Quantifying Protein–Protein Interactions by Molecular Counting with Mass Photometry

Interactions between biomolecules control the processes of life in health and their malfunction in disease, making their characterization and quantification essential. Immobilization- and label-free analytical techniques are desirable because of their simplicity and minimal invasiveness, but they struggle with quantifying tight interactions. Here, we show that mass photometry can accurately count, distinguish by molecular mass, and thereby reveal the relative abundances of different unlabelled biomolecules and their complexes in mixtures at the single-molecule level. These measurements determine binding affinities over four orders of magnitude at equilibrium for both simple and complex stoichiometries within minutes, as well as the associated kinetics. These results introduce mass photometry as a rapid, simple and label-free method for studying sub-micromolar binding affinities, with potential for extension towards a universal approach for characterizing complex biomolecular interactions.     

Fluorophenyl Bilirubins: Synthesis and Stereochemistry

Summary.  Analogs of bilirubin with vinyl groups replaced by symmetrically-disposed o-fluorophenyls (1, bis-exo, and 2, bis-endo) were synthesized and characterized spectroscopically. Their ¹H NMR spectra and NOE data are consistent with an intramolecularly hydrogen-bonded ridge-tile conformation where each propionic acid group embraces an opposing dipyrrinone. Like bilirubin, 1 and 2 exhibit negative chirality induced circular dichroism (ICD) Cotton effects in chloroform containing quinine. Unlike bilirubin, however, in aqueous buffer containing human serum albumin, 2 exhibits a negative exciton chirality ICD, whereas that of 1 is positive. Received May 22, 2001. Accepted May 29, 2001     

Computer simulation of the distribution of hexane in a lipid bilayer: spatially resolved free energy, entropy, and enthalpy profiles

The partitioning behavior of small molecules in lipid bilayers is important in a variety of areas including membrane protein folding and pharmacology. However, the inhomogeneous nature of lipid bilayers on a nanometer length scale complicates experimental studies of membrane partitioning. To gain more insight in the partitioning of a small molecule into the lipid bilayer, we have carried out atomistic computer simulations of hexane in a dioleoyl phosphatidylcholine model membrane. We have been able to obtain spatially resolved free energy, entropy, enthalpy, and heat capacity profiles based on umbrella sampling calculations at three different temperatures. In agreement with experiment, hexane partitions preferentially to the center of the bilayer. This process is driven almost entirely by a favorable entropy change, consistent with the hydrophobic effect. In contrast, partitioning to the densest region of the acyl chains is dominated by a favorable enthalpy change with a small entropy change, which is consistent with the "nonclassical" hydrophobic effect or "bilayer" effect. We explain the features of the entropy and enthalpy profiles in terms of density and free volume in the system.     

Spectroscopic determination of proton position in the proton-coupled electron transfer pathways of donor-acceptor supramolecule assemblies

A homologous set of porphyrin derivatives possessing an isocyclic five-membered ring appended with an amidinium functionality has been used to examine proton-coupled electron transfer (PCET) through well-characterized amidine-carboxylic acid interfaces. Conjugation between the porphyrin chromophore and the amidinium interface can be altered by selective reduction of the isocyclic ring of an amidinium-purpurin to produce an amidinium-chlorin. The highly conjugated amidinium-purpurin displays large spectral shifts in the visible region upon alteration of the amidinium/amidine protonation state; no such change is observed for the chlorin homologue. Analysis of the UV-vis absorption and emission profiles of the amidinium-purpurin upon deprotonation allows for the measurement of the porphyrinic-amidinium acidity constant for the ground state (pKa = 9.55 +/- 0.1 in CH3CN) and excited state (pKa)= 10.40 +/- 0.1 in CH3CN). The absorption spectrum of the purpurin also provides a convenient handle for determining the protonation state of assembled interfaces. In this way, the purpurin macrocycle provides a general tool for PCET studies because it can be used to determine the location of a proton within PCET interfaces formed from carboxylic acid electron acceptors including dinitrobenzenes (DNBs) and naphthalenediimide (NI), which have been used extensively in previous PCET studies. An amidine-carboxylic acid interface is observed for electron-rich acceptors, whereas the ionized amidinium-carboxylate interface is observed for electron-poor acceptors. The PCET kinetics for purpurin/chlorin associated to NI are consistent with an amidine-carboxylic acid interface, which is also verified spectrally.     

The biochemiresistor: an ultrasensitive biosensor for small organic molecules

New sensation: A resistance-based biosensor uses gold-coated magnetic nanoparticles (Au@MNPs) functionalized with the antibiotic enrofloxin (see picture; purple), which bind to anti-enrofloxin as analyte (blue). The Au@MNPs can be magnetically assembled between electrodes, and the measured resistance R is a function of analyte concentration.     

The effects of chain conformation in the microfluidic entry flow of polymer–surfactant systems

An associative polymer–surfactant system has been used to observe the effects of chain conformation in the entry flow through a microfabricated planar 16:1:16 contraction–expansion geometry. The well-studied system of the flexible polymer poly(ethylene oxide) (PEO) and anionic surfactant sodium dodecyl sulfate (SDS) was used. Dilute polymer solutions with increasing SDS concentration were characterized in steady and dynamic shear, as well as capillary breakup extensional rheology. Based on this characterization, the primary quantitative difference is an increase in zero-shear viscosity as a result of the PEO chain expansion brought on by association of SDS surfactant micelles. However, these quantitatively similar solutions were observed to exhibit much more qualitatively different flow patterns via fluorescent streak imaging in the entry flow. In contrast to previous work on PEO solutions, the PEO–SDS systems were observed to transition to a steady viscoelastic flow regime characterized by stable lip vortices at much lower elasticity and Weissenberg numbers. The resulting insight gained regarding the utility of microfluidic flows in elucidating effects of subtle conformational changes further illustrates the potential for using microfabricated devices as rheometric tools for measuring the properties of dilute and weakly viscoelastic fluids.