The exposed linker accounts for the difficulty in purifying full-length collagenase. The C-terminal domain provides a structural model for haemopexin and its homologues. It controls the specificity of MMPs, affecting both substrate and inhibitor binding, although its role remains obscure. These structural results should aid the design of site-specific mutants which will reveal further details of the specificity mechanism.
The automated microbatch technique developed at Imperial College has been used to establish a phase diagram for crystallization. The concentrations of the protein (carboxypeptidase G2) and precipitant (PEG 4000) were varied, while pH and temperature were kept constant. The diagram consists of an undersaturation and a supersaturation zone, the latter being subdivided into the metastable, nucleation and precipitation zones. In the metastable zone, crystals may grow but nucleation of crystals does not occur. It is the best zone for growth of X.-ray diffraction quality crystals because of the slower growth rate and the avoidance of uncontrolled nucleation, which uses up protein in the formation of tiny crystals. Nevertheless, in practice, it is rarely well defined or used because nuclei must be introduced artificially into the system. The new method used here consists of setting crystallization droplets at nucleation conditions and later diluting them to conditions where nucleation has not been observed. Single diffracting crystals of typical dimensions 0.3 × 0.3 x 0.2 mm were routinely obtained in the metastable zone, equivalent to the best (very rarely) obtained crystals in the nucleation zone.
Control of nucleation may be needed to obtain a reliable supply of large protein crystals, when standard techniques give many small or twinned crystals. Heterogeneous nucleation may be controlled by the use of fine filters, with the elimination of airborne contaminants by working under paraffin oil. The area of contact with the supporting vessel also has an important effect. A heterogeneous nucleant for lysozyme (identified earlier) has been shown to be effective for carboxypeptidase Gz. Control of homogeneous nucleation (previously demonstrated by dilutions of a nucleating sample after various times of incubation) may also be achieved by incubating a sample at 1 temperature, where nucleation can occur, and changing the temperature to conditions where there is growth but no nucleation.
The Kunitz-type soybean trypsin inhibitor (STI) has played a key role in the early study of proteinases, having been used as the main substrate in the biochemical and kinetic work that led to the de®nition of the standard mechanism of action of proteinase inhibitors. A partial structure of STI complexed with porcine trypsin has previously been reported, in which the ®rst 93 residues of the inhibitor, including the region of contact with trypsin, were relatively well de®ned, whereas for the remaining part of the peptide chain only some C atoms were located. The structure of the inhibitor in its free form has now been determined by molecular replacement to 2.5 A Ê , using the coordinates of the homologous Erythrina trypsin inhibitor as a search model. When the re®ned atomic coordinates of STI are compared with the partial model previously available, the conformation of the reactive-site loop and its position with respect to the main body of the molecule does not change when the inhibitor interacts with trypsin. There are instead, despite the high similarity in the overall tertiary structure, signi®cant differences between STI and Erythrina trypsin inhibitor (ETI) in the region which is in contact with the enzyme in the STI:trypsin crystal structure. Some of these differences can explain the unique speci®city of ETI and its ability to inhibit the ®brinolytic enzyme tissue-type plasminogen activator.
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