Christine Meyer Senior Seminar Presentation

Protein Crystallization:

A Method to Study Phenylalanine

Mutation Effects on Protein Efficiency

Presented By: Christine Meyer

Mentor: Dr. Steven Berry

Department of Chemistry & Biochemistry

Spring 2013 Senior Symposium

Protein Design: A Mechanism to Explore Electron Transfer Proteins

• Electron transfer reactions are observed widely through out biological systems– Photosynthesis

– Respiration

– Nitrogen-fixation

• Mutate specific residues to mimic native proteins and explore how subsequent structural changes effect protein functionality

Image: Biology, by Kenneth R. Miller and Joseph Levine, ©2002 by Pearson Education, Inc.

Azurin: A Member of the Blue Copper Protein Family

• Belongs to class of oxidoreductases

• Type 1 Blue Copper Proteins

• Large reduction potential variation between proteins despite similar Cu2+ binding active sites

Protein Redox Potential mV

Stellacyanin 180

Azurin 310

Rusticyanin 680 Azurin Active Site: Bound Cu2+

His 46

Cys 112

Gly 45 His 117

Met 121

Azurin versus Rusticyanin

Azurin (4AZU): 6 Phenylalanine

residues, 2 within 8Å of Cu2+ binding siteRusticyanin (1RCY): 10 Phenylalanine

residues, 5 within 8Å of Cu2+ binding site

• Phenylalanine creates a hydrophobic environment around the active site

Azurin Phenylalanine Mutant Series

• Single Mutants– Leu33Phe

– Met44Phe

– Leu86Phe

• Double Mutant:– Leu33Phe & Met44Phe

– Met44Phe & Leu86Phe

• Triple Mutant:– Leu33Phe, Met44Phe,

Leu86PheS.M. Berry et al. Journal of Inorganic Biochemistry 104 (2010) 1071-1078

Residue Substitution ΔE, mV (±5)

ΔLeu33Phe

WTL33F 12

M44FL33F/M44F 10.5

M44F/L86FL33F/M44F/L86F 16.5

ΔMet44Phe

WTM44F 32.5

L33FL33F/M44F 31

L33F/L86FL33F/M44F/L86F 39

ΔLeu86Phe

WTL86F 27.5

L33FL33F/L86F 27

L33F/M44F L33F/M44F/L86F 35

• Key residues in secondary coordination sphere were mutated to Phe to resemble native Rusticyanin secondary coordination sphere

Experimental Procedure

• Grow crystals to identify structural reason for reproducible redox potential change upon introduction of Phe residue

• Begin by growing Phe Azurin crystals using the hanging drop method

Growing Phe Azurin Crystals• Crystal Box: The Hanging Drop method

Hanging Drop Method for Crystal Growth

Cover Slip

2μL protein + 2μL reservoir solution

SiliconeGrease

Reservoir

Solution: 1000μL

• Vary reservoir conditions to optimize crystal growth

Crystal Box VariationsCondition Variations

Buffer Tris HCl Imidazole Ammonium Acetate

pH Range: 3.8 to 8.0

Precipitating Agent

50% PEG – 2K, 4K, 8K

Salt LiNO3 or CaCl2

Temperature 20°C or 4°C

Crystal Box Reservoir Variations

Decreasing [50%-PEG]

Increasing [Salt]

30% 26% 22% 18% 14% 10%

0.03 M

0.09 M

0.15 M

0.21 M

• 100 mM Tris-HCl, 5 mM CuSO4, plus varied PEG and CaCl2

Crystal Pictures

Increasing [CaCl2]

Decreasing [50%-PEG]

• Less PEG resulted in slower crystal growth• Varying CaCl2 resulted in different crystal sizes

Example of Ordered Crystals

Dehydration: Improved Crystal Order• A technique used to decrease mosaicity

– Mosaicity: skewed arrangement of molecules

– Before Dehydration: Range 1.0-1.5°

– After Dehydration: Range 0.3-0.5°

Before Dehydration, high mosaicity

After Dehydration,low mosaicity

H2O

H2O

H2O

H2O

+ Glycerol

& PEG

Crystal Pinning

• Pin crystal on loop

• Flash freeze in liquid nitrogen

• Store in liquid nitrogen

Crystal Diffraction: Rigaku Rapid II©

Nitrogen Stream

Pinned CrystalCollimator

Cu Source

Crystal Screening: Dozens of Crystals

• Screening Process:

– Collect two frames

• 5 min exposures, 0.5° rotation, 90°frames

– Measure Resolution

• Determine how far spots diffracted (<2.0 Å)

– Measure Mosaicity

• <0.5°

2.0 Å1.41 Å

Collecting Data on the Best Crystal

• Collect 400 to 500 frames

– Expose 20 minutes, 0.5° rotation

– Rotate 0.5°

– Repeat 400-500 times

Image Plate

Nitrogen Stream

Collimator Pinned Crystal

Goniometer

Solving the Crystal Structure

• CrystalClear© Software for integration

• CCP4© for molecular replacement and refinement

• Coot© for manipulating to fit electron density

CrystalClear Software CCP4 Coot

Solved Phe Azurin Crystal Structures

Wild Type1 Double Mutant:

