bolie@ifm.liu.se www.ifm.liu.se/applphys/sensor Synthetic polypetides for materials science and biosensing Professor Bo Liedberg, Molecular Physics, Linköping University, Sweden 436 th Heraeus Seminar, Bremen, June 30, 2009
2D-, 3D- Architectures for Sensing Functional Thiols and Lipids for SAMs Lipid Architectures for Sensing De Novo designed synthetic peptides Functional Sugars for SAMs Chelators for Immobilisation ~2 nm and Arraying HH H H SH N H 15 H H H N H H H H Lars Baltzer H N SH 15 H N H SH 15 15 SH Helix Loop - Helix Swedish Research Council DPN-Nanoarray K. Enander D. Aili
Supramolecular hybrid materials Helix-Loop-Helix Polypeptides A Molecular Lego Immobilization on Solid Surfaces Controlled Nanoparticle Aggregation Receptor Mimicking Binders Lys8 Lys15 Nanoparticle Plasmonic Sensing LA-42h Lys34 0,06 0,05 Biochip Extinction 0,04 0,03 0,02 0,01 0 400 450 500 550 600 650 700 750 800 λ (nm) LSPR
Basic Properties Helix-Loop-Helix Polypeptides
Synthetic polypeptide scaffolds basic properties 42-mer polypeptide No structure in solution 1 42 21 The amino acid sequence determines net charge, peptide folding characteristics, and the presence of accessible group for subsequent modification K. Enander, L. Baltzer et al., J. rg. Chem. 2002, 67, 3120-3123.
Peptide Characterization Dimerization secondary structure, dimerization and function CD experiments in solution Peptide in Bis-Tris buffer, ph 7 5000 0 Random coil + JR2E JR2K JR2E/JR2K -5 +11 Monomers random coil Dimer a-helix K d ~ 20 mm molar ellipticity (deg*m -1 *cm -1 ) -5000-10000 -15000-20000 -25000 a-helix 0.5 mm E in Bis-Tris 0.5 mm K in Bis-Tris 0.5 mm E and K in Bis-Tris 200 210 220 230 240 250 260 wavelength (nm) K. Enander et al., Langmuir 2005, 21, 2480-2487.
Homo- vs Heterodimerization four helix bundle formation Loop 42-mer Helix 42-mer Anchor position SH, CH, NH 2, Biotin, His-tag Homodimer low/high ph ~5, 9 Heterodimer neutral ph
Molecular Lego Immobilization ptical properties
A Molecular Lego Building blocks I: xidation of thiol groups in the loop to yield disulphides S S Mixed peptides Same peptide II: Modification in the loop region for attachment to surfaces SH SH S Au
Expected structures in solution ph 5 + ph 7 ph 9 + ph 5 ph 5 + ph 7 ph 9
What type of structures can be formed? Hydrodynamic radius obtained from Dynamic Light Scattering ph 7 1,2 1 dimer fiber ref ph 7 0,8 0,6 0,4 0,2 0 0,1 1 10 100 1000 10 4 radius [nm] (number weight, linearized) Strings? D. Aili, B. Liedberg et al. Angew. Chemie, 2008, 120, 5636-5638.
String Examples TEM images homodimerization ph ~5 String Ring Defects Double Single
Ring structures The relative presence of strings and rings depends on the concentration
A Molecular Lego Building blocks I: xidation of thiol groups in the loop to yield disulphides S S Mixed peptides Same peptide II: Modification in the loop region for attachment to surfaces SH SH S Au
Surface-anchored Polypeptide Scaffolds folding characteristics using FT-IR S S JR2EC + + JR2K JR2E ph 7 ph 7 S S -log(r/r 0 ) 0.028 0.024 0.020 0.016 0.012 0.008 0.004 0.000 +JR2K +JR2E JR2EC Amide I 1667 Amide II 1545 1669 1542 1669 1542 unfolded ph 7 folded native-like 23 Å 9 Å 9 Å C = Cys in the loop S JR2EC 2000 1800 1600 1400 1200 Wavenumbers v (cm -1 ) [cm -1 ] 1000 K. Enander et al., Langmuir 2005, 21, 2480-2487.
Nanoparticle aggregates - basic idea folding/dimerization as a generic glue Peptide decorated NPs Folding/dimerization + ph 7 Heterodimerization
ptical Properties of Au Nanoparticles + Heterodimerization 0,3 0,25 ph 7 Absorbance 0,2 0,15 0,1 Au NP Ø = 13 nm Note! NPs not to scale 0,05 0 400 450 500 550 600 650 700 λ λ (nm) Small Δλ Large Δλ
Nanoparticle aggregates - basic idea folding/dimerization as a generic glue Peptide decorated NPs Folding/dimerization + ph~5 Homodimerization
What about homodimerization at near neutral ph?
