Carbonic anhydrase II microcrystals suitable for XFEL studies

Lomelino, Carrie L.; Kim, Jin Kyun; Lee, Cheol; Lim, Sujeong; Andring, Jacob T.; Mahon, Brian P.; Chung, М.; Kim, Chae Un; McKenna, Robert

doi:10.1107/s2053230x18006118

Cited by 8 publications

(9 citation statements)

References 24 publications

(19 reference statements)

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“…Hence, they may be detected only in time resolved experiments employing, for instance, serial femtosecond crystallography . Although the synthesis of HCA II microcrystals, needed for such studies, has been recently reported, no further time‐resolved data are available in the literature as yet. The only relevant experimental study based on time‐resolved X‐ray absorption spectroscopy detected fast variations between tetra‐ and penta‐coordinated states at the catalytic zinc site of bacterial alcohol dehydrogenase .…”

Section: Introductionmentioning

confidence: 99%

Coordination Dynamics of Zinc Triggers the Rate Determining Proton Transfer in Human Carbonic Anhydrase II

Paul

Taraphder

2020

ChemPhysChem

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We present, for the first time, how transient changes in the coordination number of zinc ion affects the rate determining step in the enzyme human carbonic anhydrase (HCA) II. The latter involves an intramolecular proton transfer between a zinc‐bound water and a distant histidine residue (His‐64). In the absence of time‐resolved experiments, results from classical and QM‐MM molecular dynamics and transition path sampling simulations are presented. The catalytic zinc ion is found to be present in two possible coordination states; viz. a stable tetra‐coordinated state, T and a less stable penta‐coordinated state, P with tetrahedral and trigonal bipyramidal coordination geometries, respectively. A fast dynamical inter‐conversion occurs between T and P due to reorganization of active site water molecules making the zinc ion more positively charged in state P. When initiated from different coordination environments, the most probable mechanism of proton transfer is found to be deprotonation of the equatorial water molecule from state P and transfer of the excess proton via a short path formed by hydrogen bonded network of active site water molecules. We estimate the rate constant of proton transfer as kP=1.29×106s-1 from P and kT=4.37×104s-1 from T. A quantitative match of estimated kP with the experimental value, (kexp∼0.8×106s-1 ) suggests that dynamics of Zn coordination triggers the rate determining proton transfer step in HCA II.

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Section: Introductionmentioning

confidence: 99%

Coordination Dynamics of Zinc Triggers the Rate Determining Proton Transfer in Human Carbonic Anhydrase II

Paul

Taraphder

2020

ChemPhysChem

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“…With one of the fastest known biological reactions, CA is an ideal candidate for such SFX experiments to map the movements of substrate/product through the active site. Microcrystals of CA II suitable for XFEL data collection have recently been produced and confirmed to diffract at the Pohang Accelerator Laboratory XFEL [ 153 ]. A further benefit is the collection of diffraction data at room temperature before the onset of radiation damage.…”

Section: Discussionmentioning

confidence: 99%

Crystallography and Its Impact on Carbonic Anhydrase Research

Lomelino

Andring

McKenna

2018

International Journal of Medicinal Chemistry

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X-ray and neutron crystallography are powerful techniques utilized to study the structures of biomolecules. Visualization of enzymes in complex with substrate/product and the capture of intermediate states can be related to activity to facilitate understanding of the catalytic mechanism. Subsequent analysis of small molecule binding within the enzyme active site provides insight into mechanisms of inhibition, supporting the design of novel inhibitors using a structure-guided approach. The first X-ray crystal structures were determined for small, ubiquitous enzymes such as carbonic anhydrase (CA). CAs are a family of zinc metalloenzymes that catalyze the hydration of CO2, producing HCO3− and a proton. The CA structure and ping-pong mechanism have been extensively studied and are well understood. Though the function of CA plays an important role in a variety of physiological functions, CA has also been associated with diseases such as glaucoma, edema, epilepsy, obesity, and cancer and is therefore recognized as a drug target. In this review, a brief history of crystallography and its impact on CA research is discussed.

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“…32,33 . Further, special sample containers can be coupled to the setup, which can be also utilized for real-time investigations to follow the crystallization process of biomolecules 34,35 . Fortunately, there are very little limitations for different types of sample containers, which can be applied, owing to a flexible platform of the MPM system.…”

Section: Methodsmentioning

confidence: 99%

“…It offers new opportunities in structural biology, e.g., allowing data collection at room temperature and to perform time-resolved experiments 5 – 7 . The size and dimensions of protein crystals required for diffractive imaging at high-brilliance micro-focus X-ray sources is continuously decreasing, down to a few micrometers or even sub-micrometer regime 8 , 9 . In this context, the demand to produce, detect, and, in particular, to monitor and image crystal suspensions upon preparation and just prior to data collection has substantially increased 10 .…”

Section: Introductionmentioning

confidence: 99%

Protein-crystal detection with a compact multimodal multiphoton microscope

Cheng¹,

Chung

Schubert

et al. 2020

Commun Biol

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There is an increasing demand for rapid, effective methods to identify and detect protein micro- and nano-crystal suspensions for serial diffraction data collection at X-ray free-electron lasers or high-intensity micro-focus synchrotron radiation sources. Here, we demonstrate a compact multimodal, multiphoton microscope, driven by a fiber-based ultrafast laser, enabling excitation wavelengths at 775 nm and 1300 nm for nonlinear optical imaging, which simultaneously records second-harmonic generation, third-harmonic generation and three-photon excited ultraviolet fluorescence to identify and detect protein crystals with high sensitivity. The instrument serves as a valuable and important tool supporting sample scoring and sample optimization in biomolecular crystallography, which we hope will increase the capabilities and productivity of serial diffraction data collection in the future.

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Carbonic anhydrase II microcrystals suitable for XFEL studies

Cited by 8 publications

References 24 publications

Coordination Dynamics of Zinc Triggers the Rate Determining Proton Transfer in Human Carbonic Anhydrase II

Coordination Dynamics of Zinc Triggers the Rate Determining Proton Transfer in Human Carbonic Anhydrase II

Crystallography and Its Impact on Carbonic Anhydrase Research

Protein-crystal detection with a compact multimodal multiphoton microscope

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