Scott AllenPhysics DepartmentWinter 2006

Research Proposal:

Angela Belcher

CHEM*7530

[Mao, Science, 2004]

Background

Summary

research performed in the Belcher group

the goal

the Proposal

the engineered virus

optical manipulation

display technology

Outline

introduce Angela Belcher

Angela Belcher, Ph.D.

Professor of Materials Science and Engineering and Biological Engineering @ M.I.T.

BA, Creative Studies, University of California, Santa Barbara, 1991

PhD, Chemistry, University of California, Santa Barbara, 1997

Presidential Early Career Award in Science and Engineering (2000)

Du Pont Young Investigators Award (1999)

MacArthur Fellowship “Genius Grant” (2004)

Belcher Group

uses nature as a guide to develop novel electronic and magnetic materials and to pattern materials on the nanoscale

pioneered a very novel, non-covalent, self-organizing approach utilizing evolutionary peptide selection and peptide engineering to specifically recognize, bind, and grow nanoscale building blocks

using selected peptides they have successfully controlled the physical properties of nanocrystals and subsequently employed molecular recognition and self-assembly in the design of hybrid biological-inorganic materials.

http://belcher10.mit.edu/research.htm

Display technology

certain biological molecules exist that display amino acid sequences or peptides to their environment

DNA manipulation allows for the systematic variation of the peptides that are on display

a library of the display variants can be created

several display platforms currently in use are bacteria [1-5], phage [6-17] and yeasts [18,19]

Using a display library

target materials can be referenced against the library to discover peptide sequences that have specific affinity for the target

TARGET

Left with molecules having specific affinity for the target

Phage display

early work in the Belcher lab focused on the M13 phage (virus) as its main display technology

6.5 nm

880 nm

encapsulated with roughly 2800 copies of the major coat protein VIII (pVIII)

capped at one end with approximately five molecules each of pVII and pIX

other end has about five molecules each of pIII and pVI

DNA container

**not to scale

Belcher group researchà highlight

pIII coat protein engineered

combinatorial library created à ~109 12 amino acid peptide combinations

GaAs(100), GaAs(111)A, GaAs(111)B, InP(100) and Si(100)

experimented with five different single-crystal semiconductors:

identified selective peptide binding:

crystal composition à binding to GaAs but not to Si

crystalline face à binding to (100) GaAs but not (111)B GaAs

[6] Whaley et al., Nature, 2000

“Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly”

able to self assemble 3D structures

created viral films containing ZnS nanocrystals

[7] Lee et al., Science, 2002

Belcher group researchà highlight

“Ordering of Quantum Dots Using Genetically Engineered Viruses”

[8] Lee et al., Adv. Mater., 2003

Belcher group researchà highlight

“Virus-Based Alignment of Inorganic, Organic, and Biological Nanosized Materials”

any molecule covalently linked to streptavidin can be bound by M13

[15] Nam et al., Nano Lett., 2004

Belcher group researchà highlight

“Genetically Driven Assembly ofNanorings Based on the M13 Virus”

bifunctional M13 viruses displayed an anti-streptavidin peptide (pIII) and hexahistidine peptide (pIX) at opposite ends of the virus

Linker Molecule

anti-strep

strep

Ni-NTA

his peptide

[16] Huang et al., Nano Lett., 2005

Belcher group researchà highlight

“Programmable Assembly of Nanoarchitectures Using Genetically Engineered Viruses”

bifunctional viruses displayed an anti-streptavidin peptide (pIII) and a selected gold binding peptide (pVIII)

TEM images

C – streptavidin conjugated CdSe (15 nm)A,B,D,E – streptavidin conjugated Au (15 nm)

~ 5 nm Au bound to viral body

[18] Peele et al., Acta Biomater., 2005

Belcher group researchà highlight

“Probing the interface between biomolecules and inorganicmaterials using yeast surface display and genetic engineering”

[19] Peele et al., Langmuir, 2005

“Design Criteria for Engineering Inorganic Material-Specific Peptides”

first attempts at developing a fundamental understanding of the interaction between the displayed peptides and the inorganic surfaces

(1) Which amino acid functional groups are sufficient for binding short peptides to the inorganic material surfaces?

