Adaptation appeared in the March 1982 issue of the Journal of Chem ical Education ipp 173-178)... 

A typical electrospray analysis can be completed in 15 min with as little as 1 pmol of protein. An analysis of the cord blood of a baby (Figure 40.6) showed quite clearly that five globins were present, viz., the normal ones (a, (3, Gy, and Ay) and a sickle-cell variant (sickle (3). The last one is easily revealed in the mass spectrum, even at a level of only 4% in the blood analyzed. 

Electrospray mass spectra of globins from the blood of (a) a child diagnosed as having the sickle-cell anemia trait and (b) of its mother. As well as the usual p-globin sickle-cell variant at m/z 15,837.2, a new variant (P-Montreal-Chori) appears at m/z 15,879.3 and is observed in both the child and the mother. 

Beta-cydodextnn Beta-galactosidase Beta-globin Betaine... 

Gofactors. Frequendy proteins exist in their native state in association with other nonprotein molecules or cofactors, which are cmcial to their function. These may be simple metal ions, such as Fe " in hemerythrin or Ca " in calmodulin a heme group, as for the globins nucleotides, as for dehydrogenases, etc. 

Fig. 3. Representation of the nine principal folds which recur in protein stmctures, where the codes of the representative proteins taken from the Brookhaven Protein Data Bank (PDB) (17) are given in parentheses (18) (1) globin (Ithb) (2) trefoil (lilb) (3) up—down (256b) (4) immunoglobulin folds...

The automated method differs from the ICSH method chiefly in that oxidation and ligation of heme iron occur after the hemes have been released from globin. Therefore, ferricyanide and cyanide need not diffuse into the hemoglobin and methemoglobin, respectively. Because diffusion is rate-limiting in this reaction sequence, the overall reaction time is reduced from approximately three minutes for the manual method to 3 —15 seconds for the automated method. Reaction sequences in the Coulter S + II and the Technicon H 1 and H 2 are similar. Moreover, similar reactions are used in the other Coulter systems and in the TOA and Unipath instmments. 

Arthur Lesk and Cyrus Chothia at the MRC Laboratory of Molecular Biology in Cambridge, UK, compared the family of globin strucfures with the aim of answering two general questions How can amino acid sequences that are very different form proteins that are very similar in their three-dimensional structure What is the mechanism by which proteins adapt to mutations in the course of their evolution ... 

To answer the first question, Lesk and Chothia examined in detail residues at structurally equivalent positions that are involved in helix-heme contacts and in packing the a helices against each other. After comparing the nine globin structures then known, the 59 positions they found that fulfilled these criteria were divided into 31 positions buried in the interior of the protein and 28 in contact with the heme group. These positions are the principal determinants of both the function and the three-dimensional structure of the globin family. 

Lesk and Chothia did find, however, that there is a striking preferential conservation of the hydrophobic character of the amino acids at the 59 buried positions, but that no such conservation occurs at positions exposed on the surface of the molecule. With a few exceptions on the surface, hydrophobic residues have replaced hydrophilic ones and vice versa. However, the case of sickle-cell hemoglobin, which is described below, shows that a charge balance must be preserved to avoid hydrophobic patches on the surface. In summary, the evolutionary divergence of these nine globins has been constrained primarily by an almost absolute conservation of the hydro-phobicity of the residues buried in the helix-to-helix and helix-to-heme contacts. 

The proteins thus adapt to mutations of buried residues by changing their overall structure, which in the globins involves movements of entire a helices relative to each other. The structure of loop regions changes so that the movement of one a helix is not transmitted to the rest of the structure. Only movements that preserve the geometry of the heme pocket are accepted. Mutations that cause such structural shifts are tolerated because many different combinations of side chains can produce well-packed helix-helix interfaces of similar but not identical geometry and because the shifts are coupled so that the geometry of the active site is retained. 

Hemoglobin is a tetramer built up of two copies each of two different polypeptide chains, a- and (5-globin chains in normal adults. Each of the four chains has the globin fold with a heme pocket. Residue 6 in the p chain is on the surface of a helix A, and it is also on the surface of the tetrameric molecule (Figure 3.13). 

Figure 3.13 The hemoglobin molecule is built up of four polypeptide chains two a chains and two (3 chains. Compare this with Figure 1.1 and note that for purposes of clarity parts of the a chains are not shown here. Each chain has a three-dimensional structure similar to that of myoglobin the globin fold. In sicklecell hemoglobin Glu 6 in the (3 chain is mutated to Val, thereby creating a hydrophobic patch on the surface of the molecule. The structure of hemoglobin was determined in 1968 to 2.8 A resolution in the laboratory of Max Perutz at the MRC Laboratory of Molecular Biology, Cambridge, UK.

We thus have here a case where a mutation on the surface of the globin fold, replacing a hydrophilic residue with a hydrophobic one, changes important properties of the molecule and produces a lethal disease. Why has the... 

The globin fold has been used to study evolutionary constraints for maintaining structure and function. Evolutionary divergence is primarily constrained by conservation of the hydrophobicity of buried residues. In contrast, neither conserved sequence nor size-compensatory mutations in the hydrophobic core are important. Proteins adapt to mutations in buried residues by small changes of overall structure that in the globins involve movements of entire helices relative to each other. 

Bashford, D., Chothia, C., Lesk, A.M. Determinants of a protein fold. Unique features of the globin amino acid sequences. /. Mol. Biol. 196 199-216, 1987. 

Pasture, A., et al. Stmctural alignment and analysis of two distantly related proteins Aplysia limacina myoglobin and sea lamprey globin. Proteins 4 240-250, 1988. 

Pasture, A., Lesk, A.M. Comparison of the structures of globins and phycocyanins evidence for evolutionary relationship. Proteins 8 133-155, 1990. 

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