Neutron Protein Crystallography

1. Neutron Protein Crystallography Benno Schoenborn ACA Robert Bau Award 2016

2. Outline Protein neutron crystallography is a valuable technique for study of biomolecular structure. Hydrogen atoms constitute about half of all atoms in proteins and play a critical role in enzyme mechanisms, macromolecular and solvent structure. • Early Days • Binding of Anaesthetics to Proteins • Studies of Myoglobin Structure • First Protein Structure using Neutron Diffraction • Developments • Advanced Detector Systems • Data Collection and Analysis • Sample Preparation • Center for Structural Biology BNL • Protein Hydration • Protein Structure on Different Length Scale • Membranes and Membrane Proteins • Neutron Protein Crystallography on Spallation Source • Recent Example • Summary Perdeuteration Myoglobin

3. Early Days • PhD student – structures of iridolactanes (C10H16O2) • Remarkable group of crystallographers at UNSW and other labs in Australia • X-ray diffraction to study structure of insect hormones with wide variety of pharmacological activity (including cat-nip!) • High-resolution (0.8Å) low-temperature (-130ºC) required • Optical transform techniques to solve structures • Sound background in diffraction methods and small biomolecular structures • Finding: chair conformation inconsistent with planarity of lactone group The structures of the iridolactanes. Benno P Schoenborn, PhD thesis UNSW 1962

4. Single detector diffractometer at BNL (Marian Koshland/William Hamilton) • Sperm whale metmyoglobin structure 1969 • Crystal size 25mm3 – many months data collect – 4600 reflections • Crystal soaked in D2O to exchange H atoms 2θ, θ scan; data points indicated by letters Section of the Fourier map depicting the haem group • Key finding: A preliminary analysis of myoglobin proves that neutron diffraction analysis of proteins is feasible and should reveal the position of fixed hydrogen atoms First Protein Structure using Neutron Diffraction Neutron Diffraction Analysis of Myoglobin. Benno P SchoenbornNature224 143-146 1969

5. Early Career Scientist MRC 1964-1966 Major developments in biomolecular structure Active research in protein structure especially myoglobin and haemoglobin Max Perutz and John Kendrew Nobel Prize Chemistry 1962 Studies of Myoglobin Structure • At MRC posed question how to determine position of hydrogen atoms • Neutron diffraction a remote possibility – nearly impossible at the time so set about finding a way • Returned to UC to continue studies • Structure of myoglobin a major theme throughout my career Note the state-of-the-art model display

6. Early Career Scientist at University of California Medical Center and MRC Cambridge (1962-1967) • Variety of techniques including X-ray diffraction to understand binding of inert gases to proteins – specifically Xe to myoglobin • Binding site involved many hydrogen atoms • Finding: specific binding site identified – London dispersion forces – hydrogen positions required to calculate binding energy Difference Fourier syntheses (X-ray) between deoxymyoglobin-xenon and metmyoglobin(1) Xenon site; (2) negative group absent in deoxymyoglobin; (3) cavity (water site in metmyoglobin) Binding of Anaesthetics to Proteins Binding of Xenon to Sperm Whale Myoglobin. Benno P Schoenborn, Herman C Watson, John C Kendrew. Nature207 28-30 1965 Binding of Xenon to Sperm Whale Deoxymyoglobin. Benno P Schoenborn, Chris Nobbs. J Mol Pharm 2 491-498 1966

7. Membranes and Membrane Proteins Membranes are complex biological structure of fundamental importance – structure of membrane proteins major future challenge • Rod photoreceptor membranes in intact retinas • Acetylcholine receptor • Oriented multilayer membranes • Isidore Edelman, Columbia University • Pioneering work on ion transport - led to discovery of Na-K ATPase • Low angle neutron diffraction of membranes – kidney outer medulla • Small angle neutron scattering solubilised protein • Donald Caspar, Brandeis and Florida State University • Low angle neutron diffraction of nerve myelin • Development of novel multilayer monochromator (patent) Neutron scattering for the analysis of membranes. BP Schoenborn. BiochimBiophysActa457 41-55 1976 Neutron diffraction of intact retinas. M Yeager, BP Schoenborn, DE Engelman, P Moore, L Stryer. J MolBiol137 315-348 1980

8. Summary • Existing facilities at LANL, ILL, (JAERI) • New facilities at SNS, J-PARC and ESS (few days for ~5mm3 crystal) • Quasi-Laue and time-of-flight techniques optimised • Not all structures in PDB • Multiple large area detectors available • Sophisticated data analysis procedures including joint X-ray/neutron refinement • Maximum-likelihood and annealing techniques (egnCNS, PHENIX) • Expression systems for production of ‘large’ quantities of protein • Molecular biology for protein deuteration – specialist deuteration labs at facilities • Well-formulated problems in Structural Biology Front covers from selected work at PCS LANL

9. Epilogue Protein neutron crystallography is now a mature experimental technique which will continue to make a valuable contribution to the understanding of macromolecular structure. The user community is expanding rapidly and the number of facilities is increasing accordingly. As problems become more complex, the need for more detailed information becomes greater. Research will continue on specific molecular mechanisms as well as general underlying principles. Acknowledgements I would like to acknowledge all my students, Postdoctoral Fellows and colleagues who over the years worked tirelessly to help achieve goals I set early in my career. I would also like to acknowledge funding agencies which have contributed to the many activities especially the US Department of Energy and the National Science Foundation.