Presentations

Question 1

Original of the CD effect

Answer 1

Okay

Question 2

What is the light in CD composed of

So what does cd measure

Answer 2

Left handed and right handed light

They are absorbed in unequal proportions whcih makes an elliptically polarized wave

The difference in absoprption of the left and right handed light, so a shift in the elliptical wave (by some degrees)

Question 3

What happens in there is equal absorption of both L and R

Undequal

Answer 3

Equal, the light stays plane polarized (looks the same as it started with same amount of R and L CPL)

Unequal: of L and R absorbed diff, light changes and becomes elliptically polarized (shape changes to an ellipse)

Question 4

What can cause CD signals to appear

Answer 4

Inherently chiral: the molecules structure make it chiral (ex. Carbon with four different groups attached, disulfide bind

Attached to a chiral centre: molecule has a chiral part connected to it

In an assymetic environment: the 3D shape makes an uneven environment

Question 5

What does the soectropolarimeter measure

Answer 5

Delta A= AL-AR (the diff in absorbtion of the two polarized light r or l

Cd signal: More l, positive, more r, negative

If no cd signal, no chiraltiy because no r or l being absorbed so no elliptically polarized light coding to make theta

Question 6

What is elliptically polarized light

Answer 6

The magnitude of L and R are unequal (due to absorbtion of either l or r)

Changes from plane polarized (r and l equal) to elliptically polarized

The head of the resulting vector is now on an ellipse because the more intense beam (not absorbed) dominates and push the vector to not be on a plane but now on an ellipse

The ellipse is (oval shaped circle)

Question 7

What is ellipticity

What is an ellipse

Answer 7

The theta (angles) of the resulting ellipse from elliptically polarized light

A conic shape resulting from a planes intersection with a circular cone

Question 8

How does you get theta obs from delta A

Answer 8

Theta obs= 32.98 x delta A

Question 9

What are typical orders of magnitude of delta a and ellipticity

Answer 9

Very small ,

Delta A 3x10-4

Theta 10 millidegrees

Question 10

What are the methods to measure CD

Answer 10

Most common: Modulation: alternating r and l, measure diff in abs

Direct subtraction: measure abs of l then abs of r and subtract them separately

Ellipsometric: measure the ellipticity of the light

Question 11

Information available from the CD studies of protiens

Answer 11

Okay

Question 12

Sources of absorption

Answer 12

Chromooores: peptide bonds, aromatic amino acid side chains, disulfide bonds

Non protien cofactors: flavins, heme groups (depending on spin state and central fe ion coordination)

Question 13

Algorthisms used to estimate secondary structure composition

Answer 13

Dichroweb

This allows multiple formats for CD data entry , and you can choose the algorithms and databases that are used to analyze your spectra

Downside: the databases don’t include the structure of oligopeptides so cd spectra of olgiopeptide can’t be resolve found unless they have a very predominant secondary structure

Question 14

What is the problem with measure cd below 180 nm

But what is the upside

Answer 14

Water (solvent) and N2 (used to purge the machine) absorb a below 180

The intensity decrease below 180

Using the 170 nm and below region in the soectra makes estimating the secondary structure more reliable, to do this you can used SRCD

Question 15

What is SRCD

Answer 15

Synchrotron radiation CD

Uses a light source with a higher photon flux (quantity of light striking a given surface area) than a bench-top CD machine

Gives more accurate data at the 170nm below wavelengths because there is a larger signal over the background noise (the signal-to-noise ratio)

Makes it so less sample is needed when recording the spectra and there is more information content available in the data

To use SRCD you need Larger datasets that have spectral data on protiens below 170 nm

Question 16

What contributes to the near uv (260-320nm) signal

Answer 16

The fine structure of aromatic amino acids trp tyr phe

These fine structure bands are caused by vibronic transitions in the aromatic aa: both electronic and vibrational transitions at the same time, makes more complex spectra

phe (255-270) tyr (275-282) Trp (290-305)

Question 17

What does the shape and magnitude of the near UV cd signal depend on

Answer 17

Number of each type of aromatic aA in the protien

Mobility of the aromatic amino acids

Their environment (h binding, polar groups, polarizablitly)

Their spatial position in the protien

Neighbouring amino acids: if close together less than 1nm apart they can couple and as excitons and make very small contributions

