Seminars Archive

Fri 23 May, at 10:30 - Seminar Room T2

Single Crystal ABSORPTION Microspectrophotometry and X-ray Crystallography: the Perfect Marriage

Andrea Mozzarelli
Department of Pharmacy, University of Parma, Parma, Italy

More than 90,000 structures are deposited in the PDB. For the majority of these structures functional properties in the crystalline state has not been tested. Lattice forces, crystal packing and conformation selection by crystallization might cause protein structures determined in the crystal to be different from the predominant and functionally-relevant conformation(s) present in solution. Single crystal polarized absorption microspectrophotometry is an approach perfectly suited to answer to the following questions: i) is the structure in the crystal the same as in solution?, ii) is a protein active in the crystal?, iii) can meta-stable intermediates be accumulated in the crystal, thus allowing cryo-crystallography-based structural determinations and structure-based functional mechanisms to be proposed on the basis of more than just the native protein structure? iv) Can meaningful structure-function correlations be obtained from thermodynamic and kinetic measurements carried out in the crystal? Selected cases will be discussed: i) cooperativity in oxygen binding to T state hemoglobin crystals, inferred from X-ray data, was not supported by oxygen binding curves carried in the same haemoglobin crystals, ii) the microspectrophotometric characterization of the reactivity towards the natural substrate of glyceraldehyde-3-P dehydrogenase in the crystal made possible the determination of the structure of the acyl-enzyme and led to propose a novel flip-flop catalytic mechanism, iii) The combination of structural and functional data determined in the crystal has been extensively exploited for the understanding of the reactivity of pyridoxal 5’-phosphate-dependent enzymes. Overall, both “couple” fights and “couple” synergies contribute to a better understanding on how proteins work.

(Referer: P. Storici)
Last Updated on Tuesday, 24 April 2012 15:21