Chemically modified mutants

Chemically Modified Mutants, a Marriage of Chemical Modification and Protein Engineering... 

Dichlorophenylboronic acid Chemically modified mutants of subtilisin 95,96... 

Until recently, the catalytic role of Asp ° in trypsin and the other serine proteases had been surmised on the basis of its proximity to His in structures obtained from X-ray diffraction studies, but it had never been demonstrated with certainty in physical or chemical studies. As can be seen in Figure 16.17, Asp ° is buried at the active site and is normally inaccessible to chemical modifying reagents. In 1987, however, Charles Craik, William Rutter, and their colleagues used site-directed mutagenesis (see Chapter 13) to prepare a mutant trypsin with an asparagine in place of Asp °. This mutant trypsin possessed a hydrolytic activity with ester substrates only 1/10,000 that of native trypsin, demonstrating that Asp ° is indeed essential for catalysis and that its ability to immobilize and orient His is crucial to the function of the catalytic triad. 

SEA (Leu-48-Gly) and SEB (Phe-44-Ser) mutants unable to bind major histocompatibility complex class II remain emetic but lack T-cell mitogenic effects. " A disulfide loop in SEs, which is absent in the non-enterotoxic TSST-1, may be responsible for the emetic activity of SEs but that too remains controversial. Carboxymethylation of histidines on SEA or SEB generates superantigenic molecules devoid of enter-otoxicity or skin reactivity. This chemically modified SEB also inhibits, perhaps in a competitive fashion, the... 

Recognition of cognate tRNAs. Many attempts have been made to learn what part or parts of tRNA molecules are involved in recognition by aminoacyl-tRNA synthetases. Nucleotide sequences of isoacceptor tRNAs have been compared. Chemically modified and fragmented tRNA molecules have been studied, and many mutant tRNAs have been made. These... 

The potential of the method of using mutant and chemically modified Hbs to assign proton resonances of Hb is greatly enhanced when correlated with both intensity measurements and calculations from the crystal structure of Hb as determined by X-ray diffraction. A representative example of this approach is El 1 Val in HbCO A. Based on intensity measurements on a mixture of HbCO A and ferrocy-tochrome c, Lindstrom etal. (1972b) have shown for the ring-current-shifted proton resonances at —5.86, -6.48, and -6.58 ppm from HDO that each arises from one CH3 per a(3 dimer. In the spectrum of a mixture of HbCO A and HbCO Sydney [(367(E1 l)Val — Ala],... 

This lack of confirmation of an apparent assignment points out that the method of using mutant or chemically modified Hbs to assign resonances as described above can be used correctly only if the single... 

Stefanini et al. 128) have also attempted to identify which carboxyl-ates form the ferroxidase center, using horse spleen ferritin. Their study, which involved the application of various spectroscopic techniques to chemically modified ferritin, identified Asp 127 and Glu 130, groups present in the threefold channel, as the ferroxidase center. However, studies by Treffry et al. 141) of site-directed mutants of human heavy-chain ferritins indicate that the ferroxidase center is not in the threefold channels. 

Wild-type azurin including a single surface histidine at position 83 and Glnl07His/His83Gln azurin mutant were chemically modified by the luminescent rhenium(I) phenantroline complex 30. 

Haemoglobins.—Perutz and his colleagues have described a number of experiments designed to test their stereochemical interpretation of the co-operative effects in haemoglobin, presented in 1970. The experiments include A -ray studies of chemically modified and mutant haemoglobins. 

Table 1. Half Saturation Oxygen Pressure and Hill Parameter of Some Chemically Modified and Mutant Hemoglobins (from Data Collected by Baldwin (1975) and Data of Coleman (1977)). Half Saturation Oxygen Pressure is Given Relative to the Value of the Normal Hemoglobin (HbA).

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