Question 18

What do we use near uv CD for

Answer 18

Compare mutant and wild type forms, to see if differences in folding is leading to the mutant phenotype

Also use to identify highly flexible regions in a protein structure, since they tend to have very low near UV signal (why? Extremely high mobility of aa side chains)

Question 19

Why does mobility of regions lead to less signal in near uv cd

Answer 19

There a weaker chance of the aromatics retaining that induced assymetry that leads to their cd signal

Question 20

Preparation of protien samples

Answer 20

Llay

Question 21

How are protien samples made

Answer 21

By overexpression of the gene encoding the protien of interest in a host

The complexity of the host depends on the protien of interests (ex size of protien, PTMS, purification tags)

Need to remove the tags (GST, HIS, MBP) before doing the structur study of the protien

Question 22

How pure does the sample on cd need to be

How can the protiens be characterized

Answer 22

Need to be minimum 95% pure on SDs page

Can use mass spec to identify protien and the PTMs

Question 23

What needs to be done to the sample before doing CD

Answer 23

Nucleic acids and oligomucleotide fragments act as contaminants, remove them by treating with the correct nuclease during purification of protien

Dialysis, some buffers and protective agents interfere with the lights absorption, (absorbs in the far UV) ex imidazole used to remove his tag absorbs in the far UV

Should be clear have no insoluble protien aggregates , to fix use ultracentrifugation or gel permeation to remove

Question 24

Why should CD have no protien aggregates

How do these affect the soectra

Answer 24

these cause artefacts because:

the aggregates size is similar/greater than the wavelength the protien absorbs , so light scattering instead of absorbtion occurs

Absorbtion flattening due to the high concentration of protien in those aggregates (so absorbtion lower than should be because not even distribution of the protien)

The shape and magnitude of the spectra becomes distorted and decrease the signal to noise ratio

Question 25

What should the characteristics of the buffer that the protiens are in be

Answer 25

the stability of the protien depends on protien protien and protien solvent interactions Buffer: the solvent should be aqueous with narrow pH range (pka +/- 1) Should be resistant to pH changes (ex shouldn’t change even if adding highly charged ligand) Should have a certain ionic strength to disperse the surface charges on the protien

Question 26

Why are protective agent in solutions. Used and what are they What do you need to make sure

Answer 26

They lower the water concentration in buffers to make the protien environment more similar to that of crowded cytoplasm (this prevents degredation) Ex. Salts (ammonium sulphate), Protiease inhibitors, glycerol (let’s storage at -20 without freezing), DTT Make sure to remove the agents by dialysis before doing CD

Question 27

What do you do if the protien has been lyohpilized (freeze dried) to store it

Answer 27

Rehydrate it carefully to avoid denaturation

Question 28

What should determine your choice of solvent or buffer system

Answer 28

The experimental technique and the protien your using (need to think about if it’ll denature or mess up integrity of the protien ) Consider if the buffer absorbs at the UV range you’re studying, usually they only absorbe at low wavlngth ranges

Question 29

What matters more stability of protien or the signal

Answer 29

Stability matter more So if you have a buffer that stabilizes the protien but it absorbs, use small pathlength to minimize the effect of the buffer, don’t change the buffer Or if buffer needs to stay at certain ionic strength to be stable, don’t use cl since it absorbs below 200nm, use anionis like fluoride

Question 30

What are the suitable buffers for far UV

Answer 30

Phosphate, tris, borate at ph 6-10 Can use HEPES, MOPS, MES,PIPES, but absorb strongly below 200nm so you them in LOW concentration if using

Question 31

Explain the effect of buffer on protien plot

Answer 31

Lysozyme With tris, with nacl buffer, and with imidazole makes it to there is grater absorabnce below 200nm region and in the far uv region In constant sodium phosphate buffer is showing regular lysozyme signal

Question 32

What things in buffer need to be removed before analysis

Answer 32

Imidazole needs to be dialyses to less than 1mM before cd analysis becaus absrobes in far uv Urea (used to denature protiens) absorbs below 210, so need to remove

