Ion-specific effects for tuning the phase behavior of protein solutions

Protein phase behaviour is of importance in various areas of research such as structural biology, rational drug design and delivery, medicine (in particular protein condensation diseases), biotechnology, food science and cell biology. A particularly intriguing variety of phase behaviours can be induced in negatively charged, globular proteins in the presence of multivalent salts such as lanthanide (Ln) chlorides. These behaviours include reentrant condensation, crystallisation and cluster formation as well as liquid-liquid phase separation (LLPS) into a protein-rich and a protein-poor phase [1-3]. LLPS can occur upon a temperature decrease or increase, which is referred to as an upper or a lower critical solution temperature (UCST- and LCST-LLPS), respectively. Here, we present a challenging set of experiments investigating the complex phenomenon of LCST-LLPS in systems of bovine serum albumin (BSA) and multivalent salts from different perspectives including thermodynamic, (non-)equilibrium and spectroscopic studies. First, the rather unusual phenomenon of LCST-LLPS in aqueous systems consisting of BSA and yttrium chloride (YCl3) is characterised thermodynamically. Surface charge (zeta potential) and isothermal titration calorimetry (ITC) measurements show LCST-LLPS to be a hydration entropy-driven condensation [2]. This mechanistic explanation is corroborated by results obtained using extended X-ray absorption fine structure (EXAFS) spectroscopy. Based on the Y3+-induced LCST-LLPS described above, the aspect investigated subsequently is the influence that the nature of the multivalent cations used has on this phase behaviour. The experiments focus on the three multivalent salts HoCl3, YCl3 and LaCl3. A multi-technique approach including temperature-controlled UV-Vis absorbance and synchrotron small-angle X-ray scattering (SAXS) measurements shows that Ho3+ cations induce the strongest protein-protein attractions, while the interactions are weakest in the case of La3+. The overall protein-protein and protein-cation interaction strengths can therefore be ranked according to the order Ho3+ \u3e Y3+ \u3e La3+ [3]. Finally, the kinetics of LCST-LLPS of BSA in the presence of varying ratios of HoCl3 and LaCl3 is investigated using synchrotron ultra-small-angle X-ray scattering (USAXS). The growth of the characteristic length scale of the respective experimental systems as a function of time and temperature is found to be strongly influenced by the HoCl3/LaCl3 ratio. Notably, a higher volume fraction of HoCl3 preferentially drives the samples into an arrested state even at low temperatures [4]. The present study thus shows how a careful choice of multivalent ions can be used to fine-tune protein-protein interactions and the resulting phase behaviour in solution. The results are of importance not only for a fundamental understanding of soft matter thermodynamics, but also for the design of so-called “smart” materials with implications for, e.g., drug delivery or water purification. [1] Zhang et al., Phys. Rev. Lett. (101), 14810 (2008) [2] Zhang et al., J. Appl. Cryst. (44), 755-62 (2011) [3] Zhang et al., Pure Appl. Chem. (86), 191-202 (2014) [4] Matsarskaia et al., J. Phys. Chem. B (120), 7731-6 (2016) [5] Matsarskaia et al., Phys. Chem. Chem. Phys. (20), 27214-25 (2018) [6] Matsarskaia et al., J. Phys. Chem. B Article ASAP, DOI: 10.1021/acs.jpcb.8b10725 (2019

