Biophysical Chemistry seminars

Actinoporins are pore-forming proteins from cnidarians that assemble into membrane-spanning complexes. Their preference for sphingomyelin-rich membranes and enhancement by cholesterol make them useful models of protein–lipid interactions. Gašper Šolinc will discuss the actinoporin-like protein Fav from Orbicella faveolata, whose funnel-shaped pores incorporate numerous lipids, with up to fifteen resolved lipids per subunit in the upper leaflet. The findings distinguish lipids involved in initial membrane binding from those stabilizing interfaces between protein subunits. Changing membrane composition yields different pore stoichiometries while preserving the protomer fold and lipid arrangement, linking membrane thickness and lipid composition to assembly. Comparisons with FraC and StnII structures address sphingomyelin recognition through hydrogen bonding and cation–π interactions, and why these pores are better understood as protein–lipid complexes.

Fabian Soltermann and Brenda Watt examine mass photometry as a complementary method for measuring therapeutic-antibody aggregates and immune complexes. The programme compares automated mass photometry with SEC-MALS and sedimentation-velocity analytical ultracentrifugation, including quantification of monomers, dimers and larger aggregates. An IL-17A/anti-IL-17 antibody study illustrates heterogeneous target-related complexes and possible implications for immunogenicity. The discussion also considers how binding geometry and target valency influence complex formation and where a rapid, low-sample, column-free assay adds useful information.

Amino acids have long been used in biological and pharmaceutical formulations, but their stabilizing effects are often described only for particular proteins. This talk examines a broader mechanism: small molecules modify colloidal interactions in proteins, DNA and synthetic nanoparticles. Measurements of osmotic virial coefficients and potentials of mean force reveal consistent effects across these systems. A model of weakly interacting amino acids binding to patchy colloidal surfaces predicts experimentally measured dissociation constants; proline–lysozyme predictions and measurements agree within a factor of two. Experiments also test predictions that short peptides outperform the same number of free amino acids and that charged amino acids stabilize oppositely charged proteins. The consequences extend to suppression of liquid–liquid phase separation and cellular stress granules, and doubled insulin bioavailability in mice when co-formulated with proline. The results motivate treating amino acids as active regulators of colloidal behavior alongside familiar influences such as salt.

Clarice D. Aiello examines evidence that quantum effects influence biological function, including magnetic sensing used in animal navigation, cellular metabolism and enzyme activity, and the capture of light energy in photosynthesis. The talk reviews proposed biophysical mechanisms and their implications for human, plant and environmental biology, while considering the difficulty of connecting observations from nanometre scales to whole organisms. A central question is whether quantum mechanics can be established or ruled out as the explanation for physiologically relevant effects, and whether those effects can be controlled for useful applications. Potential directions include biomimetic electromagnetic probes, quantum-computing architectures that operate at room temperature, improved solar-energy devices and new therapies. Progress will require work across disciplines and scales, with theory and experiment developed together.

Diverse physical processes played important roles in the origin of life. I will review the origin of molecular homochirality, the growth of protocell membranes, and potential roles for liquid-liquid phase separation. I will then discuss the concept of functional information and its quantitative relationship with molecular function.