Question 33

How are studies in of membrane protiens done in cd

Answer 33

Need to extract them first using detergents The detergent chosen should retain the stability of the protien and not absorb in the far uv range (balance between stability and signal) Cd analysis of membrane protien am an be had because of artefacts caused by Light scattering and absorption flattening due aggregation of membrane protiens

Question 34

What detergent should be used in cd

Answer 34

Alkyl glycosides: include laulryl maltosides and octal glycosides These are good for far UV but are expensive So use triton x is want cheaper option, but this might be hard to remove which leads to high absorbance around 280 nm

Question 35

When and what type of non aqueous solvents/ organic solvents would you use for protien

Answer 35

Only used for non polar integral membrane protiens Bad: solvents with carbon chloride (absorb strongly below 230nm) ex.DMSO Use acetonitrile, ethanol, methanol (good down to 190 nm) Use TFE (dehydrates specific residue to promote helix formation)

Question 36

Overall how do we check for to experimental accuracy and protien stability

Answer 36

1. Buffer and solvent suitability: so use blank soectra to confirm the absorbance of buffer is within acceptable limits, and to detect unwanted noise from chiral companies in the buffer 2. Check for protien stability: ensure the protien stable under the experimental conditions, avoid the damage from light sources, validate that the protien is intact throughout experiment 3. Assess stability: check for loss of biological activity, check for the loss of cd signal overtime

Question 37

Experimental conditions for CD

Answer 37

Okay

Question 38

What are the experimental parapemters we consider in CD

Answer 38

Band width Time constant Scan rate Number of scans

Question 39

What do we need for the bandwidth in cd

Answer 39

Increasing bandwidth decreases resolution and increases intensity In cd less than or equal to 1nm Can jse 0.1nm to help resolve the near uv fine structure, but anything less than that you lose the peak

Question 40

What is time constant and scan rate What do we need to consider with these for cd

Answer 40

Scan rate: how fast the wavlngth changes during the scan (high means more wavelengths covered in less time) Time constsnt: the period of time over which the CD data is averaged (time it take to get cd data) Consider: Find a balance: longer time constant improve S/N but if also have high scan rate you lose resolution because with high scan rate cant collect a lot of data points Rule: Scan rate (nm/min) x time constant (s) < bandwidth Best is 0.5 s and 50 nm/min

Question 41

What to consider for Number of scans for cd

Answer 41

Increasing numeet of scans increases S/N (because more data) S/N is proportional to the root of the number of scans Overall more scans are ideal, but need to consider the stability of the sample under the experimental conditions (less stable, worse data)

Question 42

What to consider for protien concentration and pathlength

Answer 42

A<1 to minimize noise and get better S/N ratio, this absorbance is measure by the high tension voltage which is the voltage produced by the PMT (needs to be less than 700V) For higher protien concentration, use shorter pathlength With high concentration of protien, lose the shape of the cd plot, the high tension voltage is too high

Question 43

What is the purpose of measuring high tension voltage

Answer 43

The high tension voltage measures the data quality If too high, abs too high, this results in artefacts, leading to altered cd peak shape

Question 44

What contetstion range of protien can you use in cd

Answer 44

A limited range of concentrations 1 cm path length can use 0.5mg/mL (lower concentration) 0.1cm pathlength can use 5mg/mL (higher concentration)

Question 45

What to account for in temperature in CD

Answer 45

20 deg best for cd Can use changes in temp to measure the stability of the folded protien: This shows which secondary structures denature or refold first Maps out the most and least stable regions Shows mutant vs WT stability Shows regulation studies (drugs binding to protein makes it more stable)

Question 46

What is a peltier device used for in CD

Answer 46

Used to do accurate temp control when doing temp studies in cd

Question 47

What are the limitations/controls of CD at diff temperatures

Answer 47

When increasing temp, the cuvette may expand and change the pathlength The solvent at high temp can evaporate, this can change concentration Inefficient heat transfer (cuvette not be as heated as machine says it is) Change change the magnitude of the cd signals: - have diff magnitudes at diff temps

Question 48

General maintenance

Answer 48

Okay

Question 49

How is the soectropolarimeter maintained

Answer 49

The temp and humidity are kept constant , the mechanical vibrations and atmospheric dust is monitored and kept to minimum Instrument is purged with dry N2 (g) before turning on light to remove water and prevent ozone formation from O2 (damages the optics) The O2 free n2 tank has to be kept in a separate room to avoid asphyxiation, also the last 10% of the tank is unused because it has impurites concentrated at that point

