SGTools: a suite of tools for processing and analyzing large data sets from in situ X-ray scattering experiments

In situ synchrotron small-angle X-ray scattering (SAXS) is a powerful tool for studying dynamic processes during material preparation and application. The processing and analysis of large data sets generated from in situ X-ray scattering experiments are often tedious and time consuming. However, data processing software for in situ experiments is relatively rare, especially for grazing-incidence small-angle X-ray scattering (GISAXS). This article presents an open-source software suite (SGTools) to perform data processing and analysis for SAXS and GISAXS experiments. The processing modules in this software include (i) raw data calibration and background correction; (ii) data reduction by multiple methods; (iii) animation generation and intensity mapping for in situ X-ray scattering experiments; and (iv) further data analysis for the sample with an order degree and interface correlation. This article provides the main features and framework of SGTools. The workflow of the software is also elucidated to allow users to develop new features. Three examples are demonstrated to illustrate the use of SGTools for dealing with SAXS and GISAXS data. Finally, the limitations and future features of the software are also discussed.

SAXS Reveals the Stabilization Effects of Modified Sugars on Model Proteins

Many proteins are usually not stable under different stresses, such as temperature and pH variations, mechanical stresses, high concentrations, and high saline contents, and their transport is always difficult, because they need to be maintained in a cold regime, which is costly and very challenging to achieve in remote areas of the world. For this reason, it is extremely important to find stabilizing agents that are able to preserve and protect proteins against denaturation. In the present work, we investigate, by extensively using synchrotron small-angle X-ray scattering experiments, the stabilization effect of five different sugar-derived compounds developed at ExtremoChem on two model proteins: myoglobin and insulin. The data analysis, based on a novel method that combines structural and thermodynamic features, has provided details about the physical-chemical processes that regulate the stability of these proteins in the presence of stabilizing compounds. The results clearly show that some modified sugars exert a greater stabilizing effect than others, being able to maintain the active forms of proteins at temperatures higher than those in which proteins, in the absence of stabilizers, reach denatured states.

N-terminal phosphorylation regulates the activity of Glycogen Synthase Kinase 3 from Plasmodium falciparum

As the decline of malaria cases stalled over the last five years, novel targets in Plasmodium falciparum are necessary for the development of new drugs. Glycogen Synthase Kinase (PfGSK3) has been identified as a potential target, since its selective inhibitors were shown to disrupt the parasite's life cycle. In the uncanonical N‑terminal region of the parasite enzyme, we identified several autophosphorylation sites and probed their role in activity regulation of PfGSK3. By combining molecular modeling with experimental small-angle X-ray scattering data, we show that increased PfGSK3 activity is promoted by conformational changes in the PfGSK3 N‑terminus, triggered by N‑terminal phosphorylation. Our work provides novel insights into the structure and regulation of the malarial PfGSK3.

Competition between crystal growth and intracrystalline chain diffusion determines the lamellar thickness in semicrystalline polymers

The non-equilibrium thickness of lamellar crystals in semicrystalline polymers varies significantly between different polymer systems and depends on the crystallization temperature Tc. There is currently no consensus on the mechanism of thickness selection. Previous work has highlighted the decisive role of intracrystalline chain diffusion (ICD) in special cases, but a systematic dependence of lamellar thickness on relevant timescales such as that of ICD and stem attachment has not yet been established. Studying the morphology by small-angle X-ray scattering and the two timescales by NMR methods and polarization microscopy respectively, we here present data on poly(oxymethylene), a case with relatively slow ICD. It fills the gap between previously studied cases of absent and fast ICD, enabling us to establish a quantitative dependence of lamellar thickness on the competition between the noted timescales.

