Protein Crystallization in the Presence of a Metastable Liquid–Liquid Phase Separation

Maier, Ralph; Zocher, Georg; Sauter, Andrea; Vela, Stefano Da; Matsarskaia, Olga; Schweins, Ralf; Sztucki, Michael; Zhang, Fajun; Stehle, Thilo; Schreiber, Frank

doi:10.1021/acs.cgd.0c01219

Cited by 25 publications

(40 citation statements)

References 73 publications

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“…59 The protein binding sites of these cations are well studied for HSA. 60 Due to the similarity of the HSA structure to that of BSA (∼ 76%), 52,61 comparable binding sites and thus binding mechanisms, of the multivalent cations can be assumed, which are solely dened by acidic (negatively charged) surface-exposed groups. Thus, the binding mechanism between cations and proteins can be rationalised by electrostatic interactions.…”

Section: Phase Diagramsmentioning

confidence: 99%

Bulk Phase Behavior vs Interface Adsorption: Specific Multivalent Cation and Anion Effects on BSA Interactions

et al. 2021

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Proteins are ubiquitous and play a critical role in many areas from living organisms to protein microchips. In humans, serum albumin has a prominent role in the foreign body response since it is the rst protein which will interact with e.g. an implant or stent. In this study, we focused on the inuence of salts (i.e., dierent cations (Y 3+ , La 3+ ) and anions (Cl − , I − )) on bovine serum albumin (BSA) in terms of its bulk behaviour, as well as its role of charges for the protein adsorption at the solid-liquid interface in order to understand and control the underlying molecular mechanisms and 1 interactions. This is part of our group's eort to gain a deep understanding of proteinprotein and protein-surface interactions in the presence of multivalent ions. In the bulk, we found not only multivalent cation-triggered phase transitions, but also a dependence on the anions. The induced attractive interactions were observed to increase from Cl − < NO − 3 < I − , resulting in iodide preventing re-entrant condensation and promoting liquidliquid phase separation in bulk. Using ellipsometry and a quartz-crystal microbalance with dissipation (QCM-D), we obtained insight into the growth of the protein adsorption layer thickness. Importantly, we found that phase transitions at the substrate can be triggered by certain interface properties, whether they exist in the bulk solution or not.Through the use of a hydrophilic, negatively charged surface (SiO 2 ), the direct binding of anions to the interface was prevented. Interestingly, this led to re-entrant adsorption even in the absence of re-entrant condensation in bulk. However, the overall amount of adsorbed protein was enhanced through stronger attractive protein-protein interactions in the presence of iodide salts. These ndings illustrate how carefully chosen surface properties and salts can directly steer the binding of anions and cations, which guide protein behaviour, thus paving the way for specic/triggered protein-protein, proteinsalt, and protein-surface interactions.

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Section: Phase Diagramsmentioning

confidence: 99%

Bulk Phase Behavior vs Interface Adsorption: Specific Multivalent Cation and Anion Effects on BSA Interactions

et al. 2021

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“…This deviation becomes stronger for higher protein concentrations and indicates that protein–protein interactions are stronger and the system is more attractive . However, a substantial broadening of regime II when H 2 O is replaced by D 2 O, as reported recently for bovine and human serum albumin solutions in the presence of trivalent ions, is not observed for BLG. , …”

Section: Resultsmentioning

confidence: 66%

“…In the BLG-CdCl 2 system, gel-like aggregates are formed in the condensed regime II instead of LLPS. , The metastable aggregates develop a correlation peak at q ≈ 0.075 Å –1 in SANS and SAXS measurements, which is close to the first Bragg peak at q = 0.1 Å –1 , indicating the preordered nature of this MIP. In our previous work on the system HSA with CeCl 3 , a metastable LLPS with respect to crystallization was found . Thus, dense liquid droplets were identified as metastable intermediates between solution and crystals .…”

Section: Discussion On the Role And The Development Of The Mipmentioning

confidence: 90%

“…Thus, dense liquid droplets were identified as metastable intermediates between solution and crystals . However, these droplets seem to act only as a reservoir rather than precursors and no structural feature such as a correlation peak was observed in SAXS/SANS data . In BLG with ZnCl 2 solutions, amorphous aggregates form initially in regime II, but they relax into a dense liquid state.…”