Question 50

How is the soectropolarimeter maintained (2)

Answer 50

The instrument needs a 30-min warm up period to gain baseline stability, the stability of the instrument is checked throughout the day by measuring changes in baseline The life span of the light source is 1000 hours , a decreases in the lifespan is detected by an increase in the high tension voltage The first mirror (thing that gathers light from the lamp) gets damaged the quickest and leads to a decrease in the S/N ratio There a small explosive risk because there’s a 4-fold increase in pressure in the light source when the machine is turned on

Question 51

How are the cuvettes maintained

Answer 51

Cuvettes are strain free: devoid of mechanical strain. Which can depolarize light Treat cuvette carefully to minimize mechanical/thermal damage which affects the optical properties of the cuvette: don’t touch side of cuvette, dry completely Wash and dry cuvettes throughout runs: nitric acid, water, ethanol, dried with vaccum pump Avoid drying with compressed air pumps, they deposit hard to remove oil coming from the compressor

Question 52

Why is nitric acid used in washing cuvettes

Answer 52

Prevents formation of air bubbles (in short path length cuvettes) and removes sticky protien deposits (requires 1-2 hour soak)

Question 53

What is record keeping in cd

Answer 53

Basically putting all data into a place where it’s stored and organized ex. CDTOOL

Question 54

Determination of protien concentration

Answer 54

Okay

Question 55

What are the meothods to find protien concentration

Answer 55

Biuret Lowry BCA Coomasie blue binding

Question 56

What is the biuret method of finding protien concentration

Answer 56

Add copper (II) sulphate to the protien The cu and peptide bonds form a purple complex and the absorbance of this at 380-435 nm is proportional to protien concentration Uniform response: works with all types of protiens This requires large amounts of sample (0.5-5mg) so not sensitive

Question 57

What is the Lowry method of finding protien concentration

Answer 57

Add copper sulphate and the folin ciocalteu reagent The protein in complex with cu reduces the reagent and makes change from yellow to blue colour The response depends on protien composition (so won’t work with some protiens) Sensitive (only need small 5-1000 micrograms of sample) Many sources of interference (buffers and detergents)

Question 58

What is the BCA method of finding protien concentration

Answer 58

Do biuret then add BCA BCA chelates with resulting cu+ (not 2+ since one charge for interacting with peptide) this makes yellow/green product Sensitive (micrograms) Depends on protien comp

Question 59

What is the coomassie blue method of finding protien concentration

Answer 59

Coomassie blue changes from red to blue when binding to protiens Sensitive (micrograms of sample) Depends on protien composition

Question 60

What is the far UV absorbance method of finding protien concentration

Answer 60

Less common Measuing absorbance in far uv 205nm (absorabnce here is due to peptide bonds) Sensitive and uniform response Many buffers absorb in this region Small contribution form aromatics at 205nm but equation accounts for that

Question 61

What is the A280 absorbance method of finding protien concentration

Answer 61

To find A280 for a 1mg/mL solution based on trp tyr and disulfide bonds There is an equation with nw ny and nc (number of trp,tyr, and cys) / MW in Da The nc term relates to disulfide binds only (because only they contribute to abs), so if no disulphides bonds ignore the nc term The 120nc is halved to 60 to account for binded cys not single cys Answer of A280 is in cm

Question 62

What are the assumptions for the A289 method

Answer 62

Only valid if: No contribution from light scattering No other chromoohore in the protien other than the trp tyr and disulphides bonds No other absorbing contaminant A correction of applied to account fkr the diff In native vs denatured protien (because assuming this calculation is for denatured protien)

Question 63

What are the assumptions for the A289 method

Answer 63

Only valid if: No contribution from light scattering No other chromoohore in the protien other than the trp tyr and disulphides bonds No other absorbing contaminant A correction of applied to account fkr the diff In native vs denatured protien

Question 64

Where can the calculate a280 values be found

Answer 64

From protparam (from the expasy system) Gives two set of calculated values, one assuming all cys for disulphide bonds, one assuming none of them do

Question 65

In the a280 assumption that no light scattering is occurring how do we ensure this is true