Multivalent ions for tuning the phase behaviour of protein solutions

Protein phase behaviour is of importance in various areas of research such as structural biology, rational drug design and delivery, medicine (in particular protein condensation diseases), biotechnology, food science and cell biology. A particularly intriguing variety of phase behaviours can be induced in negatively charged, globular proteins in the presence of multivalent salts such as lanthanide (Ln) chlorides. These behaviours include reentrant condensation, crystallisation and cluster formation as well as liquid-liquid phase separation (LLPS) into a protein-rich and a protein-poor phase. LLPS can occur upon a temperature decrease or increase, which is referred to as an upper or a lower critical solution temperature (UCST- and LCST-LLPS), respectively. In the present thesis, the complex phenomenon of LCST-LLPS in systems of bovine serum albumin (BSA) and multivalent salts is investigated from different perspectives including thermodynamic, (non-)equilibrium and spectroscopic studies in a challenging set of experiments. In the first part of this thesis, the rather unusual phenomenon of LCST-LLPS in aqueous systems consisting of BSA and yttrium chloride (YCl3) is characterised thermodynamically. Surface charge (zeta potential) and isothermal titration calorimetry (ITC) measurements show LCST-LLPS to be a hydration entropy-driven condensation. As the Y3+ cations bind to negatively charged residues on the protein surface and bridge protein molecules, highly ordered water structures around both the surface residues as well as the cations break up and water molecules are released into the bulk solution. This leads to an increase of the overall entropy of the system. Starting from the Y3+-induced LCST-LLPS described above, the second part of this thesis is concerned with the influence that the nature of the multivalent cations used has on this phase transition. The experiments focus on the three multivalent salts HoCl3, YCl3 and LaCl3. Temperature-controlled UV-Vis absorbance measurements demonstrate that the transition temperature T_trans separating homogeneous from phase-separated states of the BSA-salt systems shifts to lower values when HoCl3 is used. In contrast, using LaCl3 leads to higher T_trans values. YCl3, used as a reference system, leads to intermediate T_trans. These findings indicate that the interprotein interactions induced by HoCl3 are much stronger than those induced by LaCl3 or YCl3. Importantly, this finding is corroborated by synchrotron small-angle X-ray scattering (SAXS) data which show the reduced second virial coefficient B_2/B_2(HS) to be lowest in BSA-HoCl3 systems, again pointing towards a stronger interprotein attraction induced by this salt. Zeta potential measurements confirm that Ho3+ has a stronger affinity to BSA than Y3+ and La3+. The overall protein-protein and protein-cation interaction strengths can therefore be ranked according to the order Ho3+ > Y3+ > La3+. Taking into account their various characteristics such as radius, electron configuration and, importantly, hydration behaviour, multivalent cations are thus shown to be a sensitive tool to fine-tune protein interactions and their resulting phase behaviours in solution. Having established the influence of cation characteristics on BSA phase behaviour, the third part is concerned with the kinetics of LCST-LLPS of BSA in the presence of varying ratios of HoCl3 and LaCl3. Using synchrotron ultra-small-angle X-ray scattering (USAXS), it is found that with an increasing HoCl3 concentration --- i.e., with increasingly attractive BSA-BSA interactions --- the growth behaviour of the characteristic system length χ(t,T) is more likely to deviate from the χ~t^{1/3} growth law. A stronger interprotein attraction, moreover, leads to arrested states at lower temperatures. The results imply that both temperature and the overall cation-mediated protein-protein interaction strength can be used to obtain multidimensional control over the kinetics of LLPS in the BSA-cation systems used. In the final part, the mechanism behind LCST-LLPS is investigated spectroscopically on the molecular scale. To this end, the change in the coordination number (CN) of Y3+ cations in BSA solutions is monitored using extended X-ray absorption fine structure (EXAFS) spectroscopy. Applying this method to protein-poor and protein-rich phases, it can be shown that the CN of Y3+ is higher in the protein-poor than in the protein-rich phase. This is attributed to the fact that in the protein-poor phase more Y3+ cations are surrounded by hydration shells and not bound to BSA or forming cation bridges between BSA molecules. The results obtained using EXAFS align well with the current rationalisation of LCST-LLPS as a hydration entropy-driven phenomenon. The results obtained indicate that a careful choice of the multivalent cation used can fine-tune protein interactions and their phase behaviour in solution. In addition, EXAFS data provide atom-level insights into the mechanism of LCST-LLPS. These findings are of strong interest not only for a fundamental understanding of protein and soft matter thermodynamics, but are also potential anchoring points for the design of stimuli-responsive “smart” materials based on polymers, colloids or proteins

