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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., (© 2022. European Biophysical Societies' Association.)

Understanding how proteins interact with DNA, and particularly the stoichiometry of a protein-DNA complex, is key information needed to elucidate the biological role of the interaction, e.g. transcriptional regulation. Here, we present an emerging analytical ultracentrifugation method that features multi-wavelength detection to characterise complex mixtures by deconvoluting the spectral signals of the interaction partners into separate sedimentation profiles. The spectral information obtained in this experiment provides direct access to the molar stoichiometry of the interacting system to complement traditional hydrodynamic information. We demonstrate this approach by characterising a multimeric assembly process between the transcriptional repressor of bacterial sialic acid metabolism, NanR and its DNA-binding sequence. The method introduced in this study can be extended to quantitatively analyse any complex interaction in solution, providing the interaction partners have different optical properties.

Pyruvate kinase catalyses the final step of the glycolytic pathway in central energy metabolism. The monomeric structure comprises three domains: a catalytic TIM-barrel, a regulatory domain involved in allosteric activation, and a lid domain that encloses the substrates. The lid domain is thought to close over the TIM-barrel domain forming contacts with the substrates to promote catalysis and may be involved in stabilising the activated state when the allosteric activator is bound. However, it remains unknown whether the lid domain is essential for pyruvate kinase catalytic or regulatory function. To address this, we removed the lid domain of Escherichia coli pyruvate kinase type 1 (PK TIM+Reg ) using protein engineering. Biochemical analyses demonstrate that, despite the absence of key catalytic residues in the lid domain, PK TIM+Reg retains a low level of catalytic activity and has a reduced binding affinity for the substrate phosphoenolpyruvate. The enzyme retains allosteric activation, but the regulatory profile of the enzyme is changed relative to the wild-type enzyme. Analytical ultracentrifugation and small-angle X-ray scattering data show that, beyond the loss of the lid domain, the PK TIM+Reg structure is not significantly altered and is consistent with the wild-type tetramer that is assembled through interactions at the TIM and regulatory domains. Our results highlight the contribution of the lid domain for facilitating pyruvate kinase catalysis and regulation, which could aid in the development of small molecule inhibitors for pyruvate kinase and related lid-regulated enzymes.

β-Lactoglobulin is the most abundant protein in the whey fraction of ruminant milks, yet is absent in human milk. It has been studied intensively due to its impact on the processing and allergenic properties of ruminant milk products. However, the physiological function of β-lactoglobulin remains unclear. Using the fluorescence-detection system within the analytical ultracentrifuge, we observed an interaction involving fluorescently labelled β-lactoglobulin in its native environment, i.e. cow and goat milk, for the first time. Co-elution experiments support that these β-lactoglobulin interactions occur naturally in milk and provide evidence that the interacting partners are immunoglobulins, while further sedimentation velocity experiments confirm that an interaction occurs between these molecules. The identification of these interactions, made possible through the use of fluorescence-detected analytical ultracentrifugation, provides possible clues to the long debated physiological function of this abundant milk protein.

Milk components, such as proteins and lipids, have different physicochemical properties depending upon the mammalian species from which they come. Understanding the different responses of these milks to digestion, processing, and differences in their immunogenicity requires detailed knowledge of these physicochemical properties. Here we report on the oligomeric state of β-lactoglobulin from caprine milk, the most abundant protein present in the whey fraction. At pH 2.5 caprine β-lactoglobulin is predominantly monomeric, whereas bovine β-lactoglobulin exists in a monomer-dimer equilibrium at the same protein concentrations. This behaviour was also observed in molecular dynamics simulations and can be rationalised in terms of the amino acid substitutions present between caprine and bovine β-lactoglobulin that result in a greater positive charge on each subunit of caprine β-lactoglobulin at low pH. The denaturation of β-lactoglobulin when milk is heat-treated contributes to the fouling of heat-exchange surfaces, reducing yields and increasing cleaning costs. The bovine and caprine orthologues of β-lactoglobulin display different responses to thermal treatment, with caprine β-lactoglobulin precipitating at higher pH values than bovine β-lactoglobulin (pH 7.1 compared to pH 5.6) that are closer to the natural pH of these milks (pH 6.7). This property of caprine β-lactoglobulin likely contributes to the reduced heat stability of caprine milk compared to bovine milk at its natural pH.