Answer 65

Can see that there is scattering if the baseline OD/absorbance increases in the 310-400nm range , should be just baseline here (caused by dust or particles) Fix by: centriguation or filtration Log log plot

Question 66

How does the log log plot work to correct for scattering

Answer 66

Log abs vs log wavlngth, linearizes the data The line obtained from 400-310 is the non absorbing region (only scattering occurs here) We can then extrapolate that line to lower wavelengths Then subtract the contribution of scattering from the measured absorbance

Question 67

For interference form nucleic acids in the A280 how do we know it’s happening and how it is fixed

Answer 67

Find by: The A280/A260, of lower means there’s nucleic acids since they Abrob more at 260nm Using dyes that bind to nucleic acids Solution: Remove the acids with a nuclease (ex DNase)

Question 68

in the A280 calculation how do we correct to native conditions (since assuming it’s denaturing cond)

Answer 68

Use parallel dilutions to Determine the ratio of A280 for native and denatured Parallel dilution: Make two dilutions of protien, one with buffer (native) and one with 8M GdmCl (denaturant) in buffer Measure the A280 for both samples and calculates ratio of native A280 / denatured a280 Use the ratio to correct and get the a280 of native

Question 69

Assessment of the reliability of secondary structure analysis

Answer 69

Okay

Question 70

What are the four main methods used to assess reliability of the structure determined by CD

Answer 70

NRMSD R value Systematic differences in analysis Use of multiple algorithms

Question 71

What are the challenges in secondary structur analysis

Answer 71

Different algorithms use distinct/different spectral ranges and data sets (your protiens secondary structural elements may not fall within the range an algorithms using) The methods of analysis are not universal (diff ways of analysis) This is why we use methods to assess the reliability of the data we got

Question 72

What is the NRMSD in assessing reliability

Answer 72

Normalized RMSD: Goodness of fit Measures how well the theorical cd spectra for that protien matches the experimental data (cd spectrum you got) Range: 0 (perfect fit) to 1 (no fit) >0.25: error in analysis <0.1 acceptable <0.05 ideal

Question 73

What is the R values in assessing reliability

Answer 73

Measure the appropriateness of the secondary structure composition you got It’s the sum of the absolute (postive) differences between the fractions of the secondary structur elements (helices beta sheets turns) in the protien Low (<0.1) is reliable analysis, high R values indicate issues with the algorithm or reference dataset

Question 74

What is the systematic differences in assessing reliability

Answer 74

Considering the systematic differences in the references dataset and experimental spectra due to the environmental conditions you used Ex. Comparing membrane protiens secondary structur against a reference of soluble protien. The membrane protien could have a shift in the wavlngth compared to the soluble due to diff in polarity

Question 75

What other ways can determine reliability

Answer 75

Using multiple algorithms: more of them providing similar estimates of the structure can improve the reliability (gives a consensus estimate) This consensus estimate can also improve the NRMSD AND R values Use larger datasets of reference protiens: more references is better for reliability Compliment the CD spectra with IR

Question 76

What are the key practices for reliable cd data Collection

Answer 76

Protien characterization and purity Suitable solvent system Instruments maintenance and calibration Instrument settings and proper cuvettes Compare outcome to known databases (asses reliability)

Question 77

Summary: Protien characterization and purity

Answer 77

Confirm the protiens identity and ensure at least 95% purity Accurately determine protien concentration

Question 78

Summary: Suitable solvent system

Answer 78

Use a solvent that stabilized the protien and minimally contributes to absorabnce in the wavlngth range you’re looking at Adapt the solvent comp to the protien requirements for both active and stability (stability is most important)

Question 79

Summary: Instruments maintenance and calibration

Answer 79

Regularly calibrate the CD instrument for amplitude and wavlngth by using reference compunds Makes sure instrument is warmed up and stable before use Make sure cuvettes are clean and palength is accurately measured

Question 80

What are the last three summary pints

Answer 80

Make sure setting on the cd instrument are shifted to get beest S/N ratio. This includes bandwidth, scan speeed, number of scans, time constant. Always blank the instrument under these cond before running experiment and monitor the high tension voltage Annotate the data effectely do you don’t lose the files you make, store and label data accuralty for later use Ensure data is reliable and reasonable and draw conclusions from it