Within the complex milieu of a cell, which comprises a large number of different biomolecules, interactions are critical for function. In this post-reductionist era of biochemical research, the ‘holy grail’ for studying biomolecular interactions is to be able to characterize them in native environments. While there are a limited number of in situ experimental techniques currently available, there is a continuing need to develop new methods for the analysis of biomolecular complexes that can cope with the additional complexities introduced by native-like solutions. We think approaches that use microfluidics allow researchers to access native-like environments for studying biological problems. This review begins with a brief overview of the importance of studying biomolecular interactions and currently available methods for doing so. Basic principles of diffusion and microfluidics are introduced and this is followed by a review of previous studies that have used microfluidics to measure molecular diffusion and a discussion of the advantages and challenges of this technique.

Protein structures may be used to draw functional implications at the residue level, but how sensitive are these implications to the exact structure used? Calculation of the effects of SARS-CoV-2 S-protein mutations based on experimental cryo-electron microscopy structures have been abundant during the pandemic. To understand the precision of such estimates, we studied three distinct methods to estimate stability changes for all possible mutations in 23 different S-protein structures (3.69 million ΔΔG values in total) and explored how random and systematic errors can be remedied by structure-averaged mutation group comparisons. We show that computational estimates have low precision, due to method and structure heterogeneity making results for single mutations uninformative. However, structure-averaged differences in mean effects for groups of substitutions can yield significant results. Illustrating this protocol, functionally important natural mutations, despite individual variations, average to a smaller stability impact compared to other possible mutations, independent of conformational state (open, closed). In summary, we document substantial issues with precision in structure-based protein modeling and recommend sensitivity tests to quantify these effects, but also suggest partial solutions to the problem in the form of structure-averaged “ensemble” estimates for groups of residues when multiple structures are available.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00249-022-01619-8.

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor belonging to the nuclear hormone receptor and has been exploited as a well-established druggable target for the treatment of diabetes mellitus (DM). Traditionally, small-molecule compounds have been developed to attack at the ligand site and Ser273 phosphorylation site of PPARγ. In this study, we derived helical peptide segments from the LXXLL motif region of coactivator proteins as antidiabetic PPARγ antagonists, which were expected to competitively disrupt the native interaction between PPARγ and its cognate coactivators by rebinding at PPARγ coactivator site. Structural analysis, dynamics simulation and energetics dissection revealed that these peptides cannot be well folded into active helical structure when splitting from the protein context of their parent coactivators and exhibit a large flexibility and intrinsic disorder in the free state, which would, therefore, incur a considerable entropy penalty upon rebinding to PPARγ. Hydrocarbon stapling strategy was employed to constrain these free coactivator peptides into ordered helical conformation, thus largely minimizing unfavorable entropy penalty but having only a moderate effect on favorable enthalpy contribution. The computational findings were further substantiated by fluorescence-based assays; the binding affinity of three potent SRC1, NCoA6 and p300 coactivator peptides to PPARγ was observed to be improved by 7.2-fold, 4.2-fold and 5.7-fold upon the stapling, which were also measured to have an efficient competitive potency with their unstapled counterparts for PPARγ coactivator site, with CC50 = 0.096, 0.12 and 0.18μM, respectively.

The skin of amphibians is widely exploited as rich sources of membrane active peptides that differ in chain size, polypeptide net charge, secondary structure, target selectivity and toxicity. In this study, two small antimicrobial peptides, temporin-Ra and temporin-Rb, originally isolated from the skin of the European marsh frog (Rana ridibunda), described as active against pathogen bacteria and presenting low toxicity to eukaryotic cells were synthesized and had their physicochemical properties and mechanism of action investigated. The temporin peptides were examined in aqueous solution and in the presence of membrane models (lipid monolayers, micelles, lipid bilayers and vesicles). A combined approach of bioinformatics analyses, biological activity assays, surface pressure measurements, synchrotron radiation circular dichroism spectroscopy, and oriented circular dichroism spectroscopy were employed. Both peptides were able to adsorb at a lipid-air interface with a negative surface charge density, and efficiently disturb the lipid surface packing. A disorder-to-helix transition was observed on the secondary structure of both peptides when either in a non-polar environment or interacting with model membranes containing a negative net charge density. The binding of both temporin-Ra and temporin-Rb to membrane models is modulated by the presence of negatively charged lipids in the membrane. The amphipathic helix induced in temporin-Ra is oriented parallel to the membrane surface in negatively charged or in zwitterionic lipid bilayers, with no tendency for realignment after binding. Temporin-Rb, instead, assumes a β-sheet conformation when deposited into oriented stacked lipid bilayers. Due to their short size and simple composition, both peptides are quite attractive for the development of new classes of peptide-based anti-infective drugs.

