A versatile approach to high-density microcrystals in lipidic cubic phase for room-temperature serial crystallography.

Serial crystallography has emerged as an important tool for structural studies of integral membrane proteins. The ability to collect data from micrometre-sized weakly diffracting crystals at room temperature with minimal radiation damage has opened many new opportunities in time-resolved studies and drug discovery. However, the production of integral membrane protein microcrystals in lipidic cubic phase at the desired crystal density and quantity is challenging.

Extracellular matrix type 0: From ancient collagen lineage to a versatile product pipeline - JellaGel™.

Extracellular matrix type 0 is reported. The matrix is developed from a jellyfish collagen predating mammalian forms by over 0.5 billion years.

High-Throughput Protein Crystallization via Microdialysis.

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug discovery. To date, X-ray crystallography remains the most successful method for solving three-dimensional protein structures at atomic resolution. With recent advances in serial crystallography, either using X-ray free electron lasers (XFELs) or synchrotron light sources, protein crystallography has progressed to the next frontier, where the ability to acquire time-resolved data provides important mechanistic insights into the behavior of biological molecules at room temperature.

Measuring Protein Aggregation and Stability Using High-Throughput Biophysical Approaches.

Structure-function relationships of biological macromolecules, in particular proteins, provide crucial insights for fundamental biochemistry, medical research and early drug discovery. However, production of recombinant proteins, either for structure determination, functional studies, or to be used as biopharmaceutical products, is often hampered by their instability and propensity to aggregate in solution . Protein samples of poor quality are often associated with reduced reproducibility as well as high research and production expenses.

Two states of a light-sensitive membrane protein captured at room temperature using thin-film sample mounts.

Room-temperature diffraction methods are highly desirable for dynamic studies of biological macromolecules, since they allow high-resolution structural data to be collected as proteins undergo conformational changes. For crystals grown in lipidic cubic phase (LCP), an extruder is commonly used to pass a stream of microcrystals through the X-ray beam; however, the sample quantities required for this method may be difficult to produce for many membrane proteins. A more sample-efficient environment was created using two layers of low X-ray transmittance polymer films to mount crystals of the archaerhodopsin-3 (AR3) photoreceptor and room-temperature diffraction data were acquired.

Structures of the archaerhodopsin-3 transporter reveal that disordering of internal water networks underpins receptor sensitization.

Many transmembrane receptors have a desensitized state, in which they are unable to respond to external stimuli. The family of microbial rhodopsin proteins includes one such group of receptors, whose inactive or dark-adapted (DA) state is established in the prolonged absence of light. Here, we present high-resolution crystal structures of the ground (light-adapted) and DA states of Archaerhodopsin-3 (AR3), solved to 1.

Membrane protein crystallography in the era of modern structural biology.

The aim of structural biology has been always the study of biological macromolecules structures and their mechanistic behaviour at molecular level. To achieve its goal, multiple biophysical methods and approaches have become part of the structural biology toolbox. Considered as one of the pillars of structural biology, X-ray crystallography has been the most successful method for solving three-dimensional protein structures at atomic level to date.

In Situ Measurements of Polypeptide Samples by Dynamic Light Scattering: Membrane Proteins, a Case Study.

Integral membrane proteins are important drug targets that are critical in supporting many biological processes. Despite that, the study of their structure-function relationships remains a major goal in structural biology, yet progress has been hampered by inherent challenges in the production for stable and homogeneous protein samples. Dynamic light scattering provides a straightforward probe of protein quality in solution, particularly in relation to stability and aggregation.

Selection of Biophysical Methods for Characterisation of Membrane Proteins.

Over the years, there have been many developments and advances in the field of integral membrane protein research. As important pharmaceutical targets, it is paramount to understand the mechanisms of action that govern their structure-function relationships. However, the study of integral membrane proteins is still incredibly challenging, mostly due to their low expression and instability once extracted from the native biological membrane.

Promiscuous and Selective: How Intrinsically Disordered BH3 Proteins Interact with Their Pro-survival Partner MCL-1.

The BCL-2 family of proteins plays a central role in regulating cell survival and apoptosis. Disordered BH3-only proteins bind promiscuously to a number of different BCL-2 proteins, with binding affinities that vary by orders of magnitude. Here we investigate the basis for these differences in affinity.

pKID Binds to KIX via an Unstructured Transition State with Nonnative Interactions.

Understanding the detailed mechanism of interaction of intrinsically disordered proteins with their partners is crucial to comprehend their functions in signaling and transcription. Through its interaction with KIX, the disordered pKID region of CREB protein is central in the transcription of cAMP responsive genes, including those involved in long-term memory. Numerous simulation studies have investigated these interactions.