Amino acid substitutions, macromolecular

In this chapter, we provide protocols to determine the ability of a peptide to mediate DNA internalization in cultured human tumor cells. Fluorescence-assisted cell sorting (FACS) analysis is used to obtain quantitative data on the time and temperature dependence of macromolecular delivery. Confocal microscopy is used to study the subcellular localization in both fixed and live cells. Fluorescently labeled transferrin and dextran are used to label the clathrin-dependent (15) and the non-clathrin, non-caveolar (16) endocytic compartments, respectively. Expression of a caveolin-l-YFP fusion protein is used to label cell surface caveolae and intracellular caveosomes (17). Finally a protocol, for the overexpression of dominant-negative dynamin [GTPase deficient dynamin-2 containing the amino acid substitution K44A (18)] is provided to evaluate the dynamin dependence of the uptake mechanism. 

We have shown that the reversible oxygen uptake of myoglobin and hemoglobin is very sensitive to the kind and sequence of a-amino acids in the macromolecular protein chain. Now it is interesting to speculate if it is possible to substitute the heme in, for example, myoglobin by other porphyrin-type compounds without loss of reversible oxygen function. This topic forms a bridge between fully natural and fully artificial/synthetic systems. 

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