How to probe a biomolecule MJ L1-2

Question 1

What region is the electromagnetic spectra?

Answer 1

10^-4 to 10^-11m

Question 2

What is the order for biological structures?

Answer 2

wavelength of 10^-10m

Question 3

What are the three interactions with EM waves?

Answer 3

Absorbance, fluorescence and scattering

In abs, an acquisition of a pocket of EM energy (photon) increases the energetic state of a molecule (reversibly).
-Either moves e-s around or causes a change in the energy state of the e-s which changes the properties/electronic structure.
In emission, a loss/release of a pocket of EM energy (photon) decreases the energy state of a molecule.

Question 4

What wavelength do electrons have?

Answer 4

a few picometers

Question 5

What wavelength do neutrons have?

Answer 5

λ of ~0.1nm

Question 6

Atomic Spectra

Answer 6

Involves precise transitions between quantised atomic states.
These spectra consist of a series of lines. Photons are emitted at very discrete energies (precise).
Lines are observed since you are moving e-s between very discrete energy states within the atom, moving electrons between orbitals. The energy gap is well defined in an individual atom between orbitals.
Energy gap will be the same in an ensemble of atoms, therefore you get a line spectrum from the photons emitted/absorbed as they all have the same energy.

Question 7

Absorbance Spectra (Complex Molecules)

Answer 7

Complex molecules have complex absorbance and emission spectra that provide spectral fingerprints.
For different atoms with different arrangements of e-s you observe different sorts of spectra.
Chl has a complex absorbance spectrum with multiple broad bands.
The whole system is dynamic/moving and the part of the molecule that interacts with light energy is this conjugated system of double bonds, links multiple atoms together.
All of the bonds are vibrating and so within the complex structure the energy is fluctuating within the molecule.
In a beaker of chlorophyll will exist an ensemble of molecules which shows a large number of energy states.
This gives multiple absorbance bands/transitions and secondly those transitions give rise to peaks/broad bands rather than individual lines.
Chlorophyll as a pigment contains an extensive system of conjugated double bonds.
In this system electrons are arranged as pairs in molecular orbitals.
When light is absorbed by a pigment an e- is promoted to a higher energy molecular orbital. The e- falls back to the ground state as light fluoresces.

Question 8

Do proteins/DNA absorb in the visible part of the spectra?

Answer 8

NO

Question 9

Nanodrop

Answer 9

Miniature spectrophotometer (2µl of liquid)
Measures in the UV abs (Peak at 260nm)
Need mass and extinction coefficient (15cm/M) to work out the concentration.

Question 10

What abs is for proteins?

Answer 10

Some protein abs at 280nm – due to Aromatic amino acids having different UV abs states
From 260-280nm ratio you can tell how clean the DNA is as contamination=distortion

Question 11

What are the different types of energy systems in a molecule?

Answer 11

For molecules one must consider not only electronic transitions but also vibrational, rotational, and translational states.

Question 12

What can the electronic state be represented by?

Answer 12

An electronic state can be represented as potential energy surface-shown as Morse potential for a diatomic molecule. (The Morse potential, is a convenient interatomic interaction model for the potential energy of a diatomic molecule.)

Question 13

Vibrational levels:

Answer 13

A molecule has vibrational sub-levels associated with each electronic state.

Question 14

How do you make a molecule vibrationally hot?

Answer 14

You can make the molecule vibrationally hot by putting energy into it.
Eg. providing infrared photons which will cause the system to move between different vibrational sublevels.

Question 15

What are the peaks like in an absorbance and emission spectra?

Answer 15

Molecular absorbance and emission spectra are broad due to multiple vibrational sublevels.

Question 16

Why is the emission spectra shifted?

Answer 16

Emission spectra are red-shifted with respect to the corresponding absorbance spectrum-STOKES SHIFT.
Spectra is shifted to a lower energy (red=lower energy) due to vibrational sublevels.
(The greater the energy, the larger the frequency and the shorter (smaller) the wavelength.)

Question 17

What is vibrational relaxation?

