Research in Biological PhysicsRama Bansil – Polymers and Gels : Scattering (light, neutrons x-rays), AFM

Shyam Erramilli – Vibrational Microscopy (SNIM, Near field Raman, Micro DLS)

Ken Rothschild – Bacteriorhodopsin; IR/Mol Bio methods

Bernard Chasan – AFM on bioploymers

B. Goldberg – Biosensing and SubCellular Imaging

H. E. Stanley - Bioinformatics, Protein folding, Alzheimers

Irving Bigio, Evan Evans (BME+Physics) – Microscopy; Proteomics

Vibrational Microscopy Vibrational Microscopy Prof. Shyam ErramilliProf. Shyam Erramilli –– Department of Physics, Department of Physics,

PhotonicsPhotonics

Novel Imaging TechniquesCombined IR/Light Scattering endoscope

Ultrafast IR dynamics

Raman Microscopy/Light Scattering Mucin

IR probes for Proteomics

CollaboratorsZiegler, Rothschild, Bansil, Bigio, Delisi, Goldberg (BU) P. T. C So (MIT)

Graduate Students

J. Celli

B. Gregor (Graduated May 2004)

J. Amsden

Xihua Wang

Contact: shyam@bu.edu

Vibrational Microscopy—Prof. Shyam Erramilli

- Contrast provided by intrinsic normal modes-- No need for fluorescent/radioactive labels

- High absorption cross sections (~ 10-18cm2)

Disadvantage: Poor spatial resolution due to diffraction limit

Scanning Near-field Infrared Microscopy(SNIM)

- Proteomics

Protein Normal modes

The peak positions of Amide I and IIare sensitive to the protein secondarystructure (α-helix, β-sheet, random coils, etc.) – Martin (LBNL)

Richardson (Duke)

ATHEROSCLEROSIS (“hardening of the arteries”)

2000 1800 1600 1400 1200 100030

35

40

45

50

55

60Intima

Cholesterol C-O1060 cm-1

Amide II1550 cm-1

Amide I1650 cm-1

Lipid C=O1735 cm-1

Tran

smis

sion

Wavenumber (cm-1)

Figure 1. An infrared spectrum of a cross-section of artherosclerotic tissue, showing

absorption bands due to the presence of ester-linked acyl chains in lipid, amide bands in proteins, and cholesterol [3]. The absorption lines serve as a “fingerprint” for localizing molecules.

Scanning Near field InfraredMicroscopy (SNIM)

Jeung et al

Quantum Cascade Laser

Free ElectronLaser

100 1000 1000010-12

10-7

10-2

103

"SNIM threshold"

CO2 laser

Free Electron LaserOPO

Synchrotron

2000 K blackbody

Max

imum

Brig

htne

ss(W

/ 0.

1%bw

/ m

m2/ s

r) QCL

108

Wavenumbers (cm-1)

G.P.Williams, G.L. CarrSource requirements for SNIM

Atherosclerotic human intima, 5 µm section

Jeung et al

Collaborations:

Bansil – Raman Microscopy/Light Scattering MucinB. Gregor, J. Celli

With P. T. C So (MIT)

Bigio – Combined IR/Light Scattering endoscopeFang Hui

Ziegler, Rothschild – Ultrafast IR dynamics

Delisi, Goldberg – IR probes for Proteomics

Hong (Physics/Photonics)

Erramilli

Polymer Physics and Biophysics Polymer Physics and Biophysics Prof. Rama BansilProf. Rama Bansil –– Department of Physics, Department of Physics,

Center for Polymer StudiesCenter for Polymer Studies

Structure and Dynamics of GelsPolymer gels

Block copolymers Biological Gels

Graduate Students

Physics Ariel Michelman Ribeiro, Minghai Li, Yongsheng Liu

Huifen Nie (graduated Fall 2004)

Cellular Biophysics Zhenning Hong (graduated May 2004)

Molecular Cellular Biology and Biochemistry Bradley Turner

Collaborators

K. Ludwig, B. Chasan, S.Erramilli, R. Mohanty (BU)

N. Afdhal, K.R. Bhaskar (Harvard Med. School)

C. Konak, M. Steinhart (IMC, Prague, Czech Republic)

Contact: rb@bu.edu

Biological and Polymer PhysicsBiological and Polymer PhysicsBiological gelsBiological gels——mucus and mucus and mucinmucin——

Preventing Preventing AutodigestionAutodigestion of the Stomachof the StomachGallstone formationGallstone formation

Electrophoresis and Smart MaterialsElectrophoresis and Smart Materials——change change shape or move in response to stimulishape or move in response to stimuli----Artificial Artificial musclemuscle