Cationic induced dimerization/folding
Folding-induced Aggregation Cation-dependent at ph 7 ph 7 EDTA Zn 2+ Zn 2+ EDTA D. Aili et al., JACS, 2008, 130, 5780 5788 Fully reversible at ph 7
Layer by Layer Assembly AFM characterization 10 nm 10 nm ptical response 100 nm 100 nm D. Aili, B. Liedberg et al., Proc. of SPIE, 2008, Vol. 6885, 688506.
Anticipated fully switchable Structures Building blocks Helix-loop-helix motifs Complex hybrid materials + Strings, junctions Controlled interparticle distance Layered structures
Receptor Mimicking Binders and Nanoparticle Plasmonics Sensing
ptical characteristics of Nanoparticles the surface plasmon band (SPB) and LSPR Sensing layer Analogous to the Mirkin approach I n SP-field Aggregation II Analyte Dl ~ 8 nm for n = 1.33-1.55 Dl ~ 0-200 nm from I to II (200 nm for large aggregates) Mirkin, C. A. et al., Nature, 1996, 382, 607-609
Sequential - site-selective modification via an unique histidine/lysine chemsitry Four helix bundle Sensing peptide Ligand histidine director 2 3 ph sensitive 1 Chemically accessable groups - lysines Fluorophore CH Surface, NP anchor Lars Baltzer, Chem. Rev. 2001, 101, 3153-3163.
Constructing a binder for CRP Binder Ranking C-Reactive Protein (CRP) an inflammatory biomarker 4C10L17 4C25L22PC6 3C25L22PC6 3C37L34PC6 4C37L34PC6 3C15L8PC6 2C25L22PC6 2C10L17PC6 4C15L8PC6 3C10L17PC6 2C37L34PC6 CRP (pentamer) 1C25C22PC6 1C37L34PC6 P - H N + N - N H - - P - - P N + CH 3 N + CH 3 CH 3 PC (K D ~5 mm) N 2 New design PC-6 (reactive ester) 1C15L8PC6 2C15L8PC6 1C10L17PC6 4-C10L17PC6 K D ~ 0. 5 nm 1E-11 1E-10 1E-09 1E-08 1E-07 1E-06 1E-05 1E-04 1E-03 estimated K D [M] Environmentally sensitive fluorescent reporter group Helena Danielson, Tony Christopeit, Uppsala University Modpro AB - Lars Baltzer, Uppsala
Receptor Mimicking combinatorial approach to obtain high affinity receptors for proteins - Human Carbonic Anhydraze (HCA II) Cellobiose Benzenesulfoneamide Galactose Lys8 Lys15 Lys34 LA-42h The complementary polypeptide is omitted for clarity
The sensor peptide KE2C-C6 for detection of Human Carbonic Anhydrase II, HCAII Mixed layer of JR2EC KE2C-C6 and on NP Nanoparticle sensing scheme Benzenesulphoneamide JR2EC (to control NP aggregation) Six carbon spacer KE2C-C6 N H 2 N H 2 N H 2 N H 2 N H 2 S S S S S N H N H N H N H N N N N N K d (mm) 3 1 0.7 0.8 0.02 HCAII (Human Carbonic Anhydrase II) 29 kda, 5x4 nm N H 2 S N H H N N H N 3 K. Enander, et al., Chemistry-A European Journal, 2004, 10, 2375-2385
Aggregation Principle Zinc is used to control folding and aggregation of particles NPs decorated with JR2EC and KE2C-C6 + Zn 2+ ph 7
A Competitive Colorimetric Assay Folding vs Recognition D. Aili, B. Liedberg, et al. Small, 2009, in press
Results - aggregation based biosensing LSPR peak vs HCA concentration 560 555 550 Aggregated without HCA λ Absmax 545 540 Dispersed with HCA 535 530 525 6 7 8 9 10 11 -log[hcaii] (M) The dynamic range can be altered by varying ligand concentration
Why not Antibodies? Sensing volume e.g. helixloop-helix
Summary Ø Helix-loop-helix polypeptides are versatile building blocks for the generation of supramolecular hybrid architectures Ø They are functionally compatible with standard immobilization strategies (e.g. Au-S-R) Ø ffers a convinient strategy for design of non-natural biorecognition molecules receptor mimicking Ø Excellent tool for controlling the aggregation behavior of optically active NPs Aggregation based sensing Ø Their size, stability and tunable affinity makes them very competivive for sensing and diagnostic applications