Phillip D. Pulsinelli, PhDProfessor Emeritus, Pharmaceutical Sciences
Dr. Pulsinelli began his research and teaching career at the University of Pittsburgh School of Pharmacy in 1973. As an Assistant Professor of Medicinal Chemistry and Adjunct Assistant Professor of Crystallography, he continued the X-ray crystallographic, spectroscopic, and theoret-ical studies on the structure and oxygen transport function of normal and abnormal hemoglobins, which he had initiated while working with Max Perutz in Cambridge, England from 1970 to 1973. His use of mutant forms of human hemoglobin to structurally probe the cooperative oxygen bind- ing mechanism of normal hemoglobin resulted in several significant 'firsts' in the fields of protein crystallography and molecular biology: His X-ray structure analysis of hemoglobin Hiroshima, for example, yielded the first three-dimensional structure of a mutant protein molecule, of any kind, let alone one that was also known to cause a serious abnormal physiological function in humans (polycythemia ).
The same structure analysis also resulted in a major correction of the chemically determined amino acid substitution that had originally been assigned to the mutant, showing for the first time, that X-ray crystallography, when done at sufficiently high resolution, could be used to directly determine, or at the very least, rigorously confirm, amino acid sequences in proteins. The detailed structure-function correlations that Hiroshima yielded not only explained its inherent abnormal behavior, it also provided the first detailed structural explanations of hemoglobin's re- markable cooperative allosteric behavior towards protons ( i.e. the Bohr effect ), CO2 , and the allosteric effector, 2,3-BPG. From a purely crystallographic standpoint, the work on Hiroshima also involved the very first application of fast Fourier transforms (FFT's) as a mathematical meth-odology for calculating electron density and difference electron density maps of proteins.
Dr. Pulsinelli's subsequent work on the abnormal hemoglobins M ( M Milwuakee, M Boston, and M Hyde Park, all of which cause severe forms of methemoglobinemia ), on hemo-globin Yakima ( a completely non-cooperative high-affinity mutant that causes compensatory polycythemia), and on chemically-prepared, ferrous-ferric, mixed-valence hybrids of normal hemoglobin, played a central role in explaining the structural and stereoelectronic origins of the cooperative subunit interactions ( heme-heme interactions ) that are displayed by normal hemo-globin, when it binds and releases oxygen.
The crystallographic and spectroscopic results on the hemoglobins M ( natural mixed-valence hybrids ), the spectroscopic work on chemically prepared hybrids, and theoretical work conducted in discussions with R.J.P. Williams at Oxford, provided the first evidence that changes in the spin-state configurations of the ferrous hemes in hemoglobin ( caused by O2-induced, axial, ligand-field perturbations of the Fe2+ valence shell electrons ) initiated conformational displacements at the hemes, which then caused a well-defined sequence of globin conformational displacements that eventually altered key sites of interaction between hemoglobin's and subunits. The structure of deoxyhemoglobin Yakima showed that the deoxy quaternary structure of hemoglobin could adopt oxy-like tertiary conformational features at key contact sites between subunits, and at the hemes, even when hemoglobin was fully deoxygenated.
At about the same time he was promoted to the rank of Associate Professor ( 1978 ), Dr. Pulsinelli initiated high-resolution infrared spectroscopic studies on variably hydrated thin films of liganded forms of myoglobin and hemoglobin. After about 5 years of painstakingly meticulous work aimed at perfecting quantitative methods for determining variations in the degree of hydra-tion of concentrated films of the proteins, he and his two graduate students at the time ( Bill Brown and Jack Sutcliffe ) were able to begin to produce computer-enhanced, multiple internal reflectance, IR spectra of the oxy, CO, and azido( N3 )met forms of sperm whale myoglobin and human hemoglobin.
The studies eventually resulted in the identification and characterization of the O=O, CαO, and N= N stretching frequencies of the heme-bound ligands in both proteins. Studies of the vibrational behavior of these heme ligands provided information about the dynamical con-formational states they could adopt at the hemes, under a wide variety of conditions; e.g. as a function of variations in the degree of hydration at the protein surface, in temperature, in the binding of allosteric effectors,...etc. Jack Sutcliffe's work on azidomethemoglobin was especially gratifying, since it rigorously confirmed that the allosteric binding interactions of 2,3-BPG and IHP, which initiate at the surface of the hemoglobin molecule, propagate to the hemes, where they shift the heme's spin configuration and the conformer equilibrium of the heme-bound azide. The work showed, in an elegant fashion, that these two dynamical processes are in fact strongly coupled and highly sensitive to the allosteric effects displayed by hemoglobin.
During this same period, Dr. Pulsinelli assembled and helped fund a small group of people with a common interest in solving the structure of the iron storage and transport protein, transferrin. A low resolution ( 5 A ) structure of hen ovo-transferrin was determined in 1981; very likely the first protein structure ever done at a school of pharmacy.
Over his entire 33-year career at Pitt, Dr. Pulsinelli has always been heavily involved in teaching, especially at the undergraduate level. He has both coordinated courses and contributed lectures in all areas of biochemistry and in selected areas of medicinal chemistry, in a number of health-related academic venues; e.g. in The Schools of Pharmacy, Dental Medicine, and Medicine.
He has also participated in the organization and presentation of graduate courses in the fields of X-ray crystallography, diffraction physics, and biophysics in the Departments of Crystallography, Physics, and Biophysics & Microbiology ( all in FAS ). In the School of Pharmacy, he coordinated a graduate tutorial course on the Molecular Biology of Drug-Receptor Interactions. In 1982, he was awarded the Hygeia Teacher of the Year Award by the School of Pharmacy Class of 1982.
Although never one to eagerly seek out committee work, Dr. Pulsinelli has at times served on various committees and panels both within the School of Pharmacy and for the University of Pittsburgh in general. His longest period of service on one committee, and one which he considers to be of particular importance, was the 16 years of service he devoted to the School of Pharmacy Academic Performance and Review committee, which monitors and evaluates the academic prog-
ress of our pharmacy students. He has also served on University of Pittsburgh Tenure Appeals Panels, Academic Integrity Boards, and a Provost's Committee on Graduate Programs in FAS.
His committee duties outside of the University have included 4 years as a member of the PA Drug Device & Cosmetic Board. On the professional side, he has served as an Organizational Chairman for the Pittsburgh Diffraction Conference and has chaired, co-chaired, or delivered lectures at international colloquia, symposia, and workshops on protein crystallography, crystallographic computing, and the structure and function of hemoglobins. At various times, he has also held memberships in a number of professional societies, namely, the Pittsburgh Diffraction Society, the American Crystallographic Association, the International Union of Crystallography, the American Institute of Physics, the American Chemical Society, and the American Association for the Advancement of Science.