Met44Phe & Leu86Phe

Triple Mutant: Leu33Phe, Met44Phe, Leu86Phe

Unit Cell Orthorhombic Orthorhombic Orthorhombic

Dimensions 57.65 x 80.93 x 110.17 Å

48.55 x 53.65 x 97.15 Å

48.46 x 53.45 x 97.43 Å

Resolution 1.93 Å 1.41 Å 1.43 Å

Space Group

P212121C2221

C2221

R factor 15.5% 18.5% 15.8%

1Journal of Molecular Biology, Volume 221, Issue 3, 5 October 1991, Pages 765-772

Backbone Structural Differences among

Phenylalanine Series

Wild Type

Double Phe Mutant: M44F L86F

Triple Phe Mutant: L33F M44F L86F

33

86

44

Residue Substitution

ΔE, mV (±5)

ΔLeu33Phe 13

ΔMet44Phe 34.2

ΔLeu86Phe 29.8

Ligand Distances & Water Content around the Cu2+ Active Site

Gly 45

His 117

His 46

Cys 112

Met 121

• Measure ligand to Cu2+ distances and water content to find variation among structures

Structural differences between MutantsWild Type (PDB ID –

4AZU)

Double Mutant:

Met44Phe & Leu86Phe

Triple Mutant: Leu33Phe, Met44Phe, Leu86Phe

Water Content: within 8 Å

2 Molecules 4 Molecules 3 Molecules

His 46: nitrogen 2.0 Å 2.0 Å 2.0 Å

His 117: nitrogen 2.1 Å 2.0 Å 2.0 Å

Cys 112: sulfur 2.3 Å 2.2 Å 2.2 Å

Met 121: sulfur 3.2 Å 3.4 Å 3.3 Å

Gly 45: oxygen (carbonyl group)

2.8 Å 3.0 Å 3.1 Å

Gly 45: carbonyl group to Cu2+ angle

133.9° 129.9 ° 133.2 °

• Copper active sites are the same for the three proteins

Conclusions• Purified and grew crystals of the double and

triple Phe azurin mutants

• Screened crystals using Rigaku Rapid II©

diffractometer

• Solved crystal structures of triple and double Phe mutant

• Compared and contrasted differences between wild-type Azurin and Phe mutants

– Backbone disruption of H-bonding network

– Water content near Cu2+

– Carbonyl ligand bonding angle

Future Directions

• Crystallize and determine structure of WT

– Purify and crystallize WT azurin

• Determine structure of single Phe azurin mutants

– Currently screening crystals of Leu86Phe

– Purify and crystallize Met44Phe azurin

• Computational analysis of dipole distribution

Acknowledgements• Dr. Berry

• Melanie Ladd

• Sarah Pedersen

• Dr. Nemykin

• Dr. Carter

• University of Minnesota Undergraduate Research Opportunities Program

• Department of Chemistry and Biochemistry

Reservoir Conditions• Buffer

• Salt

• Precipitating agent

• Copper Source

• Filtered Millipore water

Buffer: 2.0M Tris HClpH 7.07

Salt: 3.0 M CaCl2 Precipitant: 50% PEG-2000Copper Source:

0.1 M CuSO4Millipore H2O

A1 50 µL (0.1 M) 10 µL (0.03M) 600 µL (30%) 50 µL (5.0 mM) 315 µL

A2 50 µL (0.1 M) 10 µL (0.03M) 520 µL (26%) 50 µL (5.0 mM) 395 µL

A3 50 µL (0.1 M) 10 µL (0.03M) 440 µL (22%) 50 µL (5.0 mM) 475 µL

A4 50 µL (0.1 M) 10 µL (0.03M) 360 µL (18%) 50 µL (5.0 mM) 555 µL

A5 50 µL (0.1 M) 10 µL (0.03M) 280 µL (14%) 50 µL (5.0 mM) 635 µL

A6 50 µL (0.1 M) 10 µL (0.03M) 220 µL (10%) 50 µL (5.0 mM) 715 µL

Sample Conditions:

Buffer: 2.0M Tris HCl pH 7.07

Salt: 3.0 M CaCl2 Precipitant: 50% PEG-2000Copper Source:

0.1 M CuSO4Millipore H2O

B1 50 µL (0.1 M) 30 µL (0.09M) 600 µL (30%) 50 µL (5.0 mM) 295 µL

B2 50 µL (0.1 M) 30 µL (0.09M) 520 µL (26%) 50 µL (5.0 mM) 375 µL

B3 50 µL (0.1 M) 30 µL (0.09M) 440 µL (22%) 50 µL (5.0 mM) 455 µL

B4 50 µL (0.1 M) 30 µL (0.09M) 360 µL (18%) 50 µL (5.0 mM) 535 µL

B5 50 µL (0.1 M) 30 µL (0.09M) 280 µL (14%) 50 µL (5.0 mM) 615 µL

B6 50 µL (0.1 M) 30 µL (0.09M) 220 µL (10%) 50 µL (5.0 mM) 695 µL

Protein Purification

• Grow E. coli containing a plasmid that codes for Phe mutants

• Use osmotic shock to separate cells from protein

• Purify the protein through SP-sepharous (cation-exchange column) and Q-column (anion-exchange column)

• Titrate with copper

• Purify through gel size exclusion column

Double Phenylalanine Mutant

• Residues Met44 and Leu86 to Phe

• Both mutations within 8Å of active site

Phe 86

Phe 44