Answer 17

Molecules with energy that populate the higher vibrational states, after absorbance occurs, these states will relax and will lose a bit of energy. Energy is lost as heat. (VIBRATIONAL RELAXATION)

Question 18

UV and Visible Region Absorbance and Emission Spectroscopy generally probes…

Answer 18

molecular electronic transitions, with the width and shape of the spectra contributed by:
1) Homogeneous broadening due to the vibration etc substructure of the molecule and (intrinsic properties)
2) Inhomogeneous broadening due to the heterogeneity in the environment of the molecules (external properties)
eg it is bumping into other molecules or being effected by physical forces causing the molecule to buckle or electrostatic interactions with other molecules etc.

Question 19

Infrared Region Spectroscopy generally probes…

Answer 19

lower energy molecular vibrational transitions-commonly used to look for individual bonds.

Question 20

What techniques for probing biomolecules used polarised light? (Light in one direction)

Answer 20

Linear Dichroism

Circular Dichroism/CD Spectroscopy

Question 21

Describe Linear Dichroism

Answer 21

Gives information about the protein orientation or structure.
If a sample is ordered (crystallised-all molecules have the same orientation), then information on the alignment of an absorbing component can be obtained from the difference in absorbance of light that is plane polarised either parallel or perpendicular to the axis or orientation.

Light is polarised in a particular direction-spectra is dependent upon the angle of that polarisation relative to the transition dipoles of the molecules inside the rxn centre.
Strength of absorbance varies on whether you have polarisation in one direction or another. It must be an ordered sample! – Shows how the individual rxn centres are ordered within the crystal.

Question 22

Describe Circular Dichroism
The difference in absorbance of left and right circularly polarised light.

UV LIGHT

Answer 22

A molecule with structural asymmetry (eg α helix) will absorb left and right circularly polarised light differently and so give rise to a CD spectrum.
Light continuously changes its polarisation (rotating polarisation).
UV-CD is widely used for analysis of protein secondary structure and how it can be effected.
Online algorithms exist that can estimate the proportions of α-helix and β-sheet and random coil from the shape of the UV spectrum.
Can use this as a measure of integrity (trustworthy).
Measuring the UV-CD spectrum as a function of temperature or other denaturants gives us information on protein folding and stability.
Protein unravels relative to its structures, especially if it’s soluble (not as much for membrane proteins).
Spectral line shape will change (eg from an α helical spectrum to a random coil spectrum). As you reverse the expt the protein may fold back up again (drop the temperature) – reversible process.
Where half that transition occurs it gives you a melting temp and from that you can work on the effect of mutation or environment of that OR observe the effect on the interaction of two proteins on that etc.

• How much secondary structure do you have?
• What type of secondary structure is there?
• How folded is your protein?

Question 23

In Linear Dichroism how do you obtain information on the alignment of an absorbing component?

Answer 23

If a sample is ordered (crystallised-all molecules have the same orientation), then information on the alignment of an absorbing component can be obtained from the difference in absorbance of light that is plane polarised either parallel or perpendicular to the axis or orientation.

Question 24

In Linear Dichroism what is the spectra dependent upon?

Answer 24

Light is polarised in a particular direction-spectra is dependent upon the ANGLE of that polarisation relative to the TRANSITION DIPOLES of the molecules inside the rxn centre.

Question 25

What techniques use scattering of light?

Answer 25

Scattering of Laser Light: Dynamic Light Scattering / Photon Correlation Spectroscopy
Scattering of X-rays:Small Angle X-ray Scattering (SAXS)– //
X-ray Crystallography
Scattering of Neutrons: Neutron Crystallography
Scattering of Electrons: Electron Crystallography

Question 26

Scattering of Laser light: Dynamic Light Scattering / Photon Correlation Spectroscopy

Answer 26

Scattering of visible light gives information on SIZE and SIZE DISTRIBUTION of molecules in the sub-micron region.
A laser is used to illuminate molecules in a solution and a diffraction pattern is produced. The rate of fluctuation of the fine structure of the DIFFRACTION PATTERN- this yields the velocity of BROWNIAN MOTION (random motion of particles in a fluid) of the scattered particles which in turn is dependent on their hydrodynamic diameter.
Can determine If the protein is monomeric, dimeric, trimeric, tetrametic etc and you can work out the relative amounts of those and whether they are undergoing any type of transition with this technique.

