Metalloprotein Crystallography: More than a Structure

Abstract

ConspectusMetal ions and metallocofactors play important roles in a broad range of biochemical reactions. Accordingly, it has been estimated that as much as 25–50% of the proteome uses transition metal ions to carry out a variety of essential functions. The metal ions incorporated within metalloproteins fulfill functional roles based on chemical properties, the diversity of which arises as transition metals can adopt different redox states and geometries, dictated by the identity of the metal and the protein environment. The coupling of a metal ion with an organic framework in metallocofactors, such as heme and cobalamin, further expands the chemical functionality of metals in biology. The three-dimensional visualization of metal ions and complex metallocofactors within a protein scaffold is often a starting point for enzymology, highlighting the importance of structural characterization of metalloproteins. Metalloprotein crystallography, however, presents a number of implicit challenges including correctly incorporating the relevant metal or metallocofactor, maintaining the proper environment for the protein to be purified and crystallized (including providing anaerobic, cold, or aphotic environments), and being mindful of the possibility of X-ray induced damage to the proteins or incorporated metal ions. Nevertheless, the incorporated metals or metallocofactors also present unique advantages in metalloprotein crystallography. The significant resonance that metals undergo with X-ray photons at wavelengths used for protein crystallography and the rich electronic properties of metals, which provide intense and spectroscopically unique signatures, allow a metalloprotein crystallographer to use anomalous dispersion to determine phases for structure solution and to use simultaneous or parallel spectroscopic techniques on single crystals. These properties, coupled with the improved brightness of beamlines, the ability to tune the wavelength of the X-ray beam, the availability of advanced detectors, and the incorporation of spectroscopic equipment at a number of synchrotron beamlines, have yielded exciting developments in metalloprotein structure determination. Here we will present results on the advantageous uses of metals in metalloprotein crystallography, including using metallocofactors to obtain phasing information, using K-edge X-ray absorption spectroscopy to identify metals coordinated in metalloprotein crystals, and using UV–vis spectroscopy on crystals to probe the enzymatic activity of the crystallized protein.

Biophysical Examination of the Calcium-Modulated Nickel-Binding Properties of Human Calprotectin Reveals Conformational Change in the EF-Hand Domains and His3Asp Site

“…Based on an established method to evaluate the elemental composition of materials, 31 this technique can be utilized to analyze the X-ray fluorescence of elements present in single-crystal samples and can be used in tandem with collecting diffraction data on the synchrotron beamline. 32 X-rays at a fixed incident energy are used to excite elements that emit photons corresponding their K-edge energies, with Compton backscattering observed near the incident energy. Fluorescence counts of various elements are detected, and emission spectra are calibrated to a standard containing Sm, Ni, Zn, Br, and Sr. Crystals were analyzed on 100-μm loops, and measurements for the sample (X-rays positioned on crystal, red lines in Figure 1) and a blank (X-rays positioned on an empty space of the loop, black lines in Figure 1) were taken at 0.9792 Å incident energy.…”

Advances in element speciation analysis of biomedical samples using synchrotron-based techniques

“…Due to the simultaneous measurement of many important parameters, XAS is among the best techniques for determining the "structure-to-function" relationship in metalloprotein studies. XAS structural studies of metal-binding sites from purified metalloproteins, which are often carried out jointly with X-ray crystallography, will not be considered in this review, information about this procedure can be found in other articles [14][15][16]. However, the emerging development of XAS in combination with chromatographic methods to characterize proteins from complex samples will be discussed.…”

Robust Production, Crystallization, Structure Determination, and Analysis of [Fe–S] Proteins: Uncovering Control of Electron Shuttling and Gating in the Respiratory Metabolism of Molybdopterin Guanine Dinucleotide Enzymes

“…A key benefit of [Fe–S] proteins for structure determination is that you may be able to use the Fe anomalous signal to solve the phase problem (Bowman, Bridwell-Rabb, & Drennan, 2016), provided you are careful to avoid loss of the damage-sensitive anomalous signal. The Fe anomalous absorption peak is at ~7112eV (1.7433Å ), which is accessible at most of synchrotron beamlines, such as SIBYLS at the Advanced Light Source (Classen et al, 2013).…”

“…3). The proper refinement of [Fe–S] cluster structures may reveal the extend to which their distortion impacts enzymatic activity and biological function (Bowman et al, 2016). Such distortion can be further measured by Mossbauer spectroscopy (Pandelia, Lanz, Booker, & Krebs, 2015) and XAS (Einsle, Andrade, Dobbek, Meyer, & Rees, 2007; Ha et al, 2017) to evaluate the coordination geometry, solvation, and redox states of [Fe–S] clusters.…”