Molecular Biophysics

What will happen to the incident and transmitted ray if there are a lot of cells?

The incident beam will scatter, and the transmitted beam is less intense.

Can use this to measure cell density

What is diffraction?

When the incident ray is scattered at an angle

How does fluorescence occur?

The incident ray promotes an electron in the sample from a ground state to an excited state

When the electron falls back down to its ground state fluorescence occurs

This is an absorbance method

What is the Boltzmann distribution equation

Useful for NMR

u = upper energy state

l = lower energy state

What is the nuclear overhauser effect equation

Useful for NMR

measures distances which can determine structures

What is the Beer-Lambert law

A = absorbance

c = concentration

l = path length

epsilon = extinction coefficient

What is the Bragg equation

What is the equation for the association constant (Ka) involving concentrations

Where H is the host

G is the guest

and binding occurs with a 1:1 stoichometry

What is the equation for the association constant (Ka) involving rates

Where k on is the rate of binding

k off is the rate of dissociation

How does Ka relate to Kd

Kd is the inverse of Ka

If Ka = 1000

Kd = 1/1000

What is the equation for Gibbs free energy relating to Ka

What is the equation for Gibbs free energy from the 2nd law of thermodynamics

Describe the structure of proteins

1º structure: AA sequence - AAs connected by amide bonds (HN-C=O). C=O can form hydrogen bonds with NH on a different part of a chain. If the different parts of the same chain are close together then a condensed helix occurs (alpha helix). If the different parts of the same chain forming H bonds are far apart then an antiparallel structure forms (beta sheet)

2º structure: alpha helix or beta-sheet

3º structure: secondary structure undergoes quick folding to form the tertiary structure. This folding is important for the protein's function

4º structure: multiple tertiary structures fits together

What is the native protein state

Form where all secondary and tertiary structures are in place

What underpins protein folding? Show E level diagram

Thermodynamics controls the conformation and stability of the native state

Kinetics determines how fast do proteins fold and unfold

Directed process:

• unfolded is a random coil, which is floppy and has little secondary structure

• intermediate (molten globule): can measure the rate of formation of this and work out the height of the energy barrier

• native state

via 2 transition states

What can we use the molten globule form?

can measure the rate of formation of this and work out the height of the energy barrier

Describe the structure of DNA

Phosphate - Ribose Sugar - Base

Base could be A-T (2 H bonds), C-G (3 H bonds)

Helical polymer which forms a column of bases stacked upon each other (3.4 Angstroms apart)

different structures of DNA exist: left handed vs right handed (be aware)

Describe the structure of lipids

Glycerol molecule + a fatty acid tail

glycerol is the head and hydrophilic (polar group)

Fatty acid tails are long hydrocarbon chains and are hydrophobic

Self assemble into bilayers due to these properties (cell membrane)

What is common between Trp, Tyr, Phe, His and describe what affects their absorbance

All are aromatics and have absorbances below 300

Aromatics absorb very well and peptide bonds can also absorb

Peptide bonds have low absorbances but because there are so many in a protein molecule, they dominate

With a protein or DNA it is common to see absorbances below 300

Abs (λmax)/nm and ɛ/M^-1 cm^-1 for Trp

Tryptophan

280 nm

5500 cm^⁻¹

Abs (λmax)/nm and ɛ/M^-1 cm^-1 for Tyr

Tyrosine

274 nm

1400 cm⁻¹

Abs (λmax)/nm and ɛ/M^-1 cm^-1 for Phe

Phenylalanine

257 nm

200 cm⁻¹

Abs (λmax)/nm and ɛ/M^-1 cm^-1 for His

Histidine

211 nm

5700 cm^-1

Abs (λmax)/nm and ɛ/M^-1 cm^-1 for peptide bond

170 - 220 nm

~1000 cm^-1

What does the absorption profile of proteins look like?

