Presentations Flashcards
Original of the CD effect
Okay
What is the light in CD composed of
So what does cd measure
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)
What happens in there is equal absorption of both L and R
Undequal
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)
What can cause CD signals to appear
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
What does the soectropolarimeter measure
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
What is elliptically polarized light
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)
What is ellipticity
What is an ellipse
The theta (angles) of the resulting ellipse from elliptically polarized light
A conic shape resulting from a planes intersection with a circular cone
How does you get theta obs from delta A
Theta obs= 32.98 x delta A
What are typical orders of magnitude of delta a and ellipticity
Very small ,
Delta A 3x10-4
Theta 10 millidegrees
What are the methods to measure CD
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
Information available from the CD studies of protiens
Okay
Sources of absorption
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)
Algorthisms used to estimate secondary structure composition
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
What is the problem with measure cd below 180 nm
But what is the upside
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
What is SRCD
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
What contributes to the near uv (260-320nm) signal
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)
What does the shape and magnitude of the near UV cd signal depend on
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
What do we use near uv CD for
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)
Why does mobility of regions lead to less signal in near uv cd
There a weaker chance of the aromatics retaining that induced assymetry that leads to their cd signal
Preparation of protien samples
Llay
How are protien samples made
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
How pure does the sample on cd need to be
How can the protiens be characterized
Need to be minimum 95% pure on SDs page
Can use mass spec to identify protien and the PTMs
What needs to be done to the sample before doing CD
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
Why should CD have no protien aggregates
How do these affect the soectra
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
What should the characteristics of the buffer that the protiens are in be
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
Why are protective agent in solutions. Used and what are they
What do you need to make sure
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
What do you do if the protien has been lyohpilized (freeze dried) to store it
Rehydrate it carefully to avoid denaturation
What should determine your choice of solvent or buffer system
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
What matters more stability of protien or the signal
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
What are the suitable buffers for far UV
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
Explain the effect of buffer on protien plot
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
What things in buffer need to be removed before analysis
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
How are studies in of membrane protiens done in cd
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
What detergent should be used in cd
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
When and what type of non aqueous solvents/ organic solvents would you use for protien
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)
Overall how do we check for to experimental accuracy and protien stability
- 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
- 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
- Assess stability: check for loss of biological activity, check for the loss of cd signal overtime
Experimental conditions for CD
Okay
What are the experimental parapemters we consider in CD
Band width
Time constant
Scan rate
Number of scans
What do we need for the bandwidth in cd
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
What is time constant and scan rate
What do we need to consider with these for cd
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
What to consider for Number of scans for cd
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)
What to consider for protien concentration and pathlength
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
What is the purpose of measuring high tension voltage
The high tension voltage measures the data quality
If too high, abs too high, this results in artefacts, leading to altered cd peak shape
What contetstion range of protien can you use in cd
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)
What to account for in temperature in CD
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)
What is a peltier device used for in CD
Used to do accurate temp control when doing temp studies in cd
What are the limitations/controls of CD at diff temperatures
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
General maintenance
Okay
How is the soectropolarimeter maintained
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
How is the soectropolarimeter maintained (2)
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
How are the cuvettes maintained
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
Why is nitric acid used in washing cuvettes
Prevents formation of air bubbles (in short path length cuvettes) and removes sticky protien deposits (requires 1-2 hour soak)
What is record keeping in cd
Basically putting all data into a place where it’s stored and organized ex. CDTOOL
Determination of protien concentration
Okay
What are the meothods to find protien concentration
Biuret
Lowry
BCA
Coomasie blue binding
What is the biuret method of finding protien concentration
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
What is the Lowry method of finding protien concentration
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)
What is the BCA method of finding protien concentration
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
What is the coomassie blue method of finding protien concentration
Coomassie blue changes from red to blue when binding to protiens
Sensitive (micrograms of sample)
Depends on protien composition
What is the far UV absorbance method of finding protien concentration
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
What is the A280 absorbance method of finding protien concentration
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
What are the assumptions for the A289 method
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)
What are the assumptions for the A289 method
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
Where can the calculate a280 values be found
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
In the a280 assumption that no light scattering is occurring how do we ensure this is true
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
How does the log log plot work to correct for scattering
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
For interference form nucleic acids in the A280 how do we know it’s happening and how it is fixed
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)
in the A280 calculation how do we correct to native conditions (since assuming it’s denaturing cond)
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
Assessment of the reliability of secondary structure analysis
Okay
What are the four main methods used to assess reliability of the structure determined by CD
NRMSD
R value
Systematic differences in analysis
Use of multiple algorithms
What are the challenges in secondary structur analysis
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
What is the NRMSD in assessing reliability
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
What is the R values in assessing reliability
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
What is the systematic differences in assessing reliability
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
What other ways can determine reliability
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
What are the key practices for reliable cd data Collection
Protien characterization and purity
Suitable solvent system
Instruments maintenance and calibration
Instrument settings and proper cuvettes
Compare outcome to known databases (asses reliability)
Summary: Protien characterization and purity
Confirm the protiens identity and ensure at least 95% purity
Accurately determine protien concentration
Summary: Suitable solvent system
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)
Summary: Instruments maintenance and calibration
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
What are the last three summary pints
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