–– Agarose gelsAgarose gels–– Deform and orient in an EDeform and orient in an E--field field

Phase transitions in block copolymersPhase transitions in block copolymersTechniquesTechniquesScattering Scattering ––light, xlight, x--rays, neutrons rays, neutrons Optical and Atomic Force MicroscopyOptical and Atomic Force MicroscopyComputer SimulationsComputer Simulations——Brownian Dynamics Brownian Dynamics

ANSWER:

Gelation of MUCIN

(GLYCOPROTEIN)

MUCIN --A Complex PROTEIN with 80% SUGAR—Molecular Weight `several million

AFM of AFM of mucinmucin in solution in solution ----conformational change leads conformational change leads to to gelationgelation/aggregation at low pH /aggregation at low pH

Individual mucin molecules at pH 6 appear as ~200 nm long worm-like threads with an average height of 1.5nm

At pH 2 the molecules aggregate forming 50 X 20 nm bundles of heights ~ 6-7nm.

Z. Hong, B. Chasan, R. Bansil, B. Turner, K.R. Bhaskar, N. H. Afdhal, Biomacromolecules (submitted)

Micro-DLS Apparatus

A schematic and photo of the micro-DLS instrument used to measure viscoelastic properties of biological gels. The path of the light through the microscope into the photomultiplier tube (PMT) is represented with arrows. The signal from the PMT is fed into a Brookhaven Instruments correlator(BI9000) to obtain the correlation function. (Developed by Brian Gregor and Jon Celli)

The diffusion of tracer particles (109 nm diameter PS latex spheres) in PGM at pH 6 (sol) and pH 2 (gel). This data has been used to characterize the viscoelastic properties of this important protein.

101 102 103 104 105 106 107 108 109

1.00

1.02

1.04

1.06

1.08

1.10

1.12 PGM at pH 2 PGM at pH 2 with beads Beads in H2O Exponential Fit

g 2(t)time in microseconds

102 103 104 105 106

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Beads in pH 6 PGM Beads in H2O

S(q,

t)

time (microseconds)

Intensity autocorrelation data normalized to calculated baseline for PGM at pH 2 with (X) and without (+) 109nm polystyrene tracer spheres, acquired at a 9.6 degree scattering angle. Note the similarity of the slow decay of the particles in the gel (X) with the gel alone (+), and of the fast mode (which is fit to an exponential decay) to the beads in water (□).

A representative curve showing the dynamic structure factor of 109nm polystyrene spheres in PGM at pH 6 (+) and in deionized water (□). Both sets of data were acquired at a scattering angle of 9.6 degrees.

Celli, J.; Gregor, B.; Turner, B.; Afdhal, N.; Bansil, R.; Erramilli, S. To appear in Biomacromolecules

MultiblockMultiblock Copolymers in Copolymers in selective solventsselective solvents

UNASSOCIATED CHAINS ( NEUTRAL OR Below CMT, CMC)

ISOLATED MICELLES(OUTER BLOCK SELECTIVE)

BRIDGED MICELLES

(MIDDLE BLOCK SELECTIVE)

ORDERED MICELLES

(CUBIC PHASE)

Key questions:

Phase Morphology and Kinetics

Loops Vs Bridges

Techniques: Small Angle X-ray Scattering (SAXS)

Simulations

Time Evolution of SAXS Intensity Following a Jump 110Time Evolution of SAXS Intensity Following a Jump 110--145C145C

0.01 0.02 0.03 0.04 0.05 0.06 0.07

10

100

Inte

nsity

(arb

.uni

t)

q (A-1)

10 sec (HEX) 30 sec 50 sec 100sec 500sec (BCC)

500sec

100sec

Nie, Liu, Bansil, Steinhart “ Time Resolved SAXS study of HEX-BCC kinetics in Triblocks” (in

ti )

10 sec

Molecular Biophysics LaboratoryMolecular Biophysics LaboratoryProf. K.J. RothschildProf. K.J. Rothschild –– Department of Department of

Physics, Photonics CenterPhysics, Photonics CenterHow Do Membranes Proteins Work?

Rhodopsins – Vision and phototaxis

Bacteriorhodopsins- Energy Transduction and proton transport

Biomaterials- Beyond Genetic Engineering

Graduate Students

V. Bergo -chemistry J. Amsden - physicsSenior Researchers

S. Mamaev J. Olejnik S. Gite

Facilities

FTIR Lab

Raman Lab

Advanced Genetic Engineering Facility

Contact: kjr@bu.edu, Rm. 209 Sci. Center 617-353-2603

COOH

NH2

COOH

NH2

How Does Light Activate Photonic Proteins?