The catalytic activity of TCPTP is auto-regulated by its intrinsically disordered tail and activated by Integrin alpha-1

T-Cell Protein Tyrosine Phosphatase (TCPTP, PTPN2) is a non-receptor type protein tyrosine phosphatase that is ubiquitously expressed in human cells. TCPTP is a critical component of a variety of key signaling pathways that are directly associated with the formation of cancer and inflammation. Thus, understanding the molecular mechanism of TCPTP activation and regulation is essential for the development of TCPTP therapeutics. Under basal conditions, TCPTP is largely inactive, although how this is achieved is poorly understood. By combining biomolecular nuclear magnetic resonance spectroscopy, small-angle X-ray scattering, and chemical cross-linking coupled with mass spectrometry, we show that the C-terminal intrinsically disordered tail of TCPTP functions as an intramolecular autoinhibitory element that controls the TCPTP catalytic activity. Activation of TCPTP is achieved by cellular competition, i.e., the intrinsically disordered cytosolic tail of Integrin-α1 displaces the TCPTP autoinhibitory tail, allowing for the full activation of TCPTP. This work not only defines the mechanism by which TCPTP is regulated but also reveals that the intrinsically disordered tails of two of the most closely related PTPs (PTP1B and TCPTP) autoregulate the activity of their cognate PTPs via completely different mechanisms.

Structural and functional analysis of the human Cone-rod homeobox transcription factor

The cone-rod homeobox (CRX) protein is a critical K50 homeodomain transcription factor responsible for the differentiation and maintenance of photoreceptor neurons in the vertebrate retina. Mutant alleles in the human gene encoding CRX result in a variety of distinct blinding retinopathies, including retinitis pigmentosa, cone-rod dystrophy, and Leber congenital amaurosis. Despite the success of using in vitro biochemistry, animal models, and genomics approaches to study this clinically relevant transcription factor over the past 24 years since its initial characterization, there are no high-resolution structures in the published literature for the CRX protein. In this study, we use bioinformatic approaches and small-angle x-ray scattering (SAXS) structural analysis to further understand the biochemical complexity of the human CRX homeodomain (CRX-HD). We find that the CRX-HD is a compact, globular monomer in solution that can specifically bind functional cis-regulatory elements encoded upstream of retina specific genes. This study presents the first structural analysis of CRX, paving the way for a new approach to studying the biochemistry of this protein and its disease-causing mutant protein variants.

Gelation Behavior and the Stability of Multicomponent Sterol-Based Oleogels

Novel fat mimetic materials, such as oleogels, are advancing the personalization of healthier food products and can be developed from low molecular weight compounds such as γ oryzanol and β-sitosterol. Following molecular assembly, the formation of a tubular system ensues, which seems to be influenced by elements such as the oleogelators’ concentration and ratio, cooling rates, and storage periods. Sterol-based oleogels were formulated under distinct environmental conditions, and a comprehensive study aimed to assess the effects of the mentioned factors on oleogel formation and stability, through visual observation and by using techniques such as small-angle X-ray scattering, X-ray diffraction, confocal Raman spectroscopy, rheology, and polarized microscopy. The long, rod-like conformations, identified by small-angle X-ray scattering, showed that different cooling rates influence oleogels’ texture. Raman spectra showed that the stabilization time is associated with the interfibrillar aggregation, which occurred differently for 8 and 10 wt%, with a proven relationship between ferulic acid and the tubular formation. This report gives fundamental insight into the critical point of gelation, referring to the time scale of the molecular stabilization. Our results verify that understanding the structuring mechanisms of oleogelation is decisive for the processing and manufacturing of novel foods which integrate oleogels in their structure.

Morphology and Rheology of a Cool-Gel (Protein) Blended with a Thermo-Gel (Hydroxypropyl Methylcellulose)

This study investigates the morphological and rheological properties of blended gelatin (GA; a cooling-induced gel (cool-gel)) and hydroxypropyl methylcellulose (HPMC; a heating-induced gel (thermo-gel)) systems using a fluorescence microscope, small angle X-ray scattering (SAXS), and a rheometer. The results clearly indicate that the two biopolymers are immiscible and have low compatibility. Moreover, the rheological behavior and morphology of the GA/HPMC blends significantly depend on the blending ratio and concentration. Higher polysaccharide contents decrease the gelling temperature and improve the gel viscoelasticity character of GA/HPMC blended gels. The SAXS results reveal that the correlation length (ξ) of the blended gels decreases from 5.16 to 1.89 nm as the HPMC concentration increases from 1 to 6%, which suggests that much denser networks are formed in blended gels with higher HPMC concentrations. Overall, the data reported herein indicate that the gel properties of gelatin can be enhanced by blending with a heating-induced gel.