Diseases that arise from misfolding of an individual protein are rare. However, collectively, these folding diseases represent a large proportion of hereditary and acquired disorders. In fact, the term "Molecular Medicine" was coined by Linus Pauling in conjunction with the study of a folding disease, i.e. sickle cell anemia. In the past decade, we have witnessed an exponential growth in the number of mutations, which have been identified in genes encoding solute carriers (SLC). A sizable faction - presumably the majority - of these mutations result in misfolding of the encoded protein. While studying the export of the GABA transporter (SLC6A1) and of the serotonin transporter (SLC6A4), from the endoplasmic reticulum (ER), we discovered by serendipity that some ligands can correct the folding defect imparted by point mutations. These bind to the inward facing state. The most effective compound is noribogaine, the metabolite of ibogaine (an alkaloid first isolated from the shrub Tabernanthe iboga). There are 13 mutations in the human dopamine transporter (DAT, SLC6A3), which give rise to a syndrome of infantile Parkinsonism and dystonia. We capitalized on our insights to explore, if the disease-relevant mutant proteins were amenable to pharmacological correction. Drosopohila melanogaster, which lack the dopamine transporter, are hyperactive and sleepless (fumin in Japanese). Thus, mutated human DAT variants can be introduced into fumin flies. This allows for examining the effect of pharmacochaperones on delivery of DAT to the axonal territory and on restoring sleep. We explored the chemical space populated by variations of the ibogaine structure to identify an analogue (referred to as compound 9b), which was highly effective: compound 9b also restored folding in DAT variants, which were not amenable to rescue by noribogaine. Deficiencies in the human creatine transporter-1 (CrT1, SLC6A8) give rise to a syndrome of intellectual disability and seizures and accounts for 5% of genetically based intellectual disabilities in boys. Point mutations occur, in part, at positions, which are homologous to those of folding-deficient DAT variants. CrT1 lacks the rich pharmacology of monoamine transporters. Nevertheless, our insights are also applicable to rescuing some disease-related variants of CrT1. Finally, the question arises how one can address the folding problem. We propose a two-pronged approach: (i) analyzing the effect of mutations on the transport cycle by electrophysiological recordings; this allows for extracting information on the rates of conformational transitions. The underlying assumption posits that - even when remedied by pharmacochaperoning - folding-deficient mutants must differ in the conformational transitions associated with the transport cycle. (ii) analyzing the effect of mutations on the two components of protein stability, i.e. thermodynamic and kinetic stability. This is expected to provide a glimpse of the energy landscape, which governs the folding trajectory.

Intrinsically disordered proteins (IDPs), lack of a well-defined folded structure, have been recently shown to be critical to forming membrane-less organelles via liquid-liquid phase separation (LLPS). Due to the flexible conformations of IDPs, it could be challenging to investigate IDPs with solely experimental techniques. Computational models can therefore provide complementary views at several aspects, including the fundamental physics underlying LLPS and the sequence determinants contributing to LLPS. In this presentation, I will start with our coarse-grained computational framework that can help generate sequence dependent phase diagrams. The coarse-grained model further led to the development of a polymer model with empirical parameters to quickly predict LLPS of IDPs. At last, I will show our preliminary efforts on addressing molecular interactions within LLPS of IDPs using all-atom explicit-solvent simulations.

Evidence regarding protein structure and function manifest the imperative role that dynamics play in proteins, underlining reconsideration of the unanimated sequence-to-structure-to-function paradigm. Structural dynamics portray a heterogeneous energy landscape described by conformational ensembles where each structural representation can be responsible for unique functions or enable macromolecular assemblies. Using the human p27/Cdk2/Cyclin A ternary complex as an example, we highlight the vital role of intra- and intermolecular dynamics for target recognition, binding, and inhibition as a critical modulator of cell division. Rapidly sampling configurations is critical for the population of different conformational ensembles encoding functional roles. To garner this knowledge, we present how the integration of (sub)ensemble and single-molecule fluorescence spectroscopy with molecular dynamic simulations can characterize structural dynamics linking the heterogeneous ensembles to function. The incorporation of dynamics into the sequence-to-structure-to-function paradigm promises to assist in tackling various challenges, including understanding the formation and regulation of mesoscale assemblies inside cells.

This talk aims to show how new physical methods can advance biological and biomedical research. A major advance in physical chemistry in the last two decades has been the development of quantitative methods to directly observe individual molecules in solution, attached to surfaces, in the membrane of live cells or more recently inside live cells. These single-molecule fluorescence studies have now reached a stage where they can provide new insights into important biological problems. After presenting the principles of these methods, I will give some examples from our current research to probe the molecular basis of neurodegeneration. Here we have used single-molecule fluorescence to detect and analyse the low concentrations of soluble protein aggregates thought to be responsible for Alzheimer’s disease and determine the mechanisms by which they damage neurons. Lastly, I will describe how fundamental science aimed at watching single molecules incorporating nucleotides into DNA gave rise to a new rapid method to sequence DNA that is now widely used.