(2) How do neighboring amino acid functional groups and their spatial arrangement in a peptide sequence modulate binding strength?

(3) Can our results be used to predictively design peptides specific for these different material surfaces?

Belcher group researchà highlight

created a library of yeast cells displaying ~109 different peptide sequences

used yeast as the display platform

4 µm spherical cells that are easily visualized by conventionallight microscopy

looked at binding to CdS, CdSe, ZnS, and ZnSe and Au surfaces

gives relative binding affinities for each displayed peptide

Belcher group researchà highlight

through systematic genetic engineering of the specific peptide sequence displayed the Belcher group concluded that:

only histidine, tryptophan, methionine, and cysteine appeared sufficient for significant binding

spatially proximal amino acids could be altered to tune the binding strength

Proposal

undoubtedly a fantastic collection of research

BUT binding studies are semi-quantitative at best

further research will focus on:

a quantitative analysis to elucidate the forces involved in the binding of displayed peptide sequences to inorganic surfaces

the effect of environmental conditions (pH, temperature, ionic species, ionic concentration) on binding affinity

the creation of a novel and generic quantitative binding assay platform

Proposalà optical manipulation

can control single nanoparticles with angstrom precision and measure interaction forces in the sub-pico-Newton regime [20,21]

able to probe behaviour of materials in isolation

traditional experimental techniques offer ensemble average responses

movement away from the focus of the laser induces a restoring force that is proportional to the distance moved

http://www.physics.arizona.edu/~visscher/OpticalTweezers.htm

Bustamante, Nat. Rev. Mol. Cell Biol., 2000

Proposalà optical manipulation

recently single-beam optical trapping in three dimensions of gold spheres with diameters ranging from 18 to 254 nm has been demonstrated

[22] Hansen et al., Nano Lett., 2005

“Expanding the Optical Trapping Range of Gold Nanoparticles”

using past experience engineered M13 could be created to bind Au nanoparticles at pIX thereby enabling a handle for optical manipulation

http://www.nbi.dk/~pmhansen/gold_trap.htm

pIX pIII

peptidegold binding motif

Proposal

enables the study of a single M13 virus à instead of yeast

bring surface toward M13 virus à allowing pIII time to bind

pull surface away from optical trap study binding force

displacement from trap centre

Proposal à the goal

this platform will enable the systematic quantitative study of binding forces between displayed peptides and inorganic surfaces

the creation of a novel and generic quantitative binding assay can be created à via molecules conjugated to streptavidin

environmental conditions can easily be altered to study effect on binding affinity

pH, temperature, ionic species, ionic concentration

anti-streptavidingold binding motif

pIX pIII

Summary

described past work done in the lab of Angela Belcher @ MIT

introduced display technologies à in particular M13 phage display

also any molecule conjugated to streptavidin

peptide display libraries can be used to bind and order inorganic nanocrystals

Belcher lab made efforts to elucidate fundamentals of peptide-surface binding through use of yeast display semi-quantitative

presented proposal for quantitative analysis of peptide-surface binding forces à optical manipulation and genetic engineering of M13

References

[1] S. Brown, Proc. Natl. Acad. Sci. U.S.A., 89, 8651 (1992).

[2] S. Brown, Nature Biotechnol., 15, 269 (1997).

[3] S. Brown, M. Sarikaya and E. Johnson, J. Mol. Biol., 299, 725 (2000).

[4] M.A. Schembri, K. Kjaergaard and P. Klemm, FEMS Microbiol. Lett., 170, 363 (1999).

[5] K. Kjaergaard, J.K. Sorensen, M.A. Schembri and P. Klemm, Appl. Environ. Microbiol., 66, 10 (2000).

