The T2 structure of polycrystalline cubic human insulin.

The polymorphism of human insulin upon pH variation was characterized via X-ray powder diffraction, employing a crystallization protocol previously established for co-crystallization with phenolic derivatives. Two distinct rhombohedral (R3) polymorphs and one cubic (I213) polymorph were identified with increasing pH, corresponding to the T6, T3R3f and T2 conformations of insulin, respectively. The structure of the cubic T2 polymorph was determined via multi-profile stereochemically restrained Rietveld refinement at 2.7 Šresolution. This constitutes the first cubic insulin structure to be determined from crystals grown in the presence of zinc ions, although no zinc binding was observed. The differences of the polycrystalline variant from other cubic insulin structures, as well as the nature of the pH-driven phase transitions, are discussed in detail.

New perspectives in macromolecular powder diffraction using single-photon-counting strip detectors: high-resolution structure of the pharmaceutical peptide octreotide.

Advances in instrumentation, as well as the development of powerful crystallographic software have significantly facilitated the collection of high-resolution diffraction data and have made X-ray powder diffraction (XRPD) particularly useful for the extraction of structural information; this is true even for complex molecules, especially when combined with synchrotron radiation. In this study, in-line with past instrumental profile studies, an improved data collection strategy exploiting the MYTHEN II detector system together with significant beam focusing and tailored data collection options was introduced and optimized for protein samples at the Material Science beamline at the Swiss Light Source. Polycrystalline precipitates of octreotide, a somatostatin analog of particular pharmaceutical interest, were examined with this novel approach. XRPD experiments resulted in high angular and d-spacing (1.87 Å) resolution data, from which electron-density maps of enhanced quality were extracted, revealing the molecule's structural properties. Since microcrystalline precipitates represent a viable alternative for administration of therapeutic macromolecules, XRPD has been acknowledged as the most applicable tool for examining a wide spectrum of physicochemical properties of such materials and performing studies ranging from phase identification to complete structural characterization.

Considerations Regarding Activity Determinants of Fungal Polyphenol Oxidases Based on Mutational and Structural Studies.

Polyphenol oxidases (PPOs) are an industrially relevant family of enzymes, being involved in the postharvest browning of fruits and vegetables, as well as in human melanogenesis. Their involvement lies in their ability to oxidize phenolic or polyphenolic compounds, which subsequently form pigments. The PPO family includes tyrosinases and catechol oxidases, which, in spite of their high structural similarity, exhibit different catalytic activities. Long-standing research efforts have not yet managed to decipher the structural determinants responsible for this differentiation, as every new theory is disproved by a more recent study. In the present work, we combined biochemical along with structural data in order to better understand the function of a previously characterized PPO from Thermothelomyces thermophila (TtPPO). The crystal structure of a TtPPO variant, determined at 1.55 Å resolution, represents the second known structure of an ascomycete PPO. Kinetic data for structure-guided mutants prove the implication of "gate" residue L306, residue HB1+1 (G292), and HB2+1 (Y296) in TtPPO function against various substrates. Our findings demonstrate the role of L306 in the accommodation of bulky substrates and show that residue HB1+1 is unlikely to determine monophenolase activity, as was suggested from previous studies.IMPORTANCE PPOs are enzymes of biotechnological interest. They have been extensively studied both biochemically and structurally, with a special focus on the plant-derived counterparts. Even so, explicit description of the molecular determinants of their substrate specificity is still pending. For ascomycete PPOs, only one crystal structure has been determined so far, thus limiting our knowledge on this tree branch of the family. In the present study, we report the second crystal structure of an ascomycete PPO. Combined with site-directed mutagenesis and biochemical studies, we depict the amino acids in the vicinity of the active site that affect enzyme activity and perform a detailed analysis on a variety of substrates. Our findings improve current understanding of structure-function relations of microbial PPOs, which is a prerequisite for the engineering of biocatalysts of desired properties.

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.

Rapid screening of in cellulo grown protein crystals via a small-angle X-ray scattering/X-ray powder diffraction synergistic approach.

Crystallization of recombinant proteins in living cells is an exciting new approach for structural biology that provides an alternative to the time-consuming optimization of protein purification and extensive crystal screening steps. Exploiting the potential of this approach requires a more detailed understanding of the cellular processes involved and versatile screening strategies for crystals in a cell culture. Particularly if the target protein forms crystalline structures of unknown morphology only in a small fraction of cells, their detection by applying standard visualization techniques can be time consuming and difficult owing to the environmental challenges imposed by the living cells. In this study, a high-brilliance and low-background bioSAXS beamline is employed for rapid and sensitive detection of protein microcrystals grown within insect cells. On the basis of the presence of Bragg peaks in the recorded small-angle X-ray scattering profiles, it is possible to assess within seconds whether a cell culture contains microcrystals, even in a small percentage of cells. Since such information cannot be obtained by other established detection methods in this time frame, this screening approach has the potential to overcome one of the bottlenecks of intracellular crystal detection. Moreover, the association of the Bragg peak positions in the scattering curves with the unit-cell composition of the protein crystals raises the possibility of investigating the impact of environmental conditions on the crystal structure of the intracellular protein crystals. This information provides valuable insights helping to further understand the in cellulo crystallization process.

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.

In Quest for Improved Drugs against Diabetes: The Added Value of X-ray Powder Diffraction Methods.

Human insulin (HI) is a well-characterized natural hormone which regulates glycose levels into the blood-stream and is widely used for diabetes treatment. Numerous studies have manifested that despite significant efforts devoted to structural characterization of this molecule and its complexes with organic compounds (ligands), there is still a rich diagram of phase transitions and novel crystalline forms to be discovered. Towards the improvement of drug delivery, identification of new insulin polymorphs from polycrystalline samples, simulating the commercially available drugs, is feasible today via macromolecular X-ray powder diffraction (XRPD). This approach has been developed, and is considered as a respectable method, which can be employed in biosciences for various purposes, such as observing phase transitions and characterizing bulk pharmaceuticals. An overview of the structural studies on human insulin complexes performed over the past decade employing both synchrotron and laboratory sources for XRPD measurements, is reported herein. This review aims to assemble all of the recent advances in the diabetes treatment field in terms of drug formulation, verifying in parallel the efficiency and applicability of protein XRPD for quick and accurate preliminary structural characterization in the large scale.

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.