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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