1. DESIGN AND CHARACTERIZATION OF SYMMETRIC HALF BARRELS
Will Proffitt
RosettaCon July 22nd, 2008

2. Outline Introduction Design of Symmetric Half Barrels
Characterization of Symmetric Half Barrels
Future Directions

3. 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?

4. 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 ( )

5. 4-fold Symmetry of TIM BarrelsC A D
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 b-barrel

6. Outline Introduction Design of Symmetric Half Barrels
Characterization of Symmetric Half Barrels
Future Directions

7. Design Protocol

8. Imidazole Glycerol Phosphate Synthase
The TIM-Barrel protein with highest degree of symmetry in primary, secondary, and tertiary structure known
1thf (HisF from Thermotoga maritima Å )
Rosetta Minimized Native Structure gives REU per AA
Lang, D.A.,  Wilmanns, M.
1thf

9. 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

10. Energy Minimization of 62 Symmetric Variants of 1thf
REU per AA
start – end AA of original sequence

11. 94_215 (FLR) has 242 AA with -3.16 REU per AA-5 +5
REU
SQAVVVAIDAKRVDGEFMVFTYSGKKNTGILLRDWVVEVEKRGAGEILLTSIDRDGTKSGYDTEMIRFVRPLTTLPIIASGGAGKMEHFLEAFLRGADKVSINTAPSLITQIAQTFG
H 1/5
S 1/5
S 4/8
S 3/7
S 2/6
H 2/6
H 3/7
H 4/8

12. Outline Introduction Design of Symmetric Half Barrels
Characterization of Symmetric Half Barrels
Future Directions

13. 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)
The gene was constructed by amplifying the corresponding fragments from the wild-type 1thf plasmid. A concatenation protocol was used to duplicate the first half of the symmetric gene, which was then inserted into a t7/lac expression vector pBG100 (a pET27b derivative) which adds a 3C protease cleavable 6-His tag to the N-terminus of the protein. The secondary structure of the protein is confirmed by circular dichroism. FLR CD signals are nearly identical to 1thf and, as predicted. are a linear combination of the standard curves for alpha helix (minima at 208 and 222, maximum at 190) and beta sheet (minimum at 215, maximum at 195). The hydrophobic dye ANS was used to probe how well the protein is folded. A poorly packed core exposes hydrophobic sidechains which bind the dye, and give rise to large fluorescence emission signals (550nm). The use of denaturants (GuHCl) to unfold the protein show an increase in fluorescence corresponding to protein unfolding, indicating a well packed core. 1thf, the positive control, showed an identical pattern of fluorescence signal increase. Similarly a Trp in a well-packed core environment will exhibit characteristic emission profiles (Excite at 295nm Buried Trp fluoresces at 330nm, Solvent exposed Trp fluoresces between 340 and 350nm), and both FLR and 1thf demonstrated the characteristics of a buried Trp residues.

14. 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-red
HisF-blue

15. 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
Use of denaturant to determine transition from folded to unfolded state
A steep sigmoidal curve indicates cooperative unfolding and the native protein adopts a compact, well-folded structure
Compare to HisF
DLS measures the protein size distribution in solution, and FLR compared well to the predicted value from the Rosetta model)
Analytical SEC is standardized within the MW range of our protein and we find that FLR elutes at a time which corresponds to protein of approx. 30kDa (predicted is 28.8kDa)
Degradation was monitored over a period of 2 weeks at room temp by SDS-PAGE

16. Diffracting Crystals (In-house Trials)
1thf original crystallization conditions
0.1M Citrate pH 5.6, 1.0M Ammonium Phosphate
Vary protein/precipitant
concentration
The original conditions used by Lang et al. to crystallize 1thf were used as the basis for a screen which varied protein concentration (13-16mg/ml) and precipitant conc. ( M NH4PO4) in hanging drop wells. Hexagonal crystals were produced at 14-16mg/ml and M NH4PO4. The crystal shown diffracted out to 2.45 angstroms. However, as indicated by the diffraction pattern shown, the crystal is apparently multiple crystal plates stacked into the hexagonal shape (this is apparent because in several places on the diffraction pattern, the spots (reflections) are not sharp but appear to be 2 reflections slightly offset from one another, indicating more than one crystal form within a single crystal). This can be remedied by several optimization strategies which we are currently using.

17. 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)
In addition to attempting to macro or micro seed the hexagonal plates in order to obtain a single crystal form, we have also begun to screen a broader set of conditions in hopes of finding a better condition. 6 additional high quality crystals have been produced, and we are in the process of reproducing these conditions and checking the diffraction. We are also prepared to initiate NMR assignment experiments if it becomes too difficult to obtain a crystal structure.

18. Acknowledgements Carie Fortenberry and Jens Meiler
Brent Dorr, Beth Repasky, and Gillian Treadwell
Laura Mizoue
The rest of the Meiler lab

19. Near-UV CD
Near-UV (CD) signals arise from aromatic sidechains and disulfides
Their CD signals are sensitive to overall tertiary structure
Phe nm, Tyr nm, Trp nm
Trp>Tyr>Phe

20. ANS 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, ANS, 6M GuHCl
Protein, ANS, 4M GuHCl
Protein, ANS, 2M GuHCl
Protein + ANS
ANS Only
Protein Only

21. Intrinsic 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
FLR alone
FLR + 2-6M GuHCl
HisF alone
HisF + 2-6M GuHCl

22. Does it Crystallize? Outsourcing Commercial Screens
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

23. Crystal Optimization Seeding Plates Screen Woodward crystal conditions
Use a diffracting crystal to “seed” growth of large, single crystal
Streaking, Macroseeding, Microseeding, etc.
Screen Woodward crystal conditions
Collect a full data set!