Section: Discussion On the Role And The Development Of The Mipmentioning

confidence: 99%

“…We have previously shown that trivalent salts such as YCl 3 or CeCl 3 can induce a reentrant condensation (RC) phase behavior in many acidic proteins like β-lactoglobulin (BLG) or human serum albumin (HSA). − Interestingly, depending on the position in the phase diagram, classical crystallization or a nonclassical crystallization process have been observed. − , In a previous study, , the two-step nucleation process of protein crystallization in solutions was investigated by following the overall crystallization kinetics using real-time optical microscopy and small-angle X-ray scattering (SAXS). , For BLG-salt (CdCl 2 ) solutions in H 2 O at the transition zone of pseudo- c **, small aggregates formed after sample preparation. These protein aggregates were identified as a metastable intermediate phase (MIP) during crystallization.…”

Section: Introductionmentioning

confidence: 99%

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Protein Crystallization from a Preordered Metastable Intermediate Phase Followed by Real-Time Small-Angle Neutron Scattering

Maier

Sohmen

Vela

et al. 2021

Crystal Growth & Design

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We present a systematic study using real-time small-angle neutron scattering (SANS) and optical microscopy to follow the protein crystallization process in the presence of a metastable intermediate phase (MIP). Using bovine β-lactoglobulin (BLG) in the presence of the divalent salt CdCl2 as a model system, we first determine the experimental phase behavior in D2O. The protein solutions become turbid after crossing the first threshold salt concentration c*, and upon further increasing the salt concentration, the solutions become less turbid but not completely clear again. Thus, the second border is called pseudo-c**. Near pseudo-c**, crystallization follows a nonclassical process with a MIP, which is further explored with a focus on the structural evolution and the growth kinetics of the MIP prior to crystal nucleation. Real-time SANS measurements show that a correlation peak develops inside the MIP, and its peak position shifts to higher q-values with time, finally stabilizing at a characteristic length scale of d MIP ≈ 84 Å. The area of this peak (proportional to the amount of MIP in the sample) increases with time first, reaches a maximum, and then decreases quickly upon crystallization due to consumption by crystal growth. The evolution of the correlation peak indicates a “preordering” nature of the MIP as precursors of crystal nucleation, which lowers the nucleation barrier for subsequent crystallization. These results of structural evolution and the role of MIPs during a nonclassical crystallization process may be relevant for other fields ranging from structural biology to pharmacy.

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Metastable Liquid–Liquid Phase Separation and Aging Lead to Strong Processing Path Dependence in Mini‐Spidroin Solutions

Fedorov,

Sammalisto,

Harmat

et al. 2024

Adv Funct Materials

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Recombinant silk proteins provide a route toward sustainable and biocompatible materials. For making such materials, the assembly process from dilute protein into a functional material is central. The assembly mechanism in engineered materials is by necessity different from the natural ones—this poses challenges but also opens opportunities for scaling up and for developing novel properties. The phase behavior of a mini‐spidroin, NT‐2Rep‐CT is studied, which is a widely studied variant of recombinant silk. NT‐2Rep‐CT can be triggered to assemble by lowering the pH, but even at high pH—considered as storage conditions—it can be in various states, such as forming condensates, clusters, gels, and soluble protein. It is shown how its assembly phases evolve through both metastable and dynamically arrested states. The observed behavior of silk protein solutions is highly complex, and elements thereof from phase diagrams associated with polymers, colloidal systems, and globular proteins are found. Based on the characterization of cluster formation and structural intermediates, a minimalist phase diagram is proposed for NT‐2Rep‐CT and argues that the understanding and insight into silk assembly via its phase behavior, and especially the arrested states, is central for designing recombinant silk proteins and their processing for materials applications.

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Protein Crystallization in the Presence of a Metastable Liquid–Liquid Phase Separation

Cited by 25 publications

References 73 publications

Bulk Phase Behavior vs Interface Adsorption: Specific Multivalent Cation and Anion Effects on BSA Interactions

Bulk Phase Behavior vs Interface Adsorption: Specific Multivalent Cation and Anion Effects on BSA Interactions

Protein Crystallization from a Preordered Metastable Intermediate Phase Followed by Real-Time Small-Angle Neutron Scattering

Metastable Liquid–Liquid Phase Separation and Aging Lead to Strong Processing Path Dependence in Mini‐Spidroin Solutions

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