01 Lecture 16th January Preliminary Version

Study programme
Biochemistry (Master of Science)

at the Rheinische Friedrich Wilhelms Universität
Biophysics
in the winter semester 2022/2023
by Prof. Dr. Thorsten Lang (LIMES institute)

 What is biophysics? a chimera of 2 fields
 Getting a feel for spatial and temporal scales
 Detection and analysis of biological structures
 A broad spectrum of available methods
 From raw data to the understanding of biology

What is biophysics?
What is biophysics?
https://en.wikipedia.org/wiki/Biophysics
What is biophysics?
 biophysics is an interdisciplinary science that applies
approaches and methods traditionally used in physics to
study biological phenomena
What is biophysics?
organism that grows, cell that migrates or

divides, biomolecule that adopts an active
conformation or is catalytically active
What is biophysics?
studies the properties of matter
What is biophysics?
 physical quantities as e.g. electric current, temperature,

interaction with electromagnetic waves (diffraction,
absorption/emission, reflection) are studied in biological
systems
What is biophysics?
ion flux through a channel

systems
What is biophysics?
heat released upon
study biological phenomena complex formation

systems
What is biophysics?

systems
using the attenuation

of light to determine
concentrations
What is biophysics?

systems
using the emission of light to probe spatial

proximity; use fluorescence labelling for
contrast generation in microscopy
What is biophysics?

systems
determination of the distances between
crystal lattices in protein crystallography
What is biophysics?
study biological phenomena aim is from biology, any model you could picture

systems
 biophysics covers all scales of biological organization, from

molecules to organisms
Getting a feel for spatial and temporal scales
Difference in one dimension: 7 orders of magnitude

Difference in volume: 21 orders of magnitude
scale bars from 0.1 nm (100 pm) to 1000 µm (1 mm) differing

by steps of one order of magnitude
Physical Biology of the Cell. Philipps, Kondev, Theriot, Garcia, Orne. 2nd Edition, Garland Science, p. 52
Light microscopy (250 nm)
https://upload.wikimedia.org/wikipedia/commons/a
/ae/Airy_disk_spacing_near_Rayleigh_criterion.png
https://upload.wikimedia.org/wikipedia/commons/6/6
b/Bacillus_cereus_Gram.jpg
objects that are smaller than the resolution
limit still are recorded but appear larger
Rizzoli and Betz (2005), Nat. Rev. Neurosci. 6, 57-69
Electron microscopy (1 nm)
Rizzoli and Betz (2005), Nat. Rev. Neurosci. 6, 57-69
Electron microscopy (1 nm) Superresolution light microscopy (1 - 100 nm)

MINFLUX (1 nm)
PALM/STORM (10 nm)
STED microscopy (30 nm)
material density SIM (100 nm)
contrast
contrast by fluorescent
labeling

MINFLUX (1 nm)
PALM/STORM (10 nm)
SIM (100 nm)
https://upload.wikimedia.org/wikipedia/com
mons/9/98/Enterobacteria_phage_T2_trans
mission_electron_micrograph.jpg

MINFLUX (1 nm)
PALM/STORM (10 nm)
SIM (100 nm)
https://upload.wikimedia.org/wikipedia/common
s/1/18/2color-STED-example.png

MINFLUX (1 nm)
PALM/STORM (10 nm)
discovery of SIM (100 nm)
cellular
structures
complementary
Light microscopy (250 nm) more widely applied
in life sciences, life
imaging possible
X-ray crystallography (0.1 nm)

MINFLUX (1 nm)
PALM/STORM (10 nm)
SIM (100 nm)
X-ray crystallography (0.1 nm)

MINFLUX (1 nm)
PALM/STORM (10 nm)
SIM (100 nm)
https://upload.wikimedia.org/wikipedia/commons/
c/c0/Bragg_diffraction_2.svg
Difference: 12 orders of magnitude
Detection and analysis of biological structures
Detection and Analysis
When and where is a What is the structure

protein expressed? of a protein?
When and where does a What is the structure

Golgi-transport vesicle of a Golgi transport
form, how is it moving vesicle?
through the cell?
a method usually is only suitable

for either the one or the other
extra slides of electromagnetic radiation
antena or molecules could generate electromagnetic waves as the move of charges moving, generating a magnetic field. The other way arround could also happen
an asocilating dipole generate this electromagnetic radiation, could be a molecule, where the frequency generat small wavelength. The freq is the time blablabla...
use of UV or visible light: rapid change of things so no delay or waiting; visible light could propafate in water and biological fluids, so it is useful
The temperature of wavelength could be converted by the avogadros number
A broad spectrum of available methods
hundreds of methods based on a manageable

