William B. O'Dell

Organization and flexibility of cyanobacterial thylakoid membranes examined by neutron scattering.

Background: In cyanobacteria, light harvesting and photosynthesis occur in the thylakoid membranes. Results: The distances between thylakoid membranes are correlated with the size of the phycobilisome antenna and change reversibly and rapidly upon illumination. Conclusion: Thylakoid membranes have a structural plasticity tied to the regulation of photosynthesis. Significance: Characterizing the structural changes in photosynthetic membranes is crucial for understanding light harvesting and photosynthetic productivity. Cyanobacteria are prokaryotes that can use photosynthesis to convert sunlight into cellular fuel. Knowledge of the organization of the membrane systems in cyanobacteria is critical to understanding the metabolic processes in these organisms. We examined the wild-type strain of Synechocystis sp. PCC 6803 and a series of mutants with altered light-harvesting phycobilisome antenna systems for changes in thylakoid membrane architecture under different conditions. Using small-angle neutron scattering, it was possible to resolve correlation distances of subcellular structures in live cells on the nanometer scale and capture dynamic light-induced changes to these distances. Measurements made from samples with varied scattering contrasts confirmed that these distances could be attributed to the thylakoid lamellar system. We found that the changes to the thylakoid system were reversible between light- and dark-adapted states, demonstrating a robust structural flexibility in the architecture of cyanobacterial cells. Chemical disruption of photosynthetic electron transfer diminished these changes, confirming the involvement of the photosynthetic apparatus. We have correlated these findings with electron microscopy data to understand the origin of the changes in the membranes and found that light induces an expansion in the center-to-center distances between the thylakoid membrane layers. These combined data lend a dynamic dimension to the intracellular organization in cyanobacterial cells.

Neutron protein crystallography: A complementary tool for locating hydrogens in proteins.

Neutron protein crystallography is a powerful tool for investigating protein chemistry because it directly locates hydrogen atom positions in a protein structure. The visibility of hydrogen and deuterium atoms arises from the strong interaction of neutrons with the nuclei of these isotopes. Positions can be unambiguously assigned from diffraction at resolutions typical of protein crystals. Neutrons have the additional benefit to structural biology of not inducing radiation damage in protein crystals. The same crystal could be measured multiple times for parametric studies. Here, we review the basic principles of neutron protein crystallography. The information that can be gained from a neutron structure is presented in balance with practical considerations. Methods to produce isotopically-substituted proteins and to grow large crystals are provided in the context of neutron structures reported in the literature. Available instruments for data collection and software for data processing and structure refinement are described along with technique-specific strategies including joint X-ray/neutron structure refinement. Examples are given to illustrate, ultimately, the unique scientific value of neutron protein crystal structures.

Structural Evidence for Inter-Residue Hydrogen Bonding Observed for Cellobiose in Aqueous Solution

The structure of the disaccharide cellulose subunit cellobiose (4-O-β-D-glucopyranosyl-D-glucose) in solution has been determined via neutron diffraction with isotopic substitution (NDIS), computer modeling and nuclear magnetic resonance (NMR) spectroscopic studies. This study shows direct evidence for an intramolecular hydrogen bond between the reducing ring HO3 hydroxyl group and the non-reducing ring oxygen (O5′) that has been previously predicted by computation and NMR analysis. Moreover, this work shows that hydrogen bonding to the non-reducing ring O5′ oxygen is shared between water and the HO3 hydroxyl group with an average of 50% occupancy by each hydrogen-bond donor. The glycosidic torsion angles φH and ψH from the neutron diffraction-based model show a fairly tight distribution of angles around approximately 22° and −40°, respectively, in solution, consistent with the NMR measurements. Similarly, the hydroxymethyl torsional angles for both reducing and non-reducing rings are broadly consistent with the NMR measurements in this study, as well as with those from previous measurements for cellobiose in solution.

On the structure of water and chloride ion interactions with a peptide backbone in solution

The arrangement of water and chloride ions around a model peptide (glycyl-L-prolyl-glycine-NH2) was investigated using Molecular Dynamics (MD) simulations and complementary Empirical Potential Structure Refinement (EPSR) simulations which adapt the modelled structure to reproduce experimentally measured neutron diffraction data. The results are in good qualitative agreement and show a common picture for all hydrogen-containing amine and amide groups: namely that there are two common chloride interactions observed - a direct contact between Cl(-) and peptide backbone and a water-mediated interaction. The geometry of this mediation depends on the distance between chloride and nitrogen and hints towards two distinct modes of interaction between water and the ion, either along one of the O-H bonds or along the water dipole.

