In the brain, α‐synuclein (aSN) partitions between free unbound cytosolic and membrane bound forms modulating both its physiological and pathological role and complicating its study due to structural heterogeneity. Here, we use an interdisciplinary, synergistic approach to characterize the properties of aSN:lipid mixtures, isolated aSN:lipid co‐structures, and aSN in mammalian cells. Enabled by the isolation of the membrane‐bound state, we show that within the previously described N‐terminal membrane anchor, membrane interaction relies both on an N‐terminal tail (NTT) head group layer insertion of 14 residues and a folded‐upon‐binding helix at the membrane surface. Both binding events must be present; if, for example, the NTT insertion is lost, the membrane affinity of aSN is severely compromised and formation of aSN:lipid co‐structures hampered. In mammalian cells, compromised cooperativity results in lowered membrane association. Thus, avidity within the N‐terminal anchor couples N‐terminal insertion and helical surface binding, which is crucial for aSN membrane interaction and cellular localization, and may affect membrane fusion.

The understanding and characterization of protein interactions is crucial for elucidation of complicated biomolecular processes as well as for the development of new biopharmaceutical therapies. Often, protein interactions involve multiple binding, avidity, oligomerization, and are dependent on the local environment. Current analytical methodologies are unable to provide a detailed mechanistic characterization considering all these parameters, since they often rely on surface immobilization, cannot measure under biorelevant conditions, or do not feature a structurally-related readout for indicating formation of multiple bound species. In this work, we report the use of flow induced dispersion analysis (FIDA) for in-solution characterization of complex protein interactions under in vivo like conditions. FIDA is an immobilization-free ligand binding methodology employing Taylor dispersion analysis for measuring the hydrodynamic radius (size) of biomolecular complexes. Here, the FIDA technology is utilized for a size-based characterization of the interaction between TNF-α and adalimumab. We report concentration-dependent complex sizes, binding affinities (Kd), kinetics, and higher order stoichiometries, thus providing essential information on the TNF-α–adalimumab binding mechanism. Furthermore, it is shown that the avidity stabilized complexes involving formation of multiple non-covalent bonds are formed on a longer timescale than the primary complexes formed in a simple 1 to 1 binding event.

Assessment of protein stability and function is key to the understanding of biological systems and plays an important role in the development of protein-based drugs. In this work, we introduce an integrated approach based on Taylor dispersion analysis (TDA), flow induced dispersion analysis (FIDA), and in-line intrinsic fluorescence which enables rapid and detailed assessment of protein stability and unfolding. We demonstrate that the new platform is able to efficiently characterize chemically induced protein unfolding of human serum albumin (HSA) in great detail. The combined platform enables local structural changes to be probed by monitoring changes in intrinsic fluorescence and loss of binding of a low-molecular weight ligand. Simultaneously, the size of the unfolding HSA is obtained by TDA on the same samples. The integration of the methodologies enables a fully automated characterization of HSA using only a few hundred nanoliters of sample. We envision that the presented methodology will find applications in fundamental biophysics and biology as well as in stability screens of protein-based drug candidates.

In this initial research on feasibility, removal of unconjugated fluorescein isothiocyanate (FITC) after fluorescent labeling of human serum albumin (HSA) with electromembrane extraction (EME) was investigated for the first time. A 100 μL solution of 0.1 mg/mL HSA was fluorescently labeled with 0.01 mg/mL FITC in a molar ratio of 10:1 in an Eppendorf tube for 30 min under agitation and absence of light. Then the labeled solution was transferred to a 96-well EME with 3 μL 0.1% (w/w) Aliquat 336 in 1-octanol as the supported liquid membrane (SLM) and 200 μL 10 mM NaOH as waste solution. EME was performed for 10 min with a voltage of 50 V, with the anode in the waste solution and at 900 rpm agitation. Negatively charged and unconjugated FITC was extracted electrokinetically into the SLM and to the waste solution. Analysis of purified samples, by Taylor dispersion analysis (TDA), showed a 92% removal of unconjugated FITC (FITC clearance: 92%, RSD: 3%), while 79% of the HSA/FITC complex remained in the sample (protein retention: 79%, RSD: 18%). Conserved functionality of the HSA/FITC complex after EME was proven by a binding affinity study with anti-HSA using flow induced dispersion analysis (FIDA). In this real sample, the dissociation constant (K d ) and hydrodynamic radius of the complex were determined to be 0.8 μM and 5.87 nm, respectively, which was in concordance with previously reported values.