Proteins structural deductions

Observations of adsorbed Hageman factor on quartz (5) showed that the protein underwent considerable structural modification however, quantitative structural deductions were not made. Furthermore, no attempt was made to follow time-dependent changes in conformation. 

Electrospray is an unusual mass spectrometry technique in that it allows the study of the three-dimensional structure of compounds, particularly proteins, in solution as it is believed that this is relatively unchanged when ions are transferred to the vapour phase. This type of application will be discussed in more detail in Chapter 5 but attention is drawn at this point to the previous comments regarding the effect that the HPLC conditions, such as pH, may have on the appearance of an electrospray spectrum and the conformational deductions that may be made from them. 

Conventionally, the first attribute known about an enzyme used to be its function, usually in a crude extract. This property was screened for in microbial cultures or in tissue samples. The crude extract was then purified to homogeneity and the protein subjected to biochemical studies to learn of its pH and T profiles, its pi and subunit composition, catalytically important residues, and other properties. Proteolytic digestion of the protein with subsequent Edman degradation led to the primary sequence, but no information on the secondary structures such as a-heli-ces and [5-sheets or the folding in three dimensions of the polypeptide chain. The primary sequence could have been used to deduct the gene sequence but, with the degeneration of the code, several possibilities for certain amino acids occur, which makes prediction of the gene sequence a risk. 

Gradually and relentlessly, after the war, chemical and crystallographic work proved that protein molecules did not have the cyclol structure. Yet many of Dorothy Wrinch s geometrical instincts and deductions were, in general terms, correct. 

Since that time many more sequences have become available through the advent of recombinant DNA technology and the deduction of amino acid sequences from the base sequences of cloned DNA. At the present time, the primary structures (amino acid sequences) of 14 proteins of the transferrin family have been established. These include seven serum transferrins, from human 10, 36), pig (37), horse 38), rabbit 39), toad Xenopus laevis) 40), sphinx moth (M. sexta) 13), and cockroach Blaberus discoidalis 4) chicken 34, 35) and duck 41) ovotransfer-rins four lactoferrins, from human (11, 42), mouse 43), pig 44) and cattle 45, 46) and the human tumor cell melanotransferrin 47). All of these sequences are available from sequence databases such as EMBL and SWISSPROT. 

The proportions of these three conformations that exist within a complex polypeptide or protein can therefore be determined with fair accuracy (using the Chou-Fasman rules Chou and Fasman, 1974 see also Chapter 2). Results such as this protein in aqueous solution is 35% a-helical and 10% (3-structured that appear in the research literature are based on interpretation of CD spectra (Woody, 1995). A search for the particular stretches of these conformations within the polypeptide is then attempted, through deductions based on the amino-acid sequence, taking account of the locations of the hydrophilic and hydrophobic side-chains, to define the three-dimensional structure of a polypeptide. 

All methods of deduction of the relative phases for Bragg reflections from a protein crystal depend, at least to some extent, on a Patterson map, commonly designated P(uvw) (46, 47). This map can be used to determine the location of heavy atoms and to compare orientations of structural domains in proteins if there are more than one per asymmetric unit. The Patterson map indicates all the possible relationships (vectors) between atoms in a crystal structure. It is a Fourier synthesis that uses the indices, l, and the square of the structure factor amplitude f(hkl) of each diffracted beam. This map exists in vector space and is described with respect to axes u, v, and w, rather than x,y,z as for electron-density maps. 

Although spectroscopic studies led to the correct deduction of the structure of the Fe2S2 core, the synthesis of model compounds containing this core provided unequivocal confirmation. The model compounds allowed detailed structural analysis unavailable for the proteins. Moreover, by using a uniform set of peripheral ligands, properties inherent to the Fe2S2 core could be discerned. 

Figure 4.7 CD spectra of polypeptides in pure conformations as indicated (left). These form a typical basis set for the deduction of secondary structure content in a protein sample of interest by mathematical analysis of the recorded CD spectrum (right).

Figure 10.1. The central dogma. Proceeding from the DNA through the RNA to the protein level, various sequence features and modifications can be identified that can be used in the computational deduction of gene structure. These include the presence of promoter and regulatory regions, intron-exon boundaries, and both start and stop signals. Unfortunately, these signals are not always present, and, when they are present, they may not always be in the same form or context. The reader is referred to the text for greater detail.

---