Peptide bonds dominate because there are so many but at a low nm

Aromatic amino acid have a second peak at a low absorption but a high nm

Little or no absorption beyond 300 nm

What is CD

Circular Dichroism

A form of spectroscopy that measures the difference in absorption when a sample absorbs left handed vs right handed circularly polarised light

How does CD work?

CD measures the difference in absorption of left- and right-circularly polarised light by chiral molecules

1) produce light that can rapidly alternate between LCP and RCP

2) hit optically active sample (chiral (proteins) or achiral but bound in a chiral environment e.g. achiral ligand bound to a chiral protein)

3) hit detector which measure how much of each light is absorbed

The CD signal is the difference between the absorbance of LCP and RCP

can convert to change in epsillon if path length and concentration is known using the Beer-Lambert law

change in epsillon is much smaller than epsillon

pheta is molar ellipticity

How do you interpret a protein CD spectrum to find out about protein folding?

•    Key rule: [θ] is positive at 200 nm for a folded protein; negative if unfolded. This is the quickest folding check.

•   α-helix: characteristic double minima at ~208 nm and ~222 nm; positive signal at ~193 nm

•       β-sheet: minimum at ~218 nm; less negative than α-helix

•       Random coil (unfolded): minimum at ~200 nm, generally featureless in the 208–222 region

What does a protein CD spectrum look like and how to interpret

Go to a wavelength of 190 and if pheta is above 0, the protein is folded

Tends to be noisy at very small wavelengths

region between 210-290 nm deviates since most transitions in this region are due to aromatic pi-pi*

What is quantum yield? (background info)

Fluoresence quantum yield (Φ): The ratio of photons absorbed to photons emitted through fluorescence

What values do AAs have for absorbance, emission wavelength, and quantum yield? (intrinsic fluoresence)

Abs < 300 nm

Emission wavelength > 300 nm (fluorescence)

Quantum yield: dependent but is ideal for studying fluorescence e.g. Quantum yield for Trp is 0.2, so for every 100 photons absorbed, 20 of them will fluoresce (0.04 is very poor so Phe is rarely studied)

What is the quantum yield of histidine?

Histidine has no measurable fluorescence

What does GuHCl do to proteins?

Denatures them

What happens to Trp fluorescence in low polarity environments (relative to water)?

Undergoes a hypsochromic (i.e. blue) shift

• has a higher quantum yield

• so lower emission maximum

The lowest curve has less fluorescence so this is an unfolded protein

What is ANS and how does it bind to proteins? Why does the binding curve plateau?

1-anilino-naphthalene-8-sulfonic acid: an extrinsic reagent

excellent for comparing structural states and mutants

• ANS binding is driven by electrostatic (sulfonate to Lys/Arg - AA side chains) and hydrophobic interactions

• ANS has a sulfonate group (always deprotonated at pH 7.4, negatively charged) and an aromatic ring (hydrophobic)

• Binds to partially exposed hydrophobic regions on proteins (especially molten globule states) due to phenyl ring

  • ↑ fluorescence intensity and causes blue shift

Fluorescence signal and ANS conc plateaus since the ANS binding sites are saturated (lots of AMS bound to prot)

Explain interactions of ANS

ANS binding is driven by electrostatic (sulfonate to Lys/Arg) and hydrophobic interactions (aromatic ring)

What should ANS spectrum look like in physiological systems?

Curve should peak at around 500 nm because pH is 7.4 in physiological systems

How can ANS binding tell you how a protein is folded?

• fully folded protein hides hydrophobic patches → low ANS fluorescence

• fully unfolded protein exposes them all → maximum ANS fluorescence

• molten globule (intermediate) → high ANS signal (partially exposed)

What is GFP?

green fluorescent protein

can express GFP in the genome or tag it onto a sequence that encodes the protein of interest

Expressed using natural mechanisms

useful for tagging and monitoring

How do nucleic acids compare to aromatic AAs in terms of absorption?

Much better at absorption

What is DNA melting?

Often a thermal process which causes the DNA double helix (native conformation) to dissociate into its single strands (random coil conformation)

Why does DNA melting occur?

