Haemoglobin, crystal structure

Respiratory pigments similar to the vertebrate haemoglobins have also been identified in many invertebrates. These vary from small proteins with two Fe-porphyrin units to large molecules containing up to 190 Fe-porphyrin units. Myoglobin, the 02 storage protein in muscle tissue, is also a small iron-protoporphyrin protein. The crystal structures of this and a number of other porphyrin proteins are now known (Chapter 20.2, Table 11). 

In 1992 the X-ray crystal structure of porphobilinogen deaminase was solved, an enzyme that is involved with the biosynthesis of a linear tetrapyrrole precursor to protoporphyrin IX, found in haemoglobin... 

Several multivalent anions, including inositol hexaphosphate (18), lower the oxygen affinity of haemoglobin. The crystal structure of the complex of... 

Ammonium sulphate is commonly used as a precipitant because of its great solubility and ready availability. It can liberate ammonia, which reacts with heavy atom derivatives. This problem can in some circumstances be alleviated by changing the mother liquor of the crystals to phosphate. Ammonium sulphate caused further problems in the analysis of the crystal structure of oxyhaemoglobin. In the presence of this salt and under irradiation, crystals of oxyhaemoglobin were oxidised rapidly to aqua-met-haemoglobin. The problem was solved by growing the crystals under usual conditions [34] and then transferring them to 3 M phosphate [16]. 

The first protein structures to be determined were myoglobin and haemoglobin in the late 1950s by Kendrew et al (1958) and Perutz et al (1960). From then on a steadily increasing number of protein structures have become known. Nowadays, once a suitable crystal is available, a new structure of a protein or even a virus can, in favourable circumstances, be determined in a year or less. In the case where there is a closely related structure available then a new crystal structure may be obtained in as little as 1-2 weeks. 

I Q) of the unlabelled protein is denoted as In and that for the labelled protein is /jj +122 + /n, where I22 is the scattering from the markers and 1 2 is the cross-term. The experimental procedure is based on the subtraction of the curve for the native protein from that of the labelled protein to give an oscillatory curve whose periodicity gives the required distance between metal sites. The subtraction assumes A2 to be negligible test calculations show that there must be > 200 electrons in each marker for a protein of 100,000 for this to be a satisfactory approximation. Thus the distance between the tetra-mercury markers attached to Cys 93 in the two chains of haemoglobin was determined as 3.8 + 0.2 nm, in good accordance with a calculation of 3.76 nm from the crystal structure [162]. The distance between mercury labels on histidine decarboxylase was determined as 6.9 0.3 nm [162]. 

Neutron experiments were first made on haemoglobin [98,99,147,166,167] and were extended to myoglobin [44,168], lysozyme [169] and catalase [170] as models of typical globular proteins. In parallel with X-ray scattering, the haemoglobin work (mainly in H20) identified a conformational change between the oxy- and deoxy-forms which was reflected in an difference of 0.054 nm in H20 buffers. Scattering curve comparisons to <2 = 3 nm with the crystal structures verified this. 

M. Paoli, R. Liddington, J. Tame, A. Wilkinson, and G. Dodson, Crystal Structure of T State Haemoglobin with Oxygen Bound at all Four Haems. J. Mol. Biol., 256,775-792,1996. 

R.C. Liddington, Z. Derewenda, E. Dodson, R. Hubbard, and G. Dodson, High resolution crystal structures and comparisons of T state deoxy and two liganded T state hemoglobins T (aoxy) Haemoglobin and T (met) Haemoglobin. J. Mol. Biol, 228,551-579,1992. 

Paoli, M. Liddington, R. Tame, J. Wilkinson, A. Dodson, G., Crystal structure of T state haemoglobin with oxygen hound at all four haems. Journal of Molecular Biology 1996, 256, (4), 775-792. 

Globular proteins were much more difficult to prepare in an ordered form. In 1934, Bernal and Crowfoot (Hodgkin) found, that crystals were better preserved if they were kept in contact with their mother liquor sealed in thin-walled glass capillaries. By the early 1940s crystal classes and unit cell dimensions had been determined for insulin, horse haemoglobin, RNAase, pepsin, and chymotrypsin. Complete resolution of the structures required identification of the crystal axes and some knowledge of the amino acid sequence of the protein—requirements which could not be met until the 1950s. 

Single-crystal X-ray diffraction is without question the most important and powerful technique for determining crystal and molecular structures, and applications of this technique led to many of the most important scientific advances that took place during the twentieth century. Scientific landmarks such as the determination of the structure of DNA 50 years ago [ 1 ] and determination of the structure of haemoglobin [2] provide potent illustrations of this point. There is every reason to expect that the central importance of single-crystal X-ray diffraction, both in the physical and in the biological sciences, will be sustained... 

A brief look at the contents page of any recent issue of the Journal of Molecular Biology (founded by John Kendrew, protein crystal-lographer and winner of the Nobel prize for Chemistry together with Max Perutz for the 3-dimensional structures of myoglobin and haemoglobin) will clearly establish that this is not so ... 

---