Automating the application of smart materials for protein crystallization

The fabrication and validation of the first semi-liquid nonprotein nucleating agent to be administered automatically to crystallization trials is reported. This research builds upon prior demonstration of the suitability of molecularly imprinted polymers (MIPs; known as 'smart materials') for inducing protein crystal growth. Modified MIPs of altered texture suitable for high-throughput trials are demonstrated to improve crystal quality and to increase the probability of success when screening for suitable crystallization conditions. The application of these materials is simple, time-efficient and will provide a potent tool for structural biologists embarking on crystallization trials. © 2015, IUCR. All rights reserved

Droplet Microfluidics XRD Identifies Effective Nucleating Agents for Calcium Carbonate

The ability to control crystallization reactions is required in a vast range of processes including the production of functional inorganic materials and pharmaceuticals and the prevention of scale. However, it is currently limited by a lack of understanding of the mechanisms underlying crystal nucleation and growth. To address this challenge, it is necessary to carry out crystallization reactions in well‐defined environments, and ideally to perform in situ measurements. Here, a versatile microfluidic synchrotron‐based technique is presented to meet these demands. Droplet microfluidic‐coupled X‐ray diffraction (DMC‐XRD) enables the collection of time‐resolved, serial diffraction patterns from a stream of flowing droplets containing growing crystals. The droplets offer reproducible reaction environments, and radiation damage is effectively eliminated by the short residence time of each droplet in the beam. DMC‐XRD is then used to identify effective particulate nucleating agents for calcium carbonate and to study their influence on the crystallization pathway. Bioactive glasses and a model material for mineral dust are shown to significantly lower the induction time, highlighting the importance of both surface chemistry and topography on the nucleating efficiency of a surface. This technology is also extremely versatile, and could be used to study dynamic reactions with a wide range of synchrotron‐based techniques

Analysis of insulin glulisine at the molecular level by X-ray crystallography and biophysical techniques

© 2021, The Author(s). This study concerns glulisine, a rapid-acting insulin analogue that plays a fundamental role in diabetes management. We have applied a combination of methods namely X-ray crystallography, and biophysical characterisation to provide a detailed insight into the structure and function of glulisine. X-ray data provided structural information to a resolution of 1.26Å. Crystals belonged to the H3 space group with hexagonal (centred trigonal) cell dimensions a = b = 82.44 and c = 33.65Å with two molecules in the asymmetric unit. A unique position of D21Glu, not present in other fast-acting analogues, pointing inwards rather than to the outside surface was observed. This reduces interactions with neighbouring molecules thereby increasing preference of the dimer form. Sedimentation velocity/equilibrium studies revealed a trinary system of dimers and hexamers/dihexamers in dynamic equilibrium. This new information may lead to better understanding of the pharmacokinetic and pharmacodynamic behaviour of glulisine which might aid in improving formulation regarding its fast-acting role and reducing side effects of this drug

X-ray crystallographic studies of RoAb13 bound to PIYDIN, a part of the N-terminal domain of C-C chemokine receptor 5

C-C chemokine receptor 5 (CCR5) is a major co-receptor molecule used by HIV-1 to enter cells. This led to the hypothesis that stimulating an antibody response would block HIV with minimal toxicity. Here, X-ray crystallographic studies of the anti-CCR5 antibody RoAb13 together with two peptides were undertaken: one peptide is a 31-residue peptide containing the PIYDIN sequence and the other is the PIDYIN peptide alone, where PIYDIN is part of the N-terminal region of CCR5 previously shown to be important for HIV entry. In the presence of the longer peptide (the complete N-terminal domain), difference electron density was observed at a site within a hypervariable CDR3 binding region. In the presence of the shorter core peptide PIYDIN, difference electron density is again observed at this CDR3 site, confirming consistent binding for both peptides. This may be useful in the design of a new biomimetic to stimulate an antibody response to CCR5 in order to block HIV infection

RoAb13 inhibits monocyte chemotaxis.

<p>PBMC were incubated in the upper chamber of transwells, while the lower chamber contained medium alone, supernatant of unactivated macrophages, or supernatant of macrophages activated with LPS (see M&M for details). RoAb13 Fab fragment or a control Fab fragement were added to some wells as shown. Migrating cells were collected from the lower chamber after three hours and the cells phenotyped as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128381#pone.0128381.s002" target="_blank">S2 fig</a>. Migration is shown as the proportion of cells in the PBMC premigration sample which migrate to the lower chamber during the assay. Each experimental condition was set up in triplet and * shows significant difference from control (T test, p<0.05).</p

Immunisation with a chimeric peptide coding a linear CCR5 antibody epitope together with a helper epitope from tetanus toxoid can stimulate antibodies which recognize native CCR5.

<p>A, The sequence of the chimeric peptide used for immunization, showing the CCR5 B cell epitope, the linker sequence and the T cell helper epitope from tetanus toxoid. B, Sera from 5 immunized mice were collected after priming and boosting (see M&M) with the peptide shown in a), and tested in ELISA for binding to hCCR5_1–31. Binding is shown as OD at 405 nm. U: serum from an unimmunized mouse (preimmune sera showed equivalent binding). RoAb13: supernatant from the RoAb13 hybridoma diluted as shown. C, Sera from the same five mice were tested for binding to CCR5 transfectants (red line) or controls at a dilution of 1:50.</p

R&D1801 and RoAb13 recognise overlapping core epitopes in the CCR5 N-terminal domain.

<p>A, R&D1801 or RoAb13 were incubated overnight with competing peptides 1–24 (1 μg/ml) in wells coated with hCCR5_1–31, and binding (shown as OD 405 nm) of antibody was then measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128381#pone.0128381.g001" target="_blank">Fig 1a</a> using a rabbit anti-mouse second layer. C—PBS used in place of competitive peptide. B, The peptide sequence of the 24 peptides tested in a. The blue bars shows all peptides which inhibited the binding by more than 50%. C, Diagrammatic representation of the core binding epitope recognized by the two antibodies, defined by peptides which inhibited binding to the whole N-terminal peptide by more than 50%.</p

The sequence and crystals of RoAb13.

<p>A, The protein sequence of RoAb13 (lower sequence) compared to the closest germline sequence for heavy (right) and light (left) chains (as determined by the V-Quest). Conserved residues are shown in red. B, Crystals of RoAb13.</p