Saccharomyces cerevisiae single-copy plasmids for auxotrophy compensation, multiple marker selection, and for designing metabolically cooperating communities

Auxotrophic markers are useful tools in cloning and genome editing, enable a large spectrum of genetic techniques, as well as facilitate the study of metabolite exchange interactions in microbial communities. If unused background auxotrophies are left uncomplemented however, yeast cells need to be grown in nutrient supplemented or rich growth media compositions, which precludes the analysis of biosynthetic metabolism, and which leads to a profound impact on physiology and gene expression. Here we present a series of 23 centromeric plasmids designed to restore prototrophy in typical Saccharomyces cerevisiae laboratory strains. The 23 single-copy plasmids complement for deficiencies in HIS3, LEU2, URA3, MET17 or LYS2 genes and in their combinations, to match the auxotrophic background of the popular functional-genomic yeast libraries that are based on the S288c strain. The plasmids are further suitable for designing self-establishing metabolically cooperating (SeMeCo) communities, and possess a uniform multiple cloning site to exploit multiple parallel selection markers in protein expression experiments

Enhancement of the mechanical properties of lysine-containing peptide-based supramolecular hydrogels by chemical cross-linking

Exposure of lysine-containing peptide-based gelators to the cross-linking agent glutaraldehyde allows tuning of gel mechanical properties. The effect of cross-linking depends on the position of the lysine residue in the peptide chain, the concentration of gelator and the conditions under which cross-linking takes place. Through control of these factors, cross-linking leads to increased gel strength

Strikingly Different Roles of SARS-CoV-2 Fusion Peptides Uncovered by Neutron Scattering.

Funder: National Collaborative Research Infrastructure Strategy (NCRIS)Funder: ANR/NSF-PIREFunder: Science and Technology Facilities CouncilFunder: Institut Laue LangevinCoronavirus disease-2019 (COVID-19), a potentially lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of selected SARS-CoV-2 Spike fusion peptides, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin-echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium, however, reorients the peptide back to the lipid leaflet closest to the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain, which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed

Strikingly Different Roles of SARS-CoV-2 Fusion Peptides Uncovered by Neutron Scattering.

Funder: National Collaborative Research Infrastructure Strategy (NCRIS)Funder: ANR/NSF-PIREFunder: Science and Technology Facilities CouncilFunder: Institut Laue LangevinCoronavirus disease-2019 (COVID-19), a potentially lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of selected SARS-CoV-2 Spike fusion peptides, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin-echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium, however, reorients the peptide back to the lipid leaflet closest to the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain, which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed

Soft matter roadmap

Soft materials are usually defined as materials made of mesoscopic entities, often self-organised, sensitive to thermal fluctuations and to weak perturbations. Archetypal examples are colloids, polymers, amphiphiles, liquid crystals, foams. The importance of soft materials in everyday commodity products, as well as in technological applications, is enormous, and controlling or improving their properties is the focus of many efforts. From a fundamental perspective, the possibility of manipulating soft material properties, by tuning interactions between constituents and by applying external perturbations, gives rise to an almost unlimited variety in physical properties. Together with the relative ease to observe and characterise them, this renders soft matter systems powerful model systems to investigate statistical physics phenomena, many of them relevant as well to hard condensed matter systems. Understanding the emerging properties from mesoscale constituents still poses enormous challenges, which have stimulated a wealth of new experimental approaches, including the synthesis of new systems with, e.g. tailored self-assembling properties, or novel experimental techniques in imaging, scattering or rheology. Theoretical and numerical methods, and coarse-grained models, have become central to predict physical properties of soft materials, while computational approaches that also use machine learning tools are playing a progressively major role in many investigations. This Roadmap intends to give a broad overview of recent and possible future activities in the field of soft materials, with experts covering various developments and challenges in material synthesis and characterisation, instrumental, simulation and theoretical methods as well as general concepts