High-resolution neutron protein crystallography with radically small crystal volumes: application of perdeuteration to human aldose reductase

Abstract

Neutron crystallography is an important complementary technique to X-ray crystallography because it provides details of the H-atom and proton (H+) positions in biological macromolecules and, given the absence of radiation damage with neutrons, the resulting structures are 'damage-free' even at room temperature. Knowledge of the positions of the H-atoms and protons is important since details of protonation and hydration are often necessary for understanding macromolecular function at an atomic level, such as enzyme mechanisms [1,] and in drug-binding [2,]. Although, historically, the study of biological macromolecules using neutron crystallography had been limited-due to the requirement for extremely large crystals of several cubic millimetres-recent improvements to the quasi-Laue diffractometer LADI-III at the Insitut Laue-Langevin (ILL) are allowing us to extend the limits for neutron macromolecular crystallography using crystals of smaller dimensions and studying larger unit-cell systems [3]. This has resulted in a typical lower limit for useful crystal volumes of ~0.1 mm3, however, from a very recent study of human recombinant insulin it has been shown that in fact much smaller crystals can be used for high-resolution neutron diffraction studies. Neutron quasi-Laue diffraction data have been collected to 2.2 Å resolution from a crystal of human insulin with a radically small volume of 0.02-0.03 mm3 (~250-300 μm on edge) using the LADI-III instrument at the ILL. Given that the insulin crystal used for data collection was H/D-exchanged this implies that the limit for perdeuterated crystals (in which all H are replaced by its isotope deuterium) should be even smaller, possibly less than 0.01 mm3. Given that crystal volumes of this order are much more feasible to grow, a large number of potential neutron crystallography studies are now within range. Herein we will describe, in detail, the neutron structure of human recombinant insulin, which has not been determined previously using neutron diffraction, and which clearly shows details of protonation and hydration that are not attainable even with ultra-high resolution X-ray crystallography.

Quantum model of catalysis based on a mobile proton revealed by subatomic x-ray and neutron diffraction studies of h-aldose reductase

“…To better visualize the active-site residue protonation states, we produced fully deuterated samples (15) and collected both x-ray and neutron diffraction data at room temperature. Neutron diffraction is particularly efficient at locating Deuterium (D) atoms, because the scattering length of D is similar to that of C, N, and O atoms.…”

“…Refinement resulted in a model with 315 residues, NADPϩ, IDD594, two citrate molecules, and 555 water molecules. The neutron data (S3) were collected from a single crystal of volume 0.15 mm 3 (unusually small for neutron diffraction) on the Laue diffractometer, LADI, at the Institut Laue Langevin (ILL) (15,20,21). The x-ray data (S3) were collected at Swiss Light Source [(SLS) Villigen, Switzerland], to 1.75-Å resolution and were used simultaneously with the neutron data in a joint refinement using PHENIX (22).…”

Large crystal growth by thermal control allows combined X-ray and neutron crystallographic studies to elucidate the protonation states inAspergillus flavusurate oxidase

“…Hydrogen atoms attached to carbon atoms remain unexchanged. The only method to replace all the hydrogen atoms is to produce a completely perdeuterated macromolecule in vivo (Berns 1963;Gamble et al 1994;Shu et al 2000;Hazemann et al 2005). To achieve this, the macromolecule must be expressed in cells grown in deuterated media, so that perdeuterated macromolecules can be synthesized.…”

New sources and instrumentation for neutrons in biology

“…At higher resolution, perdeuteration of macromolecular systems in crystals and fibres provides a major advantage through the enhanced coherent scattering power of substituted deuterium and the elimination of hydrogen incoherent scattering. One important consequence of this is that the serious limitations in sample size and data collection times required for hydrogenated samples are alleviated [79,80]. In inelastic neutron scattering experiments, the application of specific hydrogen-deuterium labelling patterns can be used to highlight specific dynamics within a system.…”