Question 27

Resolution for Scattering of Laser light: Dynamic Light Scattering / Photon Correlation Spectroscopy

Answer 27

LOW structural resolution

Question 28

Scattering of X-rays: Small Angle X-ray Scattering (SAXS)–

Answer 28

A technique that can give lower resolution structural information on the average SIZE and SHAPE of biomolecules in the 1-25nm range (macromolecules).
By fitting algorithms to the scattering CURVE you can produce a MOLECULAR ENVELOPE from the molecules, which you can then fit known structures and bits of that molecule in order to work out what the higher order of that molecule is in solution.
A CURVE of scattering INTENSITY vs scattering ANGLE is used to construct a low resolution model of the protein SHAPE.
A related technique is called Small Angle Neutron Scattering (SANS).

Question 29

Scattering of X-rays: X-ray Crystallography

2-3Å high resolution structure

Answer 29

This technique uses DIFFRACTION PATTERNS obtained from a highly-ordered CRYSTAL of biomolecules to determine atomic structure.
X-rays interact with electron clouds around atoms to produce an electron density map.
A monochromic beam of X-rays, usually from a synchrotron source, is passed through the crystal and a diffraction pattern is recorded, the crystal is rotated, another pattern recorder and so on.
The set of 2D diffraction patterns is converted into a 3D map of electron density.

Crystal→ Beam of X-rays → Diffraction Pattern → Computer → High Resolution X-ray Crystal Structure
Uses a diamond light source in the synchrotron (in Oxfordshire).
A lot is done at liquid nitrogen temperatures as it reduces atomic motion and so you get a higher degree of order and so increased resolution. This also decreases radiation damage.
Can see hydrogen’s at v.high resolution.
LIMITATIONS: -resolution
-degree of order, need crystals
-atomic information is required from heavy atoms and light atoms (hydrogen).
A model of protein is built that fits the electron density map. (NB it is much more complicated.)
➢ Cytochrome bc1 complex resolved by X-ray crystallography showed that is is necessary that the cytochrome bc1 complex is dimeric to function.

Question 30

What is the limitations of Xray Crystallography?

Answer 30

LIMITATIONS: -resolution

	- degree of order, need crystals
	- atomic information is required from heavy atoms and light atoms (hydrogen).

Question 31

Scattering of Neutrons: Neutron Crystallography

Answer 31

A technique that uses diffraction patterns obtained from a highly-ordered crystal of a biomolecule to determine its atomic structure

Question 32

What is the limitations of Neutron Crystallography?

Answer 32

LIMITATIONS: - large samples needed

- crystals needed (most protein crystals are too small)!

Question 33

What type of crystallography can you see hydrogens in the structure?

Answer 33

Neutron

Question 34

What temperatures do you need for Xray and Neutron and Electron Crystallography?

Answer 34

Xray-Liquid nitrogen
Neutron-Room temp (causes less radiation damage than X-rays)
Electron-cryogenic temps to limit radiation damage

Question 35

Scattering of Electrons: Electron Crystallography

Answer 35

A technique that uses diffraction patterns obtained from a 2D crystal (1 molecule think, high degree of order) of a biomolecule to determine its atomic structure.
In the transmission electron microscope a beam of e-s is passed through the sample and diffraction is detected, use of cryogenic temps limiting radiation damage.
2D crystals are used because penetration of e-s is much lower than X-rays.
Particularly seen in membrane proteins which form 2D arrays.

Question 36

How thick is the sample in electron crystallography?

atomic structure

Answer 36

1 molecule thick

2D crystal

Question 37

Light and Electron Microscopy

Answer 37

Gives information on cell and organelle structure and dynamics.
Cryoelectron Microscopy (CryoEM) - At the highest resolution, can give information on the gross structure of single proteins. 
EM images of single proteins are recorded and classified into different views, arranged and the 3D structure reconstructed. The resolution can be high enough to show the surface of individual polypeptide chains.
Lots of pictures are taken at many angles of individual molecules and then a computer reclassifies them according to different orientations that they have on that surface in order to derive structural information.

Question 38

What type of microscopy gives the highest resolution?

Answer 38

cryoelectron microscopy

can give info on the gross structure of single proteins

Question 39

Light Microscopy-

Answer 39

process used widely to look at proteins and their interactions and functions in living cells and organelles.