[6] S.R. Whaley, D.S. English, E.L. Hu, P.F. Barbara and A.M. Belcher, Nature, 405, 665 (2000).

[7] S.W. Lee, C. Mao, C.E. Flynn and A.M. Belcher, Science, 296, 892 (2002).

[8] S.W. Lee, S.K. Lee and A.M. Belcher, Adv. Mater., 15, 689, (2003).

[9] R.R. Naik, S.J. Stringer, G. Agarwal, S.E. Jones and M.O. Stone, Nature Mater., 1, 169 (2002).

[10] C. Mao, C.E. Flynn, A. Hayhurst, R.Y. Sweeney, J. Qi, G. Georgiou, B. Iverson and A.M. Belcher, Proc. Natl. Acad. Sci. USA, 100, 6946 (2003).

[11] S. Wang, E.S. Humphreys, S.-Y. Chung, D.F. Delduco, S.R. Lustig, H. Wang, K.N. Parker, N.W. Rizzo, S. Subramoney, Y.-M. Chiang and A. Jagota, Nature Mater., 2, 196 (2003).

[12] C.E. Flynn, C. Mao, A. Hayhurst, J.L. Williams, G. Georgiou, B. Iverson and A.M. Belcher, J. Mater. Chem., 13, 2414 (2003).

References

[13] S.-W. Lee and A.M. Belcher, Nano Letters, 4, 387 (2004).

[14] C. Mao, D.J. Solis, B.D. Reiss, S.T. Kottmann, R.Y. Sweeney, A. Hayhurst, G. Georgiou, B. Iverson and A.M. Belcher, Science, 303, 213 (2004).

[15] K.T. Nam, B.R. Peelle, S.-W. Lee and A.M. Belcher, Nano Letters , 4, 23 (2004).

[16] Y. Huang, C.-Y. Chiang, S.K. Lee, Y. Gao, E.L. Hu, J. De Yoreo and A.M. Belcher, Nano Letters, 5, 1429 (2005).

[17] S.-K. Lee, D.S. Yun and A.M. Belcher, Biomacromolecules, 7 ,14 (2006).

[18] B.R. Peelle, E.M. Krauland, K.D. Wittrup and A.M. Belcher, Acta. Biomater., 1, 145 (2005).

[19] B.R. Peelle, E.M. Krauland, K.D. Wittrup and A.M. Belcher, Langmuir, 21, 6929 (2005).

[20] K. Dholakia and P. Reece, nanotoday, 1, 18 (2006).

[21] C.A. Mirkin, R.L. Letsinger, R.C. Mucic and J.J. Storhoff, Nature, 382, 607 (1996).

[22] P.M. Hansen, V.K. Bhatia, N. Harrit and L. Oddershede, Nano Letters , 5, 1937 (2005).

[23] M. Rief, F. Oesterhelt, B. Heymann and H.E. Gaub, Science, 275, 1295 (1997).

[24] J. Ni, S.-W. Lee, J.M. White and A.M. Belcher, J. Poly. Sci. B , 42, 629 (2004).

[25] C. Bustamante, J.C. Macosko and G.J.L.Wuite, Nat. Rev. Mol. Cell Biol., 1, 130 (2000).

Extra Slides

Fibre spinning

[13] Lee et al., Nano Lett., 2004

Belcher group researchà highlight

“Virus-Based Fabrication of Micro- and Nanofibers Using Electrospinning”

liquid crystalline M13 viruses extruded from a capillary tube into a crosslinking solution of glutaraldehyde

fibers were 10-20 µm in diameter

M13 viruses were mixed with polyvinyl pyrolidone (PVP) and electrospun into nanofibres

virus-PVP electrospun fibers maintained their ability to infect bacterial hosts after resuspending in buffer solution

Force Spectroscopy

very similar to force spectroscopy done using AFM

“Single Molecule Force Spectroscopy on Polysaccharides by Atomic Force Microscopy”

[23] Rief et al., Science, 1997