DESIGN AND DESIGN AND CHARACTERIZATION OF CHARACTERIZATION OF SYMMETRIC HALF BARRELSSYMMETRIC HALF BARRELSWill ProffittWill ProffittRosettaCon July 22RosettaCon July 22ndnd, 2008 , 2008

Outline

Introduction Design of Symmetric Half Barrels Characterization of Symmetric Half

Barrels Future Directions

Introduction

Why explore symmetric design? Advancements in the design

of larger, more complex proteins

Potential scaffold for enzyme design

Common theme in nature, evolutionary pressure?

Structurally Symmetric Superfolds?

Almost all tertiary structures can be categorized into 1 of 10 fundamental protein folds

6 of the 10 fundamental superfolds are symmetric (αβ-plait, TIM-barrel, β-trefoil, “jelly roll,” IG-like, and “up-down”)

Images taken from CATH (www.cathdb.info )

4-fold Symmetry of TIM Barrels

(A) Topology of the eight repeating units

(B) Schematic of barrel structure

(C) Ribbon diagram of HisF (1thf)

(D) Cartoon representations of the central -barrel

Outline Introduction Design of Symmetric Half Barrels Characterization of Symmetric Half

Barrels Future Directions

Design Protocol

The TIM-Barrel protein with highest degree of symmetry in primary, secondary, and tertiary structure known

1thf (HisF from Thermotoga maritima - 1.45Å - 2000)

Rosetta Minimized Native Structure gives -3.00 REU per AA

Imidazole Glycerol Phosphate Synthase

1thf

Structural Superimposition of 1thf Half Barrels

1thf: 3.40Å over 202 AA

62 positions within α-helices and β-strands superimpose spatially with high accuracy → 62 symmetric variants

Energy Minimization of 62 Symmetric Variants of 1thf

start – end AA of original sequence

REU per AA

94_215 (FLR) has 242 AA with -3.16 REU per AA

SQAVVVAIDAKRVDGEFMVFTYSGKKNTGILLRDWVVEVEKRGAGEILLTSIDRDGTKSGYDTEMIRFVRPLTTLPIIASGGAGKMEHFLEAFLRGADKVSINTAPSLITQIAQTFG

1 21 41 61 81 101 121

H 1/5S 1/5 S 4/8S 3/7S 2/6 H 2/6 H 3/7 H 4/8

-5

+5

REU

Outline Introduction Design of Symmetric Half Barrels Characterization of Symmetric Half

Barrels Future Directions

Does it express and is it folded?

Expression vector adds a cleavable, hexa-histidine tag Expresses at 20mg/L induction in BL21 pLysS

cells (37°C in LB media) Circular dichroism (CD) ANS fluorescence

Under native and denaturing conditions Intrinsic Fluorescence 2-Dimensional NMR (1H-15N HSQC)

1H-15N HSQC NMR Indicates Proper Folding

Provides the most insight into whether the fold is native-like

Each residue gives rise to a single peak

Sharp, well-dispersed peaks indicate proper folding

Compare to HisF Approx. half the

number of peaks Peaks overlay Similar dispersion

FLR-redHisF-blue

Is FLR Stable and Monomeric?

Fold Stability Chemical Denaturation

Oligomeric State Dynamic Light Scattering

FLR estimated at 50±20Å (compared to predicted of 54Å)

Size Exclusion Chromatography FLR elutes as monomeric, ~30kDa protein

FLR is not Prone to Degradation or Precipitation

Initiate structural determination

Diffracting Crystals (In-house Trials)

1thf original crystallization conditions 0.1M Citrate pH 5.6, 1.0M Ammonium

Phosphate Vary protein/precipitant

concentration

Future Directions

Continue seeking optimal crystal conditions for 94_215 (FLR)

NMR Assignments? Explore Symmetric Quarter Barrel

Designs of 1v5x (Will Proffitt) Continue characterizing alternate half-

barrel designs (Gillian Treadwell)

Acknowledgements

Carie Fortenberry and Jens Meiler Brent Dorr, Beth Repasky, and

Gillian Treadwell Laura Mizoue The rest of the Meiler lab

Near-UV CD Near-UV CD Near-UV (CD) signals arise from aromatic sidechains and disulfides Their CD signals are sensitive to overall tertiary structure

Phe 250-270 nm, Tyr 270-290 nm, Trp 280-300 nm Trp>Tyr>Phe

ANS FluorescenceANS Fluorescence

ANS- hydrophobic dye used to probe the protein’s surface

Fluorescence increases as the dye binds hydrophobic patches

Monitor fluorescence under native and denatured conditions Protein Only

ANS OnlyProtein + ANS

Protein, ANS, 2M GuHClProtein, ANS, 4M GuHClProtein, ANS, 6M GuHCl

Intrinsic FluorescenceIntrinsic Fluorescence

Probes Trp environment Excite at 295nm Buried Trp fluoresces at 330nm Solvent exposed Trp fluoresces between 340 and 350nm HisF contains 1 Trp,

while FLR has 2

HisF alone

HisF + 2-6M GuHCl

FLR + 2-6M GuHClFLR alone

Does it Crystallize? Does it Crystallize? Outsourcing

Screen 1000’s of conditions for low cost Pros- High-throughput, low commitment Cons- Difficulty to reproduce in-house

Commercial Screens Premixed conditions (96 per kit) Pros- Medium-throughput, high reproducibility Cons- Can be “too narrow,” require multiple

kits Previously proven conditions

Crystal OptimizationCrystal Optimization

Seeding Plates Use a diffracting crystal to “seed” growth of

large, single crystal Streaking, Macroseeding, Microseeding,

etc. Screen Woodward crystal conditions Collect a full data set!