Question 40

NMR Spectroscopy
High resolution

Advantage of NMR is that they deliver the TIMESCALE of transitions together with atomic resolution.

Dynamic information is extracted from relaxation of nuclei after excitation using a variety of NMR exits to span dynamics on timescales from picoseconds to seconds to assess several types of nucleus 1H, 2H, 13C, 15N site specifically

Answer 40

-Information on the structure and mechanism of biomolecules can also be obtained with magnetic and electric fields.
Depending on the local chemical environment, different PROTONS in a nucleus resonate at slightly different frequencies. They display different ‘chemical shifts’.
Analysis of the chemical shifts in a spectrum provides structural information about the molecule.
Identifies what atoms are involved in your molecule and how they are connected together.
A principle use of NMR in molecular life sciences is the determination of atomic structures for small proteins

Question 41

What isotopes are used for NMR

Answer 41

Magnetic nuclei such as 1H, 13C or 15N exposed to a magnetic field absorb and RE EMIT electromagnetic radiation at a frequency characteristic of the isotope.

Question 42

Does NMR need crystals?

Answer 42

Proteins are in solution (attractive technique due to this property) so no need to use crystals and….intrinsically unstructured proteins can be studied.

Question 43

Electron Paramagnetic Resonance (EPR) or Electron Spin Resonance (ESR) Spectroscopy

Answer 43

The basic concepts of EPR are analogous to those of nuclear magnetic resonance (NMR), but it is electron spins that are excited instead of the spins of atomic nuclei.
Involves analysis of chemical species with one or more unpaired electrons (paramagnetic centre), such as organic free radicals or transition metal ions. (Transition metals can occupy lots of different charged states).
EPR is the absorption of microwave energy by paramagnetic centres in an external magnetic field - the size of the field is varied and at certain values energy is absorbed as unpaired electrons in the paramagnetic centre move between their two possible spin states (which have different energies in the applied magnetic field).
Therefore as you vary the magnetic field you end up with e-s flipping spin states. The energy in which they do depends upon what group they’re attached to and what environment it is.
EPR can give very precise information on paramagnetic centres to study structure, mechanism, stability and dynamics.

Question 44

How can the usefulness of EPR be extended?

Answer 44

The usefulness of EPR can be extended by attaching spin molecules to biomolecules.
Adding labels with an unpaired electron. Add 2 labels to observe the interaction between those 2 centres.
Reaction centres often have a positive and negative charge at opposite ends of the molecule. The distance between these two centres can be measured by EPR.
You can look at the static structure, how the distance between those 2 centres varies during its mechanism, use it to study protein stability and the general dynamic flexibility of proteins.
As an example a nitroxide group with an unpaired electron on the NO group can be attached to a native or engineered CYSTEINE side chain.
You could also change the environment eg warm the sample up and see whether the distance gets greater or smaller, you could also look at the conformational flexibility. You could add a ligand to see if the conformational flexibility changes.

Question 45

What type of energy is absorbed in EPR?

Answer 45

Microwave energy
EPR is the absorption of microwave energy by paramagnetic centres in an EXTERNAL magnetic field - the size of the field is varied and at certain values energy is absorbed as unpaired electrons in the paramagnetic centre move between their two possible spin states (which have different energies in the applied magnetic field).

Question 46

What is AFM?

Answer 46

Direct Probing
In AFM, variations in the physical interaction between a very sharp probe and a sample on an atomically-flat substrate/biomolecule are used to map the topology of the sample.
Sometimes it can be done in LIQUID or AIR.
The sample is moved under the probe (tip) held by a flexible cantilever and deflections of the cantilever are monitored by a laser.
The sharp tip is able to interact with your sample either directly or indirectly at a fixed distance away from the sample because you have employed some feedback loop.
Laser→ Detector
Fine stage allows sample to move very small distances.
The probe can knock/damage samples so you can use a variety of different modes

Question 47

What 3 modes are their in AFM?