Cells need to access single stranded DNA for reading in transcription

has to be able to unwind in DNA melting

Is also used in PCR to make templates (thermal process)

What is the Tm?

Melting temp of DNA: the tempeature at which half the DNA is single stranded (where unfolding is happening most rapidly e.g. if were to differentiate and then observe at what temperature the peaks occur)

Dependent on H bonds e.g. Tm will be higher if DNA is rich in C-G

If one person has a complementary sequence and the other person is a slight variant, the person with the complementary sequence has a higher Tm

How can Tm be measured using absorbance?

UV-Vis

dsDNA has a low absorbance

ssDNA has high absorbance

dsDNA is hypochromic (stacked bases reduce absorption).

• On melting: hyperchromicity — A260 increases as bases unstacked.

• Sigmoidal curve vs T; midpoint = Tm.

How can Tm be measured using the extinction coefficient?

CD

Useful for telling you if other structures of DNA helix is present e.g. A form or Z form

CD signal of DNA changes as helix unwinds with ↑ temp.

Also monitors A→B→Z-form transitions.

Need molecule that absorbs and is chiral (nucleic acids tick both).

More sensitive than UV-vis

Why is dsDNA hypochromic (decrease in intensity of absorption (εmax)) compared to ssDNA?

Because of proximity of transition dipoles in pi-stacked base pairs - affects E

What is FRET, and how does it occur?

Förster Resonance Energy Transfer

Involves E transfer from donor → acceptor ( through-space dipole–dipole coupling (NOT emission/re-absorption)_

• Detected by: quenching of donor fluorescence (decrease in intensity/lifetime) + appearance of acceptor emission

Occurs if the donor emission overlaps with the acceptor excitation

e.g. if close enough in the spectrum, the emission of GFP will excite RFP

only effective at short distances - overlap is essential

•       Rate of energy transfer kET ∝ r⁻⁶ (very steep distance dependence: r ≤ 5 nm; r₀ ≈ 5 nm for CFP)

How can Tm be measured using Fluorescence

FRET

Even more sensitive than CD

One strand labelled with donor, complementary strand with acceptor.

• In dsDNA: strands close → FRET seen.

• On heating above Tm: strands separate → loss of FRET.

• Sigmoidal curve midpoint = Tm.

When the distance between fluorophores is less than the critical distance, FRET occurs

Produces a spectrum where the donor peak is a lot smaller than the acceptor peak and there is overlap in wavelength

Measuring fluorescence decay gives a better signal: noise ratio than if fluorescence intensity was measured since samples scatter light

What property of DNA enables things to slide between the bases

Bases are stacked in the middle of the double helix structure so things can slide between them

absorbance is affected by the space between them

What FRET do you get if single stranded DNA is tagged with a fluorophore (one strand per fluorophore)

No FRET

only donor emission

What FRET do you get if double stranded DNA is tagged with a fluorophore (one strand per fluorophore)

FRET

donor and acceptor emission so 2 peaks

What is intercalating FRET (iFRET)? State one application

Excess of donor so can slide in between DNA bases when the DNA strand is tagged with the acceptor

• used to look at mutations

• only 1 strand needs to be tagged

Donor = intercalating dye (flat aromatic, inserts between base pairs of dsDNA; cationic → interacts with phosphates)

Acceptor = fluorophore on 3' end of probe strand

•       Donor can only intercalate when dsDNA is present → FRET only occurs when probe has hybridised to its target

•       Only the probe needs to be labelled — target genomic DNA can be unmodified

Application: screening genomic DNA populations for specific sequences or SNPs

• If target contains mismatch: lower Tm (weaker hybridisation) → separate sigmoidal curve at lower temperature in first-derivative plot

How can iFRET be used to m

Intercalator acts as donor; loss of intercalation at Tm removes FRET. Only probe needs labelling.

What happens to nucleic acids below the Tm?

double stranded

helical (normal DNA structure)

What happens to nucleic acids above the Tm?

