high-throughput-protein-crystallization-via-microdialysis

High-Throughput Protein Crystallization <em>via</em> Microdialysis

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug ...

National Physical Laboratory

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. Nevertheless, milligrams of pure, stable, and functional protein are always required for biochemical and structural studies. Many modern biophysical tools are available today that provide critical information regarding to the characterisation and behaviour of integral membrane proteins in solution. These biophysical approaches play an important role in both basic research and in early-stage drug discovery processes. In this review, it is not our objective to present a comprehensive list of all existing biophysical methods, but a selection of the most useful and easily applied to basic integral membrane protein research.

Selection of Biophysical Methods for Characterisation of Membrane Proteins.

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. However, the necessity to use large amounts of sample and the low-throughput nature of the analysis remain as major bottlenecks of the technique.Here, we present a protocol for dynamic light scattering measurements that are executed in a fully automated fashion for low-volume samples, in situ. The protocol offers a generic pre-screening method for downstream structural studies of biomolecules using higher-resolution approaches such as X-ray crystallography, electron microscopy, small-angle X-ray scattering, and NMR .

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

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. It is however limited by the success in obtaining well-ordered protein crystals that diffract at high resolution. This is especially true for challenging targets such as membrane proteins (MPs). Understanding structure-function relationships of MPs at the biochemical level is vital for medicine and drug discovery as they play critical roles in many cellular processes. Though difficult, structure determination of MPs by X-ray crystallography has significantly improved in the last two decades, mainly due to many relevant technological and methodological developments. Today, numerous MP crystal structures have been solved, revealing many of their mechanisms of action. Yet the field of structural biology has also been through significant technological breakthroughs in recent years, particularly in the fields of single particle electron microscopy (cryo-EM) and X-ray free electron lasers (XFELs). Here we summarise the most important advancements in the field of MP crystallography and the significance of these developments in the present era of modern structural biology.

Membrane protein crystallography in the era of modern structural biology.

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 1.1 Å and 1.3 Å resolution respectively. We observe significant differences between the two states in the dynamics of water molecules that are coupled via H-bonds to the retinal Schiff Base. Supporting QM/MM calculations reveal how the DA state permits a thermodynamic equilibrium between retinal isomers to be established, and how this same change is prevented in the ground state in the absence of light. We suggest that the different arrangement of internal water networks in AR3 is responsible for the faster photocycle kinetics compared to homologs.

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

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. By using transparent and opaque polymer films, two structures, one corresponding to the desensitized, dark-adapted (DA) state and the other to the ground or light-adapted (LA) state, were solved to better than 1.9 Å resolution. All of the key structural features of AR3 were resolved, including the retinal chromophore, which is present as the 13-cis isomer in the DA state and as the all-trans isomer in the LA state. The film-sandwich sample environment enables diffraction data to be recorded at room temperature in both illuminated and dark conditions, which more closely approximate those in vivo. This simple approach is applicable to a wide range of membrane proteins crystallized in LCP and light-sensitive samples in general at synchrotron and laboratory X-ray sources.

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

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. Several biophysical methods are available for measuring protein aggregation and stability. Yet, discovering and developing means to improve protein behaviour and structure-function integrity remains a demanding task. Here, we discuss workflows that are made possible by adapting established biophysical methods to high-throughput screening approaches. Rapid identification and optimisation of conditions that promote protein stability and reduce aggregation will support researchers and industry to maximise sample quality, stability and reproducibility, thereby reducing research and development time and costs.

Article	Year
Selection of Biophysical Methods for Characterisation of Membrane Proteins. International journal of molecular sciences\| PubMed ID: 31137900	2019
In Situ Measurements of Polypeptide Samples by Dynamic Light Scattering: Membrane Proteins, a Case Study. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 32856264	2021
Membrane protein crystallography in the era of modern structural biology. Biochemical Society transactions\| PubMed ID: 33170253	2020
Structures of the archaerhodopsin-3 transporter reveal that disordering of internal water networks underpins receptor sensitization. Nature communications\| PubMed ID: 33504778	2021
Two states of a light-sensitive membrane protein captured at room temperature using thin-film sample mounts. Acta crystallographica. Section D, Structural biology\| PubMed ID: 34981761	2022
Measuring Protein Aggregation and Stability Using High-Throughput Biophysical Approaches. Frontiers in molecular biosciences\| PubMed ID: 35651816	2022

Tristan O. C. Kwan

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