Nano-mechanics of Biostructures. --from energy to force in biochemistry--

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1 Nano-mechanics of Biostructures --from energy to force in biochemistry-- Substantial parts of the AFM presentation is a kind gift to Biomeasurement technologies from professor Atsushi Ikai Tokyo Institute of Technology

2 To understand the mechanical hierarchy of the life supporting structures, we do: on Protein: Stretch or compress single protein molecules to probe for their internal mechanical heterogeneity. (Enzymes cannot be just soft to function). on Cell: Measure the force to extract membrane proteins to probe for the mechanics of protein-lipid interaction. on Information Transfer: Measure the force to disrupt protein-protein and ligand-receptor interactions.

3 a commercial AFM instrument for imaging and force measurement

4 basics of force measurement

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6 use of covalent crosslinkers

7 sheet structure of BCA II

8 knot free extension of BCA II (Gln253Cys) c C SPDP 253rd amino acid residue N SPDP

9 Extension Curve of Type I (knotted) and Type II (unknotted) BCA II Alam et al., FEBS letters 2001

10 mechanical heterogeneity and local change of protein compliance implied in thermal factors in crystallography human carbonic anhydrase with inhibitor

11 hardness of BCA II Afrin et al (2004) Apparent Young s Modulus (MPa) Native Variously denatured state 系列 1 系列 Depth 2 系列 of 3 Indentation 系列 4 系列 5 (nm) 系列 6 系列 7

12 stretching of the tandemly repeated giant proteins H.E.Gaub and his collaborators (1997)

13 Stretching of tandemly repeated giant proteins, such as the muscle protein Titin: Gaub & coworkers

14 Titin kinase can be pulled apart stepwise Puchner et al., PNAS 2008

15 Ligand bindning alters the appearance of the force curves

16 apocam Calmodulin charged with different ligands: Which peaks belong to which molecular events? holocam holocam-mlt holocam-tfp Hertadi et al pn

17 Membrane Protein Extraction with AFM by using covalent crosslinking system to -NH 2 shown below (DSS) AFM tip crosslinker NH 2 live cell with membrane proteins substrate

18 Different bacterial membrane proteins: Disulfide linked hexamers, non-covalent hexamers, bakterierhodopsin (series of helices): which is which?

19 distribution of final rupture force how strong is the cell surface? Frequency Rupture Force (nn) Force to sever a single covalent (Si- C) bond Frequency Rupture Force Ranges (nn)

20 If you force tip to break into cytoplasm---

21 Harvesting mrna from cytoplasm (Uehara et al. 2004)

22 AFM - summary We can scan a surface on a molecular level investigate internal molecular forces within proteins study the strength of ligand binding and protein interactions study membrane interactions manipulate single molecules or molecular structures in single cells

23 Surface plasmon resonance (SPR)

24 SPR- the phenomenon If the metal film is much thinner than the wavelength we get 1) an evanescent field 2) a total internal reflection at a certain angle where 3) the light energy escapes via an evanescent quantum mechanical wave created in the metal film. 4) The TIR angle is proportional to the dielectricity constants in the two materials adjacent to the metal film.

25 How can we utilize this to look at binding between moecules? By altering the mass / density on the surface of the gold chip, the dielectricity constant is affected and thereby the TIR angle, which can be measured by a detector measuring the intensity of the refleted light as a function of.

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27 Immobilization of proteins to the chip surface

28 Unique invention at LiU how to utilize surface plasmon resonance to detect affinities between biomolecules fundamental BIACORE technology

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30 The surface and the flow system is critical technology in BIACORE.

31 Affinity of antibodies on maturation

32 Analysis of mutational effects

33 Measuring binding affinities

34 What errors can we have?

35 Not every interaction is perfect

36 Applications Biacore / SPR

37 Summary SPR Kinetics (k on, k off ) Affinities (equilibrium binding constants) No probe is needed Detects changes in mass via change in TIR (total internal reflection) Sensitive to conditions (buffer, flow etc)