number of physical phenomena
dependeing on the energy of photon, the change of molecules or matter is directed.
Absorption: the absorption of light, that could be converted into geat in some solutions or could also could generate light (fluorescence/phosphorescence).
this absorption depends on the molecule that absorbs.
Phosphorence: emitts some minutes later and fliorescence is more quickly. fluorescence has higher snr than absoprtion.
Photobleaching of fluorephore: photodestruction of the fluorophore.
change of plane of polarization could be used for measuirng the size of moleucles (the plane does not change after emission of the molecule), but if there is a roation
of the molecules that emitts, the plane change, so low anisotroyp(decrease of polarized light) molecules are small and high anisotropy cannot rotate so much, so they
are not that rotate, this is not change a polarized.
interference: waves in phase is when the maximum of both are at the same, so the total wave is increased (construtive), and if there are inverted (destructive) that is.
circularized polarized light_: useful for quiral molecules. also useful for DNA helix
useful for the golgi ap for example for vesicle transport and trafficking
A spectrum of available methods
proteins and so also fluorescence, atlhough more in the UV

but some could emit in the visible light
UV, visible light
Modern Biophysical Chemistry. P. J. Walla, Wiley-VCH

if a molecule has a characteristic absorption band (after labeling or if it is the only

component in the solution) we can follow its concentration
molecules in water rotate less the bigger they are, complex formation increases
size and diminishes rotational diffusion, labeling with fluorescence required

UV, visible light

probing spatial proximity of two fluorophores; reports on binding provided fluorophores

attach to different molecules, and conformational change if attached to one molecule
one of Lang's favourite methods
aparrent difussion coefficient: movility and difusion of a molecules

molecules exchange by diffusion; bleaching of fluorescence in one area and observation of
repopulation with unbleached molecules by diffusion yields apparent diffusion coefficient
different absorption of circular polarized light correlates with amount of secondary

structure that may change upon complex formation; no labeling is required
visible light
infrared
radio frequency waves
microwaves
visible light
not based on the interaction of matter with

electromagnetic waves
From raw data to the understanding of biology
1. change in electric current

raw data 2. change in temperature
3. diffraction pattern
1. change in electric current

raw data 2. change in temperature
3. diffraction pattern
1. ion channel activity

knowledge 2. binding enthalpy
3. protein structure
methods with only limited

insight/methods raw data
technically
imperfect/wrong
assumptions
complementary/
knowledge refined methods
methods with only limited

insight/methods raw data
technically
imperfect/wrong
assumptions
complementary/
knowledge refined methods
sometimes working decades

on one topic
Example: atom positions from diffraction patterns
https://proteopedia.org/wiki/images/d/d4/X_r
ay_diffraction_flow_chart_Splettstoesser.png
Protein Data Bank (PDB)
archive
mostly protein crystallography
search for pdb xx (xx protein name)
go to website for
downloading
download file
Swiss PdbViewer
Atom positions from diffraction patterns: GFP
two chains
GFP crystallizes as a dimer

in each chain an oxygen was missing

the missing oxygen was added

click and drag mouse for repositioning

click and roll mouse for zoomin in and out

click and drag mouse for rotating

chain A numbered AA
chain B last AA
GFP amino acid sequence
Exchanged by Ala1
1 mskgeelftg vvpilveldg dvnghkfsvs gegegdatyg kltlkfictt gklpvpwptl

61 vttfsygvqc fsrypdhmkq hdffksampe gyvqertiff kddgnyktra evkfegdtlv
121 nrielkgidf kedgnilghk leynynshnv yimadkqkng ikvnfkirhn iedgsvqlad
181 hyqqntpigd gpvllpdnhy lstqsalskd pnekrdhmvl lefvtaagit hgmdelyk
missing
Thr230
Modified according to Brejc et al. (1997) Proc. Natl. Acad. Sci. USA 94, 2306-2311
chain B deselected select

red = oxygen
blue = nitrogen
orange = sulfur
deselect select
tyrosine side chain side chains deselected
interstrand H-bonds
stabilizing  sheet
reconstructed O
all but 3 AA
Ser65-Tyr66-Gly67
deselected
Tyr66
Gly67
Ser65
reconstructed O
reconstructed O
Gly67
Tyr66
Ser65
Modified according to Mizuno et al. (2003) Molecular Cell 12, 1051-1058

real structure
Tyr66
Ser65
Modified according to Mizuno et al. (2003) Molecular Cell 12, 1051-1058

electron density map
of GFP chromophore
region
https://proteopedia.org/wiki/images/d/d4/X_r Ormö et al. (1996) Science

