Fajun Zhang, Roland Roth, Marcell Wolf, Felix Roosen-Runge, Maximilian W. A. Skoda, Robert M. J. Jacobs, Michael Stzuckie

and Frank Schreiber

Universität Tübingen, Institut für Angewandte Physik - 1st December 2011

Soft Matter, 2012, 8, 1313

Stijn KoshariChemical and Biomolecular Engineering

/352OVERVIEW

/353OVERVIEW

/354OVERVIEW

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0. Overview 1. Introduction 2. Approach 3. SAXS 4. Discussion 5. Crystallization 6. Conclusion

5CHARGE-CONTROLLED LLPS

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Charge-controlled phase separationMetastable liquid-liquid phase separation (LLPS)

6CHARGE-CONTROLLED LLPS

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Protein solutions stabilized by (DLVO):◦ van der Waals’ forces◦ Electrostatic forces (surface charges)

Change of ion concentration in solution can affect surface charge Effective interactions

Hence, may lead to aggregation Compare to colloids, but more complex

7INTRODUCTION

/358INTRODUCTION

F. Zhang, et al. Phys. Rev. Lett., 2008, 101, 148101.

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Two liquid phases:◦ Protein-rich Condensation (gel)◦ Protein-poor Crystallization

Metastable

9INTRODUCTION

/3510INTRODUCTION

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Two liquid phases:◦ Protein-rich Condensation (gel)◦ Protein-poor Crystallization

Metastable:◦ Merge and grow◦ Density fluctuations

Important phenomenon:◦ Universal, but not often observed◦ Prerequisite for protein aggregation diseases◦ Crystallization (controlled)

11INTRODUCTION

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MaterialsMethods

12CHARGE-CONTROLLED LLPS

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Protein: ◦ Human serum albumin (HSA)◦ Globular medium sized (67kDa)◦ Negatively charged

Salt:◦ Yttrium chloride YCl3◦ Trivalent salt◦ Cation binds to acidic residues (Asp, Glu)◦ Charge inversion

13APPROACH

/3514APPROACH

F. Zhang, et al. Phys. Rev. Lett., 2008, 101, 148101.

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Determine phase diagram for a fixed temperature

Vary protein and salt concentration Observation techniques◦ Optical microscopy◦ X-ray and ultra-violet light absorption◦ Small-angle X-ray spectroscopy (SAXS)

15APPROACH

/3516APPROACH

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ConceptDilute samplesConcentrated samplesLLPS criteria

17CHARGE-CONTROLLED LLPS

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Small-angle X-ray spectroscopy X-ray EM wave interacts with orbital electrons Fluctuations in sample lead to structure Information about gross characteristics

18

A. Sali, et al. (http://www.cgl.ucsf.edu/Research/sali), (2013).

SAXS

/3519SAXS

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Particle positions are uncorrelated Form factor gives general information about

particle:◦ Low : Guinier limit Radius of gyration◦ High : Porod limit Fractal information◦ Overall shape Particle shape

20SAXS

/3521SAXS

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Particle positions are correlated(rotation, position)

Form factor gives general information about particle determine from dilute sample and remove

Structure factor gives general information about particle interactions:◦ Low Compressibility◦ Overall shape Particle distribution/structure

22SAXS

/3523SAXS

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Structure factor fitting gives information about particle interactions:◦ Assume sticky hard sphere (SHS), short ranged

square well◦ Fitted stickiness parameter is directly related to

the second virial coefficient Second virial coefficient is a measure for

strength of interactions Both and have critical values which

indicate occurrence of LLPS

24SAXS

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ResultsOrigin of interactions

25CHARGE-CONTROLLED LLPS

/3526DISCUSSION

/3527DISCUSSION

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Physical origin of short-range attraction remains to be identified:◦ van der Waals◦ Solvation◦ Structural forces

Protein-Y3+ complexes can be highly complex:◦ Non-spherical shape◦ Nonhomogeneous surface charge pattern◦ Complex hydration properties

28DISCUSSION

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Crystal growth controlCrystallization criterion

29CHARGE-CONTROLLED LLPS

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Possibility for high quality protein single crystals

Two-step or single-step procedure at same temperature

Play with ion concentration Changing temperature can further help

optimize crystallization conditions Already used

30CRYSTALLIZATION

/3531CRYSTALLIZATION

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Second virial coefficient good predictor of protein crystallization

Shown to have almost constant value at critical point

Thermodynamic criterion

32CRYSTALLIZATION

/3533CHARGE-CONTROLLED LLPS

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Use trivalent ions to control phase behavior of proteins

Consistent and comprehensive understanding Experimental and thermodynamic evidence Universal applicability

34CONCLUSION

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