CytoplasmicSide

Extracellular Side

R A

Light

Central Question: How does Light activate rhodopsin in the process of vision and

phototaxis?

DeGrip, W. J., and Rothschild, K. J. (2000). In "Molecular Mechanisms oinVisual Transduction" (D. G. Stravenga, W. J. de Grip, and E. N. Pugh, eds.), Vol. 3, pp. 1-54. Elsevier Science B.V, Amsterdam.

Central Question: How does the energy of a photon driven the active transport of a proton against an electrochemical

gradient?

N OH

Rothschild, K. J., and Sonar, S. (1995). In "CRC Handbook of Organic Photochemistry and Photobiology" (W. M. Horspool and P.-S. Song, eds.), pp. 1521-1544. CRC Press, Inc., London.

Biophysical and Molecular Engineering Approaches

• FTIR Difference Spectroscopy

• Raman Spectroscopy

• Time-Resolved Spectroscopy

• Genetic Engineering

• TRAMPE-tRNA mediated Protein Engineering (Advanced Genetic Engineering Developed in Rothschild Laboratory)

FTIR Difference Spectroscopy can detect Small Conformational Changes in Complex

Macromolecules such as Proteins

A B

C=O HNCOO-→ COOH∆A

Wavenumbers (cm-1)1800 1700

OHC

O

Rothschild, K. J., Cantore, W. A., and Marrero, H. (1983). Science 219, 1333-5.

FTIR Difference Spectroscopy Can Study How Key Proteins Such as Sensory Receptors Function

Bergo, V., Spudich, E. N., Spudich, J. L., and Rothschild, K. J. (2002). PhotochemPhotobiol 76, 341-9.

Methods in Molecular Biology are Used to Assign FTIR Bands

OH

OHOH

OH

Native Protein Uniform Isotopic Label

Site-directed Mutagenesis

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH OH

Bergo, V., Mamaev, S., Olejnik, J., and Rothschild, K. J. (2003). Biophysical J.84, 960-966.

Advanced Methods in Biophysics and Molecular Engineering Can Lead to Development of

Diagnostic Assays for CancerGite, S., Lim, M., Carlson, R., Olejnik, J., Zehnbauer, B., and Rothschild, K. (2003). Nat Biotechnol 21, 194-7.

ELISA-PTTGite et al., Feb 2003 Nature Biotechnology

FTIR Difference Spectroscopy has recently FTIR Difference Spectroscopy has recently detected the movements of the sensory rhodopsindetected the movements of the sensory rhodopsin--transducer complex, ubiquitous in many forms of lifetransducer complex, ubiquitous in many forms of life

Bergo V, Spudich EN, Spudich JL, Rothschild KJ. 2003. Conformational changes detected in a sensory rhodopsin II-transducer complex. J Biol Chem 278(38):36556-62.

Prof. Goldberg--- Optical Biosensing Using Micro-ring Resonators

1542 1546 1550Wavelength (nm)

FSR=4.2nm

FWHM=0.126nm

• High Q ↔ sensitivity to added molecules• Uses 1.5 µm telecom technology• Non-labeled, high sensitivity, high throughput, low cost, and compact biosensors.

inout

input/output waveguides

inout

Glass microring resonator nλ=2πr ↔ ultra-narrow resonance ↔ high Q

Ayça Yalçın (Physics, ECE, BME collaboration)

AvidinAvidin--Biotin Binding ExperimentsBiotin Binding Experiments

Break due to data acquisition

Binding

0.00

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0.04

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0.08

0.10

Inte

nsity

(V(r

ms)

)

Time (s)0 2000 4000 6000 8000 10000 12000 14000 16000

DI BL

DI

pH

DI

Binding

BLDI: deionized H2OBL: Biotin-LectinpH: pH-7 buffer

input output

Nanoscale Imaging of Subcellular ProcessesNanoscale Imaging of Subcellular ProcessesFluorescein emission

17000 18000 19000 20000Wavenumber 1/λ (cm-1)

w/ mirror

w/out

Large round trip distance causes spectral fringesSpectral fringes encode (Fourier transform) distance above mirrorDistances of molecules determined with nm precisionMicroscope slideMirror

Si MirrorSiO2

Lipid biLipid bi--layers and DNA nanoscale imaging layers and DNA nanoscale imaging

4 nm

8 nm

12 nm

Fraction of hybridization of double-strand0 5 10 15

frequency

0

2

4

6

8

10

12

14

fluor

opho

re h

eigh

t (nm

)

Average height of tags on double stranded DNA from SSFM

DNA conformation determined with nanometer resolution.

Physical system → trans membrane protein imaging