Biophysical reviews最新文献

Nucleic acids are primary therapeutic targets, and understanding drug-DNA interactions is essential to the discovery of new clinical agents. In recent years, the desire to develop therapies with specific biological targets has produced new molecules that preferentially interact with complex nucleic acid sequences and structures. As such, the targeting of non-canonical nucleic acids, including DNA triplexes, G-quadruplexes, i-motifs, three-way junctions and Holliday junctions, have emerged due to their roles in gene regulation, genome stability and cellular stress responses. Characterising the interactions of these non-canonical structures with new ligands and metal complexes has led to the discovery of promising agents with therapeutic potential. Biophysical techniques including spectroscopic methods, crystallography and biomolecular assays have been critical to probing these interactions. This review describes recent advancements in the analysis of higher-order drug-DNA interactions for the rational design of targeted therapeutics.

Integral membrane proteins are crucial molecules ubiquitous to all cell types, coordinating cell signalling and facilitating the tightly regulated transport of essential nutrients across plasma membrane. Defects in membrane proteins are associated with disease, emphasising the need to understand the structural, mechanistic and regulatory mechanisms which control integral membrane proteins. Recent technological advances in optical microscopy have allowed appropriate study of these small proteins using tools with molecular resolution which can non-invasively observe their native organisation in the plasma membrane in situ. Complimentarily, by utilising photochemical phenomena and analyses, single-molecule detail can be elucidated from conventional microscope systems. In this review, we firstly overview the methodologies used for studies of membrane proteins and then review the biophysical results gleaned from their application with an emphasis on membrane transporters. We show that single molecule studies of integral membrane proteins are beginning to unveil striking new regulatory mechanisms with wide applicability across many distinct fields of biological research.

Microscopy of the brain has been facing problems of contrast and thick tissue imaging. Second harmonic generation (SHG) is a non-linear effect of the light interaction with the imaged material, resulting in photon emission at half the wavelength of the absorbed light. SHG microscopy provides an unprecedented opportunity for imaging collagen and other noncentrosymmetric protein fibrils in unstained thick tissue samples and in the live brain via a regular multiphoton setup. This opens a remarkable methodological window for imaging pathological processes of high importance, including brain trauma, fibrosis, tumorigenesis, and neuroimplant-induced foreign body response. Moreover, SHG is a valuable tool for imaging astrocytes and nerve fiber microtubules. Third harmonic generation enhanced by three-photon resonance with the Soret band of hemoglobin is combined with SHG to resolve the microstructure of blood vessel walls and astrocyte-process endfeet on gliovascular interfaces. Here, we review current state-of-the-art methods in the field of brain imaging applications of SHG, including research on brain and spinal cord injury, glioma, ischemia, Alzheimer's disease, neuroimplantation, and brain meninges. We then address the method development perspective in the broader context of other tissue pathologies. Finally, we account for recent progress in artificial intelligence applications for SHG microscopy data analysis.

This review focuses on spectral shift analysis as a tool to study macromolecular interactions and describes its current place among the available biophysical methods. NanoTemper's Dianthus platform facilitates a plate-based, microfluidics-free, mass-independent, and immobilisation-free high-throughput screening platform for protein-ligand, protein-protein, and protein-nucleic acid interactions, as well as ternary complexes, for example in proteolysis targeting chimera (PROTAC) design. In addition to spectral shift, the Dianthus offers an orthogonal method, temperature-related intensity change (TRIC). Both methods are presented alongside fluorescent labelling techniques. Specific examples with practical tips for spectral shift methods for diverse binding partners are provided. Finally, current and future applications of spectral shift methods in the drug discovery process are discussed in the context of high-throughput screening, fragment-based drug discovery, and hit-to-lead optimisation.

The nucleus is a highly compartmentalized organelle and this spatial organization reflects gene-regulatory environments. Chromatin exists in two distinct forms: transcriptionally active, euchromatin and silenced, compacted heterochromatin. The spatial organization of chromatin along with its transcriptional activity is governed by biomolecular assemblies (BAs). Gene regulatory assemblies form and operate through highly dynamic protein-protein and protein-DNA interactions often established via their recruitment by non-coding RNAs. The formation of BAs is essential for retaining diffusible regulatory proteins at specific genomic regions, enabling local confinement and precise gene regulation. Phase separation, particularly in the form of liquid-liquid condensation, is suggested to play a crucial role in transcriptional regulation, serving as a key driver of biomolecular assembly formation. However, some studies indicate that phase separation may also be a non-essential byproduct of the crowded nuclear environment or may not be involved in certain BAs. Despite extensive investigations into these macromolecular crowding phenomena, the precise mechanisms underlying both the formation of gene-regulatory BAs and how these localized protein concentrations function to regulate chromatin structure and gene expression remain unclear. This review highlights progress made in elucidating the mechanisms of chromatin-modifying BAs, highlighting how super-resolution microscopy and single-molecule technologies are proving essential for probing these nuclear structures in situ, within their native cellular context.