Spatial confinement of excitons in the nano-crystalline region of semiconducting nanostructures differ significantly from the optoelectronic properties exhibited by the bulk material. We report spike-like absorption observed in the UV spectrum of a phenylalanine hexamer peptide [(Ff)3-OH] nano-assembly, which may be attributed to the spatial confinement of electrons to the dimension of quantum dots. Interdependency of the UV and PLE spectrum of the peptide confirms the existence of quantum confinement in (Ff)3-OH nano-assemblies.

Rotations of the flagella control the movement of a peritrichous (multiflagellar) bacterium in fluids, the run and tumble events being caused through modulations in the flagella's collective rotation speed and pattern. Observing such modulations is a challenge in free swimming bacteria. In this work, we present a setup to measure the collective flagellar rotational features of an optically confined Bacillus subtilis bacterium. We adopt a Continuous Wavelet Technique (CWT) while monitoring the rotational patterns in frequency and time, thus achieving optimal resolution in both the domains. This enables in marking the events wherein subtle changes in the flagellar rotational pattern occur. These studies unravel a fact, hitherto unknown, that variations in swimming speed that are seen in pure run sequences are also caused by modulations in the rotating flagella. Further, we have monitored the flagellar rotation for durations over a minute and observe a gradual slowing down of the rotation before ceasing completely due to the trapping laser induced photodamage. We have observed a significant alteration in the rate of rotational fall off in real time with changes in pH or the nutrient concentration in the fluid. This work serves to demonstrate the advantage of optical confinement of a bacterium in its pristine form for carrying out such studies and can serve as a marker for work that assesses membrane photodamage in active matter. Details on the role of flagella in propulsion and on other factors influencing the rotations, can be of significance in the design of artificial microswimmers.

Human serum albumin binds a wide variety of drugs with different structure and affinity to two main binding sites, drug site 1 (DS1) and drug site 2 (DS2), which partially or totally overlap with fatty acid (FA) sites. Although multiple binding sites are available for endogenous compounds, FAs are the primary physiological ligands of albumin and their competition in the occupancy of DS1 and DS2 affects the binding of exogenous molecules, with a possible impact on drug delivery. In this work, we have investigated the simultaneous binding of oleic acid, warfarin and ibuprofen to albumin using differential scanning calorimetry and fluorescence to evaluate the impact on the conformational stability of the protein. The two drugs are widely used for their anticoagulant (warfarin) and anti-inflammatory (ibuprofen) properties, and can be also considered as site markers to probe DS1 and DS2, respectively. Oleic acid is one of the most important fatty acids from a physiological point of view for its role as a source of energy for cells, and also it binds albumin with the highest association constant. When complexed with oleic acid the calorimetric profile of albumin shows a biphasic trend whose line shape depends on the ligand concentration. The binding capacity of either warfarin or ibuprofen to albumin is modulated by oleate molecules in a concentration-dependent mode being synergic cooperative (warfarin) or competitive-like (ibuprofen). The overall results provide insights on the dynamics of albumin/ligands complex, which in turn may have important pharmacokinetic and pharmacodynamic implications.

The effects of the chemical environment of menaquinones (all-trans MK-4 and all-trans MK-7) incorporated in lipid monolayers on mercury electrodes have been studied with respect to the thermodynamics and kinetics of their electrochemistry. The chemical environment relates to the composition of lipid films as well as the adjacent aqueous phase. It could be shown that the addition of all-trans MK-4 to TMCL does not change the phase transition temperatures of TMCL. In case of DMPC monolayers, the presence of cholesterol has no effect on the thermodynamics (formal redox potentials) of all-trans MK-7, but the kinetics are affected. Addition of an inert electrolyte (sodium perchlorate; change of ionic strength) to the aqueous phase shifts the redox potentials of all-trans MK-7 only slightly. The formal redox potentials of all-trans MK-4 were determined in TMCL and nCL monolayers and found to be higher in nCL monolayers than in TMCL monolayers. The apparent electron transfer rate constants, transfer coefficients and activation energies of all-trans MK-4 in cardiolipins have been also determined. Most surprisingly, the apparent electron transfer rate constants of all-trans MK-4 exhibit an opposite pH dependence for TMCL and nCL films: the rate constants increase in TMCL films with increasing pH, but in nCL films they increase with decreasing pH. This study is a contribution to understand environmental effects on the redox properties of membrane bond redox systems.Graphical abstract

The determination of a suitable buffer environment for a protein of interest is not an easy task. The requirements of advanced techniques, the demands on the biological material and the researcher time needed for buffer optimization, as well as personal inflexibility, lead frequently to the use of sub-optimal buffers. Here, we demonstrate the design of a 48-condition buffer screen that can be used to determine an appropriate environment for downstream studies. By the combination of several techniques (differential scanning fluorimetry, dynamic light scattering, and bio-layer interferometry), we are able to assess the protein stability, homogeneity and binding activity across the screen with less than half a milligram of protein in 1day. The application of this screen helps to avoid unsuitable conditions, to explain problems observed upon protein analysis and to choose the most suitable buffers for further research. The screen can be routinely used as a primary screen for buffer optimization in labs and facilities.