DNA becomes single stranded

H bonds break

base stacking disrupted

strands become random coils

What is nuclear spin characterised by?

spin quantum number I

What does nuclear spin give the nucleus?

a magnetic moment (μ)

this magnetic moment can align with or against an external magnetic field (B₀) with a magnetic quantum number between ml (-l and +l)

What is the spin lattice relaxation time

T1 - z direction

What is the spin-spin relaxation time

T2 - x,y direction

what is the relationship between T1 and T2

T2 is less than T1 for biomacromolecules

T1 and T2 are similar for small organic molecules

What is tau(c)

the correlation time for random motions in solution

What are some practical decisions for biomolecular NMR

small proteins need 2D

large proteins need 2D/3D NMR but the maximum size limit isn't actually very much

need a certain amount of H1

some atoms can be isotopically enriched

need to assign chemical shifts to structural components

partial assignments may be sufficient for dynamics or ligand binding

What does 2D NMR give info about

connectivity or proximity

what does the 2D NMR spectrum of a folded protein look like

a broad spread across the whole spectrum with similar intensities is indicative of a folded protein

What are the 2 dimensions in 2D NMR

t1 and t2

What can the data be thought as in 2D NMR

a stack of 1D files, each differing by a change in t1

What does a HMQC spectrum look like

at pH 2, proteins are in molten globular state

at pH 8, states are beginning to fold into native states

at pH 8 after some time, the proteins are fully folded and have reached the native state

What is HNCO

3D method of biomolecular NMR

magnetisation transferred from the H amide to the N and then to the C=O and back again

confirms secondary structure (from C=O shifts) and aids backbone assignment

What is HNCA

3D method of biomolecular NMR

magnetisation transferred from the H amide to the N and then to the alpha C and back again

useful for backbone assignment

What is an example of a 3D NMR spectrum

each slip correlates to each other

if blob is strong (big and black), there is a strong shift for the alpha carbon

What is NOESY

useful for backbone assignment and structure determination

get proximity effect between neighbouring residues- determine secondary structure of proteins

measures changes in intensity depending on spatial arrangement

energy transfer is dipole-dipole bases relaxation through space

how can you determine the secondary structure of a protein through NOESY data

strong nOe between NH of residue i and NH of residue i + 1 for an alpha helix, but not in beta helix

strong nOe between proton on alpha C or residue i and the NH residue of i+1 in beta sheet

can be used to set distance restraints but need a reference distance for residues

What is H/D exchange

H atoms involved in H bonding exchange slowly particulary inside a folded protein

How is host-guest binding measured

using K: K can be measured by monitoring any observable property that changes as the host and guest concs are varied

What does the chemical exchange profile of DMF look like

at low temps, there are 2 sharp peaks due to methyl groups (no significant rotation around CN so bonds are distinct)- slow exchange

as temp increases, signals broaden but stay at the same chemical shift - intermediate exchange

at high temps, there is rotation around the amide bond so get fast exchange and see 1 signal- fast exchange

a difference in peak intensity reflects equilibrium intensities

What does the NMR spectrum of protein- ligand binding looks like

at the bottom, there are weak signals since there is a small conc in guest

the magnitude of shift change tells you how close it is to the binding site, NOT how strongly it binds (K)

Where are resonances that move likely to be located

close to the binding site

a dot = free protein

top of arrow = high conc of ligand

LHS = noise

RHS = no noise depicted by smoother circles

How to interpet a protein-ligand NMR

If there shifts are well separated e.g. 54, then there is fast exchange, indicating the residue is close to the binding site (there is a progressive change in chemical shift

If it seems like there is only one spot (the one at the bottom), the residue hardly shifted so far away from the binding site

Residues with big shift changes are closer to the binding site

Why is protein-ligand NMR useful

the interactions at the binding site of the protein e.g. which residues are involved in binding, are being revealed by chemical shift changes

What is another example of protein-ligand NMR

G98 is a free protein because there is a big shift change and progressive contours, which means there is fast exchange

