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Ren Z.,University of Chicago | Ren Z.,Renz Research Inc.
PLoS ONE | Year: 2013

Hemoglobin transports molecular oxygen from the lungs to all human tissues for cellular respiration. Its α2β2 tetrameric assembly undergoes cooperative binding and releasing of oxygen for superior efficiency and responsiveness. Over past decades, hundreds of hemoglobin structures were determined under a wide range of conditions for investigation of molecular mechanism of cooperativity. Based on a joint analysis of hemoglobin structures in the Protein Data Bank (Ren, companion article), here I present a reverse engineering approach to elucidate how two subunits within each dimer reciprocate identical motions that achieves intradimer cooperativity, how ligand-induced structural signals from two subunits are integrated to drive quaternary rotation, and how the structural environment at the oxygen binding sites alter their binding affinity. This mechanical model reveals the intricate design that achieves the cooperative mechanism and has previously been masked by inconsistent structural fluctuations. A number of competing theories on hemoglobin cooperativity and broader protein allostery are reconciled and unified. © 2013 Zhong Ren. Source

Munshi P.,Oak Ridge National Laboratory | Munshi P.,Middle Tennessee State University | Munshi P.,Shiv Nadar University | Snell E.H.,State University of New York at Buffalo | And 6 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2014

Ketol-isomerases catalyze the reversible isomerization between aldoses and ketoses. d-Xylose isomerase carries out the first reaction in the catabolism of d-xylose, but is also able to convert d-glucose to d-fructose. The first step of the reaction is an enzyme-catalyzed ring opening of the cyclic substrate. The active-site amino-acid acid/base pair involved in ring opening has long been investigated and several models have been proposed. Here, the structure of the xylose isomerase E186Q mutant with cyclic glucose bound at the active site, refined against joint X-ray and neutron diffraction data, is reported. Detailed analysis of the hydrogen-bond networks at the active site of the enzyme suggests that His54, which is doubly protonated, is poised to protonate the glucose O5 position, while Lys289, which is neutral, promotes deprotonation of the glucose O1H hydroxyl group via an activated water molecule. The structure also reveals an extended hydrogen-bonding network that connects the conserved residues Lys289 and Lys183 through three structurally conserved water molecules and residue 186, which is a glutamic acid to glutamine mutation. © 2014 International Union of Crystallography. Source

Ren Z.,University of Chicago | Ren Z.,Renz Research Inc.
PLoS ONE | Year: 2013

Structural motions along a reaction pathway hold the secret about how a biological macromolecule functions. If each static structure were considered as a snapshot of the protein molecule in action, a large collection of structures would constitute a multidimensional conformational space of an enormous size. Here I present a joint analysis of hundreds of known structures of human hemoglobin in the Protein Data Bank. By applying singular value decomposition to distance matrices of these structures, I demonstrate that this large collection of structural snapshots, derived under a wide range of experimental conditions, arrange orderly along a reaction pathway. The structural motions along this extensive trajectory, including several helical transformations, arrive at a reverse engineered mechanism of the cooperative machinery (Ren, companion article), and shed light on pathological properties of the abnormal homotetrameric hemoglobins from α-thalassemia. This method of meta-analysis provides a general approach to structural dynamics based on static protein structures in this post genomics era. © 2013 Zhong Ren. Source

Perry S.L.,University of Massachusetts Amherst | Perry S.L.,University of Chicago | Perry S.L.,University of Illinois at Urbana - Champaign | Guha S.,University of Illinois at Urbana - Champaign | And 7 more authors.
Journal of Applied Crystallography | Year: 2014

Renewed interest in room-temperature diffraction has been prompted by the desire to observe structural dynamics of proteins as they function. Serial crystallography, an experimental strategy that aggregates small pieces of data from a large uniform pool of crystals, has been demonstrated at synchrotrons and X-ray free-electron lasers. This work utilizes a microfluidic crystallization platform for serial Laue diffraction from macroscopic crystals and proposes that a collection of small slices of Laue data from many individual crystals is a realistic solution to the difficulties in dynamic studies of irreversible biochemical reactions. © 2014 International Union of Crystallography. Source

Pawate A.S.,University of Illinois at Urbana - Champaign | Srajer V.,University of Chicago | Schieferstein J.,University of Illinois at Urbana - Champaign | Guha S.,University of Illinois at Urbana - Champaign | And 8 more authors.
Acta Crystallographica Section:F Structural Biology Communications | Year: 2015

Serial methods for crystallography have the potential to enable dynamic structural studies of protein targets that have been resistant to single-crystal strategies. The use of serial data-collection strategies can circumvent challenges associated with radiation damage and repeated reaction initiation. This work utilizes a microfluidic crystallization platform for the serial time-resolved Laue diffraction analysis of macroscopic crystals of photoactive yellow protein (PYP). Reaction initiation was achieved via pulsed laser illumination, and the resultant electron-density difference maps clearly depict the expected pR1/pRE46Q and pR2/pRCW states at 10μs and the pB1 intermediate at 1ms. The strategies presented here have tremendous potential for extension to chemical triggering methods for reaction initiation and for extension to dynamic, multivariable analyses. © 2015 International Union of Crystallography. Source

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