Answer 47

Contact, Non-Contact and Tapping

Question 48

3 modes:

Answer 48

In contact mode AFM to avoid damage to the sample the force between the tip and the surface is kept constant through a feedback mechanism while the tip is moved across the sample.
The angle which the laser bounces off changes and from this you can get info about the height of the sample.
In non-contact mode the tip is oscillated at a certain frequency and with an amplitude of a few nm above the sample - the height of the sample affects this frequency and amplitude, providing information from which to construct a topology map.
In tapping mode the tip is moved much further (100-200 nm) and an image is formed from measurement of the force of the intermittent contacts of the tip with the sample surface – this has the least destructive effect on the underlying sample.

Question 49

AFM is used to measure what?

Answer 49

AFM is used to image individual macromolecules (large proteins), 2D crystals and entire MEMBRANE systems. Produces detailed information.

Question 50

What can AFM tips be functionalised with?

Answer 50

AFM tips can also be functionalised with proteins, ligands and antibodies to study specific interactions, or even functionalised with cells to look at surface interactions.

Question 51

Single molecule spectroscopic techniques?

Answer 51

AFM
Single Particle CryoEM
Scanning Probe Microscopy

Question 52

Pros of single molecule spectroscopy?

Answer 52

There is rapid development in techniques geared towards studying the properties of single molecules,
or extracting information from them.
One applied aim is to be able to read the base sequence of a single DNA molecule.
Single molecule experiments can reveal details of mechanism that will be hidden in an “ensemble” experiment where the average properties of a population of molecules are measured.

Question 53

Scanning Probe Microscopy

Single Molecule Spectroscopy

Answer 53

Some forms of Scanning Probe Microscopy involve measuring the optical properties of individual molecules eg you can functionalise your tip with a protein or antibody in order to get information on the strength of that interaction, looking at its physical properties.
You can have OPTICALLY and ELECTRICALLY adapted tips.

Question 54

What distance in FRET is able to be detected?

Answer 54

When fused CFP and YFP approach within 10nm FRET can be detected.
FRET occurs because there is overlap between the emission spectrum of CFP and the absorbance spectrum of YFP.
FRET is v.sensitive over a small range.
The efficiency of this system is dependent on distance.

Question 55

What is the Forster distance?

Answer 55

R0 is the ‘Forster distance’ at which the efficiency is 50%.
Ranges between 2-7nm – depends on the details of the system from pigment to pigment.
Energy can propagate very efficiently when the pigments are very close together.
If you see evidence between 2 proteins you know that they have to be interacting very closely - may have to be bound together/interacting in a significant manner to see FRET.

Question 56

FRET

Answer 56

Widely used in conjunction with light microscopy or fluorescence microscopy to study protein structure, protein-protein interactions, protein cleavage and protein dynamics.
Requires 2 chromophores that have appropriately matched absorbance and fluorescence properties.
Chromophores can be suitably matched dyes or pairs of fluorescent proteins such as CFP/GFP/YFP added by gene fusion.
It is important that this tagging doesn’t interfere with the protein properties.

Question 57

Difference between Chl and BChl?

Answer 57

The main light harvesting pigment in purple photosynthetic bacteria is not chlorophyll but bacteriochlorophyll (BChl), a closely related magnesium porphyrin that has a more saturated tetrapyrrole ring (Figure 1A). This causes BChl to absorb at significantly longer wavelengths than chlorophyll in the near infrared, the absorbance spectrum being dictated by the details of the conjugated electron system of the macrocycle.
Light harvesting is also carried out by a variety of carotenoids that provide the main pigmentation in the visible region of the spectrum, and so make purple bacteria purple (or a variety of other colours)

Question 58

Slow step 875nm-870nm LH1->RC

Answer 58

The slowest step is the transfer of the excited state from the B875 BChls to the P870 BChl dimer (referred to as ‘trapping’), which, due to the relatively large distance, takes place in around 30-50 ps. This arrangement, where the charge-separating Chls or BChls of the RC are separated from the light-harvesting Chls or BChls of the antenna by an exclusion zone formed by the protein scaffold, is also a feature of the structure of both PS1 and PS2 of oxygenic photosynthesis. This architecture appears to have two principal functions, to ensure that unproductive back transfer of excitation energy (detrapping) is slow compared to productive charge-separation (which takes place in a few ps), and to ensure that the efficiency of membrane-spanning charge separation is not interfered with by unwanted electron transfer reactions between RC and antenna BChls.