What do you always say after progressive change in shift in protein-ligand NMR

there is fast exchange

What do you say if the shift change has a large magnitude

large magnitudes = close to the binding site

What does 1D NMR spectra look like

What does 2D NMR spectra look like

in vivo spectra are hard to interpret since there is lots of noise

What is isothermal titration calorimetry

measures heat exchange from induced ligand-protein binding

the guest (ligand) solution is titrated into a solution of the host (protein) in the sample cell

power is supplied to maintain the reference and sample cell temperature

gives info about delta H and Ka, and therefore delta S

can be used to determine the binding stoichiometyr

What does the data look like from isothermal titration calorimetry and how to interpret

the first peak is unusual since there are often bubbles in the syringe

before the first injection there is no ligand at all, so binding is small since [ligand] is small but [protein] is massive

if you integrate under the peaks, you get the amount of heat released per injection in kilo J (the bottom graph)

the bottom graph is in kJ and you look half way in the curve (-5) and go down to 1, which means there is 1:1 binding

the control would be to inject a buffer instead of a ligand

What could they ask you in exam about isothermal titration calorimetry

BE AWARE: COULD BE IN KILO CALORIES RATHER THAN KILO JOULES

integrate data to get the sigmoidal curve

at low/ medium conce, the most heat is produced (maximal binding)

at high concs all binding sites are saturated

if know the Kd, know delta G and therefore delta H and delta S

Also know the stoichiometry

LIMITATION: binding must be strong enough to produce enough heat

Interpret this example for ITC

at -5 kcal, the molar ratio is 1, so the stoichiometry is 1:1 (bottom graph)

quantitative data matches qualitative data well

What is induced circular dichroism

some samples e.g. berenil are achiral so no CD on its own

Bind to DNA (chiral) to get a CD signal as it has benefitted from being in a chiral environment

flat line in top graph= no binding

see signals as DNA conc increases

How does berenil bind to DNA

it is an intercalator

at pH 7, both ends of berenil are charged so can interact with the negative charge on the DNA phosphate backbone

it is planar so can slide inbetween the stack of bases

it can either strengthen the interactions (and increase Tm) or disrupt the structure and lower the Tm (like adding salt to water)

can use the changes in Tm to determine binding

What are sphingolipids

there is a fatty acyl group attached to the amine of the amide

What are sterols

They are lipids that do not consist of glycerol or fatty acids. They are composed of four connecting rings of carbon and hydrogen, with a polar OH group

the polar OH group means sterols cannot dissolve in water

they are responsible for regulating the fluidity of the lipid menbrane

What are liposomes

closed-lipid bilayer spheres

they are used as models of the phospholipid bilayer but they are much simpler

What are cell membranes made out of

a complex mixture of sterols, (glyco)lipids, (glyco)proteins

fluid mosaic model

have transport mechanisms since need to transport polar molecules into cells without touching the inside of the bilayer (cannot directly diffuse across)

How can you separate the lipid bilayer for analysis

use freeze fracture

What are the three types of diffusion across the cell membrane

Rotational diffusion: D(R)

Lateral diffusion: D(L) - diffuse in the plane of the bilayer

Transbilayer diffusion: flip (move to the inside) /flop (move to the outiside) - phospholipid moves between leaflets

what are the typical rates of D(R) for proteins and lipids

proteins: 10^4 s^-1

lipids: 10^6-10^7 s^-1

what are the typical rates of D(L) for proteins and lipids

proteins: 1 - 5 micro m^2 s^-1

lipid: 20-30 micro m^2 s^-1

important for cell signalling (membrane remodels in response to cellular events)

what are the typical rates of flip flop for proteins and lipids

proteins do not flip flop

lipids flip flop slowly (every 200 - 1500 min)

How does a dichroic mirror work

only transmits fluorescence (some wavelengths) and reflects others

the focal volume determines the resolution

can detect from the bottom of the slide (inverted microscopy)