Cellular activity depends on constant flux of ions across biological membranes. Artificial membrane models like planar lipid bilayers and liposomes are ideal for studying membrane transport phenomena as they are free of the structural complexity of cells and allow examination of transport processes under tightly controlled conditions. Over the last decades, artificial membrane-based techniques like single-channel recording and fluorescent monitoring of transport through bulk lipid vesicle suspensions have revealed many molecular mechanisms of transport. Recently, giant unilamellar vesicles (GUVs), cell-sized liposomes, have emerged as an important tool for studying cellular processes, including ion transport. The principal advantage of GUVs derives from their micron scale, which enables ease of visualisation and manipulation using microscopy and microhandling. For that reason, GUVs have also become the state-of-the-art for recapitulating a host of cell structures and functions for the purpose of developing artificial cells. Taken together, GUVs represent a promising biomimetic system to elucidate ion transport mechanisms and unravel the association between ion fluxes and various cellular processes such as neuronal transduction, nutrient uptake, electrochemical gradient development. Nevertheless, despite their great potential as a model system, the use of GUVs in ion transport studies is still limited. The aim of this review is to outline recent GUV-based ion transport studies, describe the current techniques for measuring ion transport in GUVs, compare the utility of GUVs relative to other available techniques such as single-channel current recording, and explore the potential of using GUVs to investigate complex ion transport processes.

To change shape, move, grow and divide, cells employ various motor and non-motor proteins that convert chemical energy into the generation of mechanical force. Force spectroscopy tools that allow the measurement of these forces generated by individual molecules revolutionised our understanding of single-molecule mechanics over the past three decades. These techniques, however, remain largely confined to studies with purified components outside cells. A critical, unresolved challenge lies in deciphering how these force-generating and force-sensing molecules coordinate their activities inside living cells. In this review, we discuss advances in magnetic tweezers designed to measure and apply mechanical forces intracellularly. We highlight recent progress in magnetic tweezers that began to provide an understanding of how active mechanical forces drive rearrangements of biological structures. We also discuss challenges associated with applying forces locally and precisely. We identify two key areas that hold potential for the development of tools for direct mechanical manipulations of specific molecules inside living cells: (1) instrument design to generate and control magnetic gradients at the single-cell scale, and (2) development of magnetic biofunctionalised particles capable of targeting specific structures. The integration of these advances should enable unprecedented ability to manipulate intracellular forces, opening new avenues to study intracellular organisation, mechanotransduction pathways, cell division and migration. By addressing current limitations in specificity and resolution, next-generation magnetic tweezers may finally bridge the gap between single-molecule biophysics in vitro and cell-scale mechanobiology in living cells.

X-ray diffraction enables determination of biomolecular structure but requires well-diffracting crystals that are notoriously difficult to grow. Porous materials can aid the crystallization of refractory proteins and, since knowledge of the mode of action of such materials may contribute to finding new crystallization inducers, this process has been studied thoroughly. It was established that, even under conditions where heterogeneous nucleation on flat surfaces is absent, a synergistic diffusion-adsorption effect inside a sufficiently narrow pore can increase the protein concentration to a level sufficient for crystal nucleation. The formation of a protein crystal in a pore begins with the assembly of molecules into a crystalline layer of monomolecular thickness, which is stabilized by its cohesion with the pore wall. We highlight thermodynamic considerations that provide an estimate of the importance of the protection due to the pore walls for crystal stability. In addition, molecular-kinetic considerations reveal further details of protein crystal nucleation assisted by porous materials. The observation that protein crystals nucleated by means of porous materials often display improved X-ray diffraction is of practical importance for structural studies. It is hoped that this review will guide scientists in their efforts to grow crystals of target proteins, complementing the usual trial-and-error strategies.

Purified proteins are sitting in a mostly aqueous environment, with normally some buffer and salt making up the conditions. This is vastly different from their natural habitat, and protein are often affected by this difference, showing signs of destabilisation or aggregation. A common method to improve the protein solubility and homogeneity is adding small molecules to the buffer conditions, as these can aid protein stability and keep the protein in solution at a concentration which is within that needed for the experiments that are to be undertaken. This review is detailing some of the small molecules that are routinely used, with a focus on them being readily available and affordable for all labs. Some of the more common small molecule additives described in this paper are (1) amino acids, like arginine or glycine, (2) sugars, like sucrose, or (3) other osmolytes, such as glycerol. The second part is covering some of the methods that can be utilised to determine whether a small molecule improves the stability of a particular protein.