Hydrogen/deuterium exchange behavior in tetragonal hen egg-white lysozyme crystals affected by solution state

Kita, Akiko; Morimoto, Yukio

doi:10.1107/s1600576720005488

Cited by 5 publications

(15 citation statements)

References 30 publications

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“…To unambiguously identify the positions occupied by caged H atoms in the protein structure, neutron crystallography or NMR experiments are required. An indication of relevant positions is found in a reverse setup, where 20 H atoms were exchanged to D using unfolding and refolding processes of H-HEWL in D 2 O (Kita & Morimoto, 2016), as observed in the neutron structure deposited in the PDB (PDB entry 6k8g; Kita & Morimoto, 2020).…”

Section: Discussionmentioning

confidence: 96%

Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme

Ramos

Laux

Haertlein

et al. 2021

Int Union Crystallogr J

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This structural and biophysical study exploited a method of perdeuterating hen egg-white lysozyme based on the expression of insoluble protein in Escherichia coli followed by in-column chemical refolding. This allowed detailed comparisons with perdeuterated lysozyme produced in the yeast Pichia pastoris, as well as with unlabelled lysozyme. Both perdeuterated variants exhibit reduced thermal stability and enzymatic activity in comparison with hydrogenated lysozyme. The thermal stability of refolded perdeuterated lysozyme is 4.9°C lower than that of the perdeuterated variant expressed and secreted in yeast and 6.8°C lower than that of the hydrogenated Gallus gallus protein. However, both perdeuterated variants exhibit a comparable activity. Atomic resolution X-ray crystallographic analyses show that the differences in thermal stability and enzymatic function are correlated with refolding and deuteration effects. The hydrogen/deuterium isotope effect causes a decrease in the stability and activity of the perdeuterated analogues; this is believed to occur through a combination of changes to hydrophobicity and protein dynamics. The lower level of thermal stability of the refolded perdeuterated lysozyme is caused by the unrestrained Asn103 peptide-plane flip during the unfolded state, leading to a significant increase in disorder of the Lys97–Gly104 region following subsequent refolding. An ancillary outcome of this study has been the development of an efficient and financially viable protocol that allows stable and active perdeuterated lysozyme to be more easily available for scientific applications.

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Section: Discussionmentioning

confidence: 96%

Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme

Ramos

Laux

Haertlein

et al. 2021

Int Union Crystallogr J

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“…The protonation states of Glu35 and Asp52 observed in the present analysis were compared with those in previous neutron structures (Niimura et al, 1997;Bon et al, 1999;Kita & Morimoto, 2020), as summarized in Table 6. The neutron structure analysis of the tetragonal HEWL crystal at pH 7.0 (Niimura et al, 1997) showed no protonation of Glu35 and Asp52.…”

Section: Figurementioning

confidence: 85%

“…In addition, the low occupancy of W42 supports protonation of the OE2 atom. As summarized in Table 5, the W42 molecule was observed in the highest resolution X-ray structure (Sauter et al, 2001) and in one of the neutron structures (Niimura et al, 1997), while the W31 molecule was conserved in all of the neutron structures (Niimura et al, 1997;Bon et al, 1999;Kita & Morimoto, 2020) and in the two highest resolution X-ray structures (Sauter et al, 2001;Chiniadis et al, 2021). In the highest resolution X-ray structure, the occupancies of W31 and W42 were 0.78 and 0.46, respectively (Sauter et al, 2001).…”

Section: Overviewmentioning

confidence: 95%

“…However, the X-ray analyses could not identify an H atom at the carboxyl group of Glu35 in the active site. Neutron crystallography was performed to locate H atoms, and three medium-resolution structures were obtained (Niimura et al, 1997;Bon et al, 1999;Kita & Morimoto, 2020): structures from tetragonal crystals at pH 7.0 (PDB entry 1io5; Niimura et al, 1997) and pH 4.6 (PDB entry 6k8g; Kita & Morimoto, 2020) determined at 2.0 A ˚resolution and a structure from a triclinic crystal at pH 4.7 determined at 1.7 A ˚resolution (PDB entry 1lzn; Bon et al, 1999). These structures showed the different protonation states of HEWL, particularly at Glu35 and Asp52.…”

Section: Introductionmentioning

confidence: 99%

“…† pK a value determined by two-dimensional 1 H-NMR(Bartik et al, 1994). ‡ References for PDB entries 1lzn, 1io5 and 6k8g areNiimura et al (1997),Kita & Morimoto (2020) andBon et al (1999), respectively. § Atomic refinement and map calculation were conducted at 2.0 and 1.8 A ˚resolution, respectively. }…”

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Protonation states of hen egg-white lysozyme observed using D/H contrast neutron crystallography

Chatake¹,

Tanaka²,

Kusaka³

et al. 2022

Acta Cryst Sect D Struct Biol

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Hen egg-white lysozyme (HEWL) is an enzymatic protein with two acidic amino acids, Glu35 and Asp52, in its active site. Glu35 acts as a proton donor to the substrate and Asp52 interacts with the positively charged substrate, suggesting different protonation states of these residues. However, neutron crystallographic studies thus far have not provided a consistent picture of the protonation states of these residues. Only one study succeeded in observing the active protonation states of Glu35 and Asp52 in the triclinic crystal system. However, their active states in the most widely studied tetragonal crystal system are still unknown. The application of the D/H contrast technique in neutron crystallography improves the ability to locate exchangeable D/H atoms in proteins. In the present study, D2O and H2O solvent crystals were prepared. Each neutron data set was collected for only five days by combining a time-of-flight diffractometer (iBIX) and the spallation neutron source at the Japan Proton Accelerator Research Complex. The D/H contrast map provided better visualization of the D/H atoms in HEWL than the conventional neutron scattering length density map. The neutron D/H contrast map demonstrated the alternative protonation of the OE1 and OE2 atoms in the carboxyl group of Glu35. This alternative protonation occurs in the absence of a substrate, where high selectivity of the protonation site does not occur. In this case, only the OE1—HE1 bond attacks the substrate in an equilibrium between OE1—HE1 and OE2—HE2, or the H+ ion of the OE2—HE2 bond moves to the OE1 atom just before or after substrate binding to initiate the catalytic reaction. In contrast, the carboxyl group of Asp52 is not protonated. Protonation of the carboxyl group was not observed for other Asp and Glu residues. These results are consistent with results from NMR spectroscopy and explain the protonation states at the active site in the apo form of HEWL.

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Hydrogen/Deuterium Exchange Behavior During Denaturing/Refolding Processes Determined in Tetragonal Hen Egg-White Lysozyme Crystals

Kita

Morimoto

2022

Mol Biotechnol

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The crystal structure analyses of hen egg-white lysozymes containing traces of hydrogen/deuterium exchange during denaturing/refolding processes in solutions were performed using a joint X-ray and neutron refinement method. Differences in hydrogen/deuterium exchange were observed depending on the denaturing methods, acidic condition, basic condition, or thermal condition. This study describes the use of occupancy values determined by a crystallographic method for the analysis of protein denaturation.

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Hydrogen/deuterium exchange behavior in tetragonal hen egg-white lysozyme crystals affected by solution state

Cited by 5 publications

References 30 publications

Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme

Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme

Protonation states of hen egg-white lysozyme observed using D/H contrast neutron crystallography

Hydrogen/Deuterium Exchange Behavior During Denaturing/Refolding Processes Determined in Tetragonal Hen Egg-White Lysozyme Crystals

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