Time-dependent analysis of K₂PtBr₆binding to lysozyme studied by protein powder and single crystal X-ray analysis

Abstract

Abstract. Multi heavy atom cluster compounds like K 2 PtBr 6 offer a way forward to solve, de novo, unknown protein structures by powder diffraction involving dispersive (measured at two X-ray wavelengths) and isomorphous intensity differences as a complement to micro-crystallography or by using both approaches in combination. Towards this end, using the ESRF high resolution synchrotron X-ray powder diffraction beamline ID31, we have recorded high quality protein powder diffractograms at the platinum LIII and bromine K absorption edges, as well as reference wavelengths, for K 2 PtBr 6 bound to lysozyme. These experiments were conducted at 80K to protect the sample against X-radiation damage as much as possible and also to fix the K 2 PtBr 6 bound state, which seemed to show instability at room temperature. With multiple powder pattern analysis we extracted intensities and showed the PtBr 6 bound in lysozyme using 'omit electron density maps'. In addition the wavelength dispersive Fourier around the Br K edge shows up the bromine signal at PtBr 6 binding site 1 in one of the six samples tested. To better understand the chemical properties of this heavy atom compound we have elucidated the detailed binding behaviour using single crystal analyses with time-resolved freeze quenching after soak times of 10, 90 and 170 minutes. Whilst the quick soaking of 10 to 30 minutes, used at ESRF ID31 shows clear binding, there is increasing binding strength with increasing soak time. Thus, these time-resolved analytical chemistry results show that further heavy atom signal optimizations are possible. Prospects for extending our approach to the yet larger isomorphous and wavelength dispersive signal case of Ta 6 Br 12 bound to lysozyme are also described.

High-resolution powder X-ray data reveal the T6hexameric form of bovine insulin

“…One common phenomenon is an irreversible radiation-induced lattice expansion, although the mechanism by which this occurs is not fully understood (Ravelli et al, 2002). In many cases, lattice changes are anisotropic and can thus be exploited to improve the resolution of overlapping reflections in a powder pattern (Basso et al, 2005;Von Dreele, 2007;Besnard et al, 2007;Margiolaki, Wright, Wilmanns et al, 2007;Wright et al, 2008;Basso et al, 2010;Helliwell et al, 2010). In the case of powder diffraction data, significant changes in the lattice parameters accompanied by a gradual increase of peak broadening and a significant loss of intensity are common characteristics of radiation-damage effects (Margiolaki, Wright, Wilmanns et al, 2007).…”

The binding of platinum hexahalides (Cl, Br and I) to hen egg-white lysozyme and the chemical transformation of the PtI6octahedral complex to a PtI3moiety bound to His15

“…In particular, we envisage that they could become important, for example, in X-ray laser experiments that are striving to work with ever smaller samples, currently at the microcrystal/nanocrystal size but anticipated to transition to nanoclusters (see Helliwell, 2013a). These compounds also offer a way to solve unknown protein structures by powder diffraction involving dispersive and/or isomorphous intensity differences (Helliwell et al, 2010). K 2 PtCl 6 and K 2 PtBr 6 have both been studied before using soaking crystallization conditions into pre-grown hen egg-white lysozyme (HEWL) crystals; Sun and coworkers described a quick soak ($10 min) approach and test using K 2 PtCl 6 (Sun et al, 2002), while Helliwell and coworkers undertook a time-dependent analysis of K 2 PtBr 6 binding to lysozyme studied by protein powder and singlecrystal X-ray analysis (up to 3 h soak time; Helliwell et al, 2010).…”

X-ray resonant powder diffraction

“…As an example of seeking to apply such an approach to protein powders, recently Helliwell et al [84] recorded high quality synchrotron X-ray protein powder diffractograms (ID31, ESRF) from K 2 PtBr 6 bound to lysozyme at 80 K to protect against X-radiation damage as much as possible and also to trap i.e. fix the K 2 PtBr 6 heavy atom compound bound state.…”