Structural studies of Neurospora crassa LPMO9D and redox partner CDHIIA using neutron crystallography and small-angle scattering

Sensitivity to hydrogen/deuterium and lack of observable radiation damage makes cold neutrons an ideal probe the structural studies of proteins with highly photosensitive groups such as the copper center of lytic polysaccharide monooxygenases (LPMOs) and flavin adenine dinucleotide (FAD) and heme redox cofactors of cellobiose dehydrogenases (CDHs). Here, neutron crystallography and small-angle neutron scattering are used to investigate Neurospora crassa LPMO9D (NcLPMO9D) and CDHIIA (NcCDHIIA), respectively. The presence of LPMO greatly enhances the efficiency of commercial glycoside hydrolase cocktails in the depolymerization of cellulose. LPMOs can receive electrons from CDHs to activate molecular dioxygen for the oxidation of cellulose resulting in chain cleavage and disruption of local crystallinity. Using neutron protein crystallography, the hydrogen/deuterium atoms of NcLPMO9D could be located throughout the structure. At the copper active site, the protonation states of the side chains of His1, His84, His157 and Tyr168, and the orientation of water molecules could be determined. Small-angle neutron scattering measurements provided low resolution models of NcCDHIIA with both the dehydrogenase and cytochrome domains in oxidized states that exhibited elongated conformations. This work demonstrates the suitability of neutron diffraction and scattering for characterizing enzymes critical to oxidative cellulose deconstruction.

Crystallization of a fungal lytic polysaccharide monooxygenase expressed from glycoengineered Pichia pastoris for X-ray and neutron diffraction

Lytic polysaccharide monooxygenases (LPMOs) are carbohydrate-disrupting enzymes secreted by bacteria and fungi that break glycosidic bonds via an oxidative mechanism. Fungal LPMOs typically act on cellulose and can enhance the efficiency of cellulose-hydrolyzing enzymes that release soluble sugars for bioethanol production or other industrial uses. The enzyme PMO-2 from Neurospora crassa (NcPMO-2) was heterologously expressed in Pichia pastoris to facilitate crystallographic studies of the fungal LPMO mechanism. Diffraction resolution and crystal morphology were improved by expressing NcPMO-2 from a glycoengineered strain of P. pastoris and by the use of crystal seeding methods, respectively. These improvements resulted in high-resolution (1.20 Å) X-ray diffraction data collection at 100 K and the production of a large NcPMO-2 crystal suitable for room-temperature neutron diffraction data collection to 2.12 Å resolution.

Structural investigation of cellobiose dehydrogenase IIA: Insights from small angle scattering into intra- and intermolecular electron transfer mechanisms

BACKGROUND Cellobiose dehydrogenases have gained interest due to their potential applications in sectors from biofuel production to biomedical devices. The CDHIIA variant is comprised of a cytochrome domain (CYT), a dehydrogenase domain (DH), and a carbohydrate-binding module (CBM) that are connected by two flexible linkers. Upon cellobiose oxidation at the DH, intramolecular electron transfer (IaET) occurs from the DH to the CYT. In vivo, CDHIIA CYT subsequently performs intermolecular electron transfer (IeET) to a lytic polysaccharide monooxygenase (LPMO). The relevant solution-state CDH domain conformations for IaET and IeET have not been fully characterized. METHODS Small-angle X-ray and neutron scattering measurements of oxidized CDHIIA from Myriococcum thermophilum and Neurospora crassa were performed to investigate the structural landscape explored in solution by MtCDHIIA and NcCDHIIA in response to cations, pH, and the presence of an electron acceptor, LPMO9D from N. crassa. RESULTS The scattering data complemented by modeling show that, under oxidizing conditions, MtCDHIIA undergoes global conformational rearrangement in the presence of Ca2+. Oxidized NcCDHIIA exhibits conformational changes upon pH variation and, in the presence of NcLPMO9D, primarily adopts a compact conformation. CONCLUSIONS These results demonstrate different conformational responses of oxidized MtCDHIIA and NcCDHIIA to changes in environment. The results also reveal a shift in the oxidized NcCDHIIA conformational landscape toward interdomain compaction upon co-incubation with NcLPMO9D. GENERAL SIGNIFICANCE The present study is the first report on the structural landscapes explored in solution by oxidized cellobiose dehydrogenases under various cation concentrations, pH conditions and in the presence of an electron-accepting LPMO.