At the time he received the letter, Max had been wondering how allosteric modulation evolved in hemoglobins and had formulated testable hypotheses for the origins of the allostery based on his crystal structures of hemoglobin, which accounted for the protein’s hallmark cooperativity. The tests involved site-directed mutagenesis and generation of recombinant protein. This might seem a straightforward endeavor today, but four decades ago there were virtually no procedures for engineering and expressing proteins, yet alone a multi-subunit protein assembly. The protocols and reagents required development almost entirely from scratch. Max had an eye for talent and immediately wrote back to Kiyoshi, inviting him to return to Cambridge and explore methods for making and characterizing the first-ever recombinant mutant hemoglobins. Kiyoshi gave up a secure lecturing post to undertake a risky project with the temporary support of a two-year fellowship and returned to Cambridge with his young family in 1981 to work again with his beloved mentor.
Through imagination, hard work, intelligence and inspired late-night discussions with Hans Thogersen and other colleagues, Kiyoshi invented methods to overexpress human hemoglobin subunits in bacteria as fusion proteins. The key to success was the liberation of hemoglobin from the fusion with a sequence-specific endopeptidase. The material was then reconstituted from a denatured state to fold with heme. This successfully produced recombinant hemoglobins with directed mutations that could be used in functional assays with allosteric modulators and for X-ray crystal structure analyses to visualize their impact on stereochemistry. In subsequent studies, Kiyoshi used this method to understand the molecular origins of divergent allosteric effects in hemoglobins of different species. The methodology revolutionized hemoglobin studies and was adopted internationally with Kiyoshi’s help.
