Single-Molecule Biophysics. Physical Cell Biology Guest lecture

Transcription

1 Single-Molecule Biophysics Physical Cell Biology Guest lecture Liviu Movileanu Syracuse University Web:

2 Single-molecule versus bulk-phase measurements Bulk-phase determinations provide average of experimentally determined quantities. Single-molecule measurements offer precise distributions of measured quantities.

3 Single-molecule biophysics Four single-molecule methods: 1. Atomic Force Microscopy (AFM) 2. Laser/Optical single-molecule traps 3. Fluorescence single-molecules. 4. Single-channel electrical recordings (patch-clamp).

4 NOBEL PRIZE IN PHYSICS (2018) Half of the prize goes to Ashkin for a device called optical tweezers for grabbing and manipulating small objects and its application to biological systems. This work was first published in Physical Review Letters. The other half is jointly awarded to Mourou and Strickland for their method of generating highintensity, ultra-short optical pulses. Both inventions represent major breakthroughs in the use of laser beams for practical purposes. CREDIT: 11

5 Hershey-Chase Experiment 8

6 Lifestyle of bacterial viruses 10

7 Bacteriophage and Modern Biophysics 11

8 Single-molecule experiment of DNA packaging 12

9 Single-molecule studies of transcription 13

10 Atomic force microscopy Copyright Garland Science 2014 Copyright Syracuse University 2018 Biological and Medical Physics - Lecture #8

11 Magnetic tweezers used to probe torsion in DNA Copyright Garland Science 2014 Copyright Syracuse University 2018 Biological and Medical Physics - Lecture #8

12 Pipette-based forced apparatus Copyright Garland Science 2014 Copyright Syracuse University 2018 Biological and Medical Physics - Lecture #8

13 Force-extension curves Copyright Garland Science 2014 Copyright Syracuse University 2018 Biological and Medical Physics - Lecture #8

14 Genetically engineered Green Fluorescent Protein (GFP) variants Courtesy to Prof. Vali Raicu (UWM) Tsien (2008) Extracted from the jellyfish Aequorea Victoria and characterized by Shimomura et al. (1962) Cloned in other organisms (Chalfie et al, Science, 1994) Mutations-induced spectral variants (Roger Tsien)

15 FRAP experiment showing recovery of a GFP-labeled protein

16 Schematic of the FCS technique

17

18 AFM view of heptameric hemolysin with Jie Yang, U Vermont

19 Perforin is produced by T lymphocytes to punch holes into a targeted virus-infected cells, enabling granzyme toxins to navigate into and destroy the targeted cell. (Image credit: Mike Kuiper, VPAC)

20 Insertion of DNA in a Nanopore DNA in Pore Time (ms) Current (na) Position (µm) F = k ( Z 1 - Z 0 ) U. F. Keyser et al. Nature Physics 2,473 (2006)

21 Silicon Nitride-based Nanopores in a tiny sub 10-nm thick membrane A B 50 nm 100 nm C D 5 nm 10 nm

22 Single-molecule and Membrane Biophysics

23 Membrane Protein Engineering Lab

24 Cornell Nanoscience and Engineering Facility and Cornell Center of Materials Research

25 Courtesy: Oxford Nanopore Technologies

26 Single-channel electrical recording cis Current (pa) DV = 40 mv 700 ps = 30 pa 1 M KCl trans

27 Single-molecule stochastic sensing frequency of events ---> concentration of analyte specific signature: t, Dg conductance etc. ---> identity of analyte alternative single-molecule detection and manipulation techniques ---> single-molecule fluorescence, single-molecule force spectroscopy

28 Kinetic information is determined from the single-channel electrical trace t on = 1/k on [A] t off = 1/k off K d = k off / k on

29 Could we do this? We need an angling rod.

30 Our fishing tool 3.4 kda biotinylated PEG

31 Molecular Model Analyte/cis A Analyte/trans B

32 Design and optimization of a synthetic receptor A Barnase, Bn Barstar, Bs Barnase, Bn Tether Adapter, O β Turns Cis Tether Adapter, O + ᴪ t-fhua Trans t-fhua B Current (pa) OBn(GGS) 2 t-fhua - Bs Loops Current (pa) OBn(GGS) 2 t-fhua + Bs Release Binding τ on O on O off Time (s) τ off Time (s) τ off

33 Are these single-molecule captures of the protein ligand by the receptor? nm Bs was added to the cis side of the chamber s Current (pa) O on O off

34 Current (pa) 0 nm Bs O on Current (pa) Current (pa) 12.6 nm Bs To acquire a better understanding of the singlemolecule kinetic details of the Bn-Bs interactions, we gradually increased the Bs O on O off concentration. The duration of the release on events, t on, decreased from ~5.1 s for 12.6 nm Bs to ~120 ms for nm Bs (Fig. 1B; Supplementary Table 50.61). nm The Bs frequency of the Bn-Bs binding events increased with Bs concentration. This finding was observed with 12.6, 50.6, and nm Bs (Fig. 2A). O on O off Current (pa) nm Bs O on O off

35 Single-molecule protein detection in a mammalian serum sample Current (pa) 0-5 Current (pa) - Bs and - FBS oon s nm Bs and - FBS Current (pa) 4s oon ooff 4s nm Bs and + 5% FBS IFBS oon ooff