Understanding structure and function of GFP
 basic structure is -barrel

with coaxial helix that
contains the chromophore
 autocatalytic chromophore
formation
 chromophore is rigid
through hydogen bonds,
increasing the quantum
yield
Brejc et al. (1997) Proc. Natl. Acad. Sci. USA 94, 2306-2311
not few but lots of studies (field of GFP research)

have led to our current picture of GFP
X-ray crystallography is a very strong method but:
in the crystal the protein may adopt an artificial

conformation; no dynamic studies in solution
no diffraction at H-atoms because of too low

electron density; H-atoms are reconstructed
based on chemical knowledge
possibly wrong assumptions when fitting the

protein chain into the electron density map
we obtain just a model
https://proteopedia.org/wiki/images/d/d4/X_r
Aims of the course
 getting a feel for spatial and temporal scales in

biology
 knowing to choose suitable methods
 knowing the limitations of the methods
Aims of the course
 getting a feel for spatial and temporal scales in

biology
 knowing to choose suitable methods
 knowing the limitations of the methods
work out these points in the seminars

01 Lecture 16th January Preliminary Version

Uploaded by

Copyright:

Available Formats

You might also like

01 Lecture 16th January Preliminary Version

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

01 Lecture 16th January Preliminary Version

Uploaded by

Copyright:

Available Formats

Study programme

Biochemistry (Master of Science)

by Prof. Dr. Thorsten Lang (LIMES institute)

 Getting a feel for spatial and temporal scales

 Detection and analysis of biological structures

 A broad spectrum of available methods

 From raw data to the understanding of biology

organism that grows, cell that migrates or

studies the properties of matter

 physical quantities as e.g. electric current, temperature,

 physical quantities as e.g. electric current, temperature,

 physical quantities as e.g. electric current, temperature,

 physical quantities as e.g. electric current, temperature,

using the attenuation

 physical quantities as e.g. electric current, temperature,

using the emission of light to probe spatial

 physical quantities as e.g. electric current, temperature,

 physical quantities as e.g. electric current, temperature,

 biophysics covers all scales of biological organization, from

Difference in one dimension: 7 orders of magnitude

scale bars from 0.1 nm (100 pm) to 1000 µm (1 mm) differing

Light microscopy (250 nm)

Light microscopy (250 nm)

Light microscopy (250 nm)

Rizzoli and Betz (2005), Nat. Rev. Neurosci. 6, 57-69

Electron microscopy (1 nm)

Light microscopy (250 nm)

Rizzoli and Betz (2005), Nat. Rev. Neurosci. 6, 57-69

Electron microscopy (1 nm) Superresolution light microscopy (1 - 100 nm)

Electron microscopy (1 nm) Superresolution light microscopy (1 - 100 nm)

Light microscopy (250 nm)

Electron microscopy (1 nm) Superresolution light microscopy (1 - 100 nm)

Light microscopy (250 nm)

Electron microscopy (1 nm) Superresolution light microscopy (1 - 100 nm)

X-ray crystallography (0.1 nm)

Electron microscopy (1 nm) Superresolution light microscopy (1 - 100 nm)

Light microscopy (250 nm)

X-ray crystallography (0.1 nm)

Electron microscopy (1 nm) Superresolution light microscopy (1 - 100 nm)

Light microscopy (250 nm)

Detection and Analysis

When and where is a What is the structure

Detection and Analysis

When and where does a What is the structure

Detection and Analysis

a method usually is only suitable

A broad spectrum of available methods

hundreds of methods based on a manageable

proteins and so also fluorescence, atlhough more in the UV

UV, visible light

Modern Biophysical Chemistry. P. J. Walla, Wiley-VCH

if a molecule has a characteristic absorption band (after labeling or if it is the only

Modern Biophysical Chemistry. P. J. Walla, Wiley-VCH

UV, visible light

Modern Biophysical Chemistry. P. J. Walla, Wiley-VCH

probing spatial proximity of two fluorophores; reports on binding provided fluorophores

one of Lang's favourite methods

aparrent difussion coefficient: movility and difusion of a molecules

different absorption of circular polarized light correlates with amount of secondary