Insulin polymorphism induced by two polyphenols: new crystal forms and advances in macromolecular powder diffraction.

This study focuses on the polymorphism of human insulin (HI) upon the binding of the phenolic derivatives p-coumaric acid or trans-resveratrol over a wide pH range. The determination of the structural behaviour of HI via X-ray powder diffraction (XRPD) and single-crystal X-ray diffraction (SCXRD) is reported. Four distinct polymorphs were identified, two of which have not been reported previously. The intermediate phase transitions are discussed. One of the novel monoclinic polymorphs displays the highest molecular packing among insulin polymorphs of the same space group to date; its structure was elucidated by SCXRD. XRPD data collection was performed using a variety of instrumental setups and a systematic comparison of the acquired data is presented. A laboratory diffractometer was used for screening prior to high-resolution XRPD data collection on the ID22 beamline at the European Synchrotron Radiation Facility. Additional measurements for the most representative samples were performed on the X04SA beamline at the Swiss Light Source (SLS) using the MYTHEN II detector, which allowed the detection of minor previously untraceable impurities and dramatically improved the d-spacing resolution even for poorly diffracting samples.

Exploring the complex map of insulin polymorphism: a novel crystalline form in the presence of m-cresol.

In this study, the first crystal structure of a novel crystal form of human insulin bound to meta-cresol in an acidic environment is reported. The combination of single-crystal and powder X-ray diffraction crystallography led to the detection of a previously unknown monoclinic phase (P21). The structure was identified from the powder patterns and was solved using single-crystal diffraction data at 2.2 Šresolution. The unit-cell parameters at pH 6.1 are a = 47.66, b = 70.36, c = 84.75 Å, ß = 105.21°. The structure consists of two insulin hexamers per asymmetric unit. The potential use of this insulin form in microcrystalline drugs is discussed.

Revisiting the structure of a synthetic somatostatin analogue for peptide drug design.

Natural or artificially manufactured peptides attract scientific interest worldwide owing to their wide array of pharmaceutical and biological activities. X-ray structural studies are used to provide a precise extraction of information, which can be used to enable a better understanding of the function and physicochemical characteristics of peptides. Although it is vulnerable to disassociation, one of the most vital human peptide hormones, somatostatin, plays a regulatory role in the endocrine system as well as in the release of numerous secondary hormones. This study reports the successful crystallization and complete structural model of octreotide, a stable octapeptide analogue of somatostatin. Common obstacles in crystallographic studies arising from the intrinsic difficulties of obtaining a suitable single-crystal specimen were efficiently overcome as polycrystalline material was employed for synchrotron and laboratory X-ray powder diffraction (XPD) measurements. Data collection and preliminary analysis led to the identification of unit-cell symmetry [orthorhombic, P212121, a = 18.5453 (15), b = 30.1766 (25), c = 39.798 (4) Å], a process which was later followed by complete structure characterization and refinement, underlying the efficacy of the suggested (XPD) approach.

High-performance small- and wide-angle X-ray scattering (SAXS/WAXS) experiments on a multi-functional laboratory goniometer platform with easily exchangeable X-ray modules.

Small-angle X-ray scattering (SAXS) is a well-established, versatile technique for the analysis of nanoscale structures and dimensions, e.g., in liquid dispersions, thin solid objects or powder samples. When combined with wide-angle X-ray scattering (WAXS), complementary information about the atomic structure can be obtained. SAXS experiments traditionally require dedicated instruments to achieve the desired angular resolution, sensitivity, stability, and speed of measurement. Here we demonstrate how a multi-functional laboratory goniometer platform, as widely being used for powder X-ray diffraction and for a variety of related techniques, can be configured with pre-aligned X-ray modules that enable advanced SAXS/WAXS experiments, without compromising the exceptional versatility of the instrument. Line and point collimation setups, as well as quick and easy switching between them, are readily possible. Key components are a detachable, evacuated beam path and a high-resolution, low-noise hybrid pixel area detector, in combination with a hardware interface design that allows to configure the instrument with different X-ray modules without the need for re-alignment. Software for SAXS data reduction and analysis was developed. The good SAXS/WAXS performance and the derived analytical results were verified on various test samples, such as gold nanoparticles, colloidal silica, liposomes, dilute protein solutions, and solid polymer samples. It is believed that this novel approach to SAXS/WAXS instrumentation will help to make this powerful structure analysis technique more widely accessible and affordable for multi-user laboratories.

Coxsackievirus B3 protease 3C: expression, purification, crystallization and preliminary structural insights.

Viral proteases are proteolytic enzymes that orchestrate the assembly of viral components during the viral life cycle and proliferation. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis are presented of protease 3C, the main protease of an emerging enterovirus, coxsackievirus B3, that is responsible for many cases of viral myocarditis. Polycrystalline protein precipitates suitable for X-ray powder diffraction (XRPD) measurements were produced in the presence of 22-28%(w/v) PEG 4000, 0.1 M Tris-HCl, 0.2 M MgCl2 in a pH range from 7.0 to 8.5. A polymorph of monoclinic symmetry (space group C2, unit-cell parameters a = 77.9, b = 65.7, c = 40.6 Å, ß = 115.9°) was identified via XRPD. These results are the first step towards the complete structural determination of the molecule via XRPD and a parallel demonstration of the accuracy of the method.