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290 Cards in this Set
- Front
- Back
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What is spectroscopy?
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Study of the interaction between electromagnetic radiation and matter
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What is spectrometry?
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Use of spectroscopic techniques to measure the quantity of substances
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What is electromagnetic radiation formed by? How is it characterized?
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EM radiation is formed by photons that are characterized by their wavelength
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What range is visible light seen?
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400-700 nm
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What is the equation that determines the energy of a photon?
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E=hv
h=Planck's constant (6.626x10^-34 J/s or 1.583x10^-34 cal/s) v = frequency (c/wavelength) c= speed of light (3x10^8 m/s) |
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What is the corresponding energy (in kcal/mol) of a mol of photons for the following wavelengths?
220, 280, 400, 700, 1500 nm |
220 nm = 130 kcal/mol
280 nm = 102 kcal/mol 400 nm = 71 kcal/mol 700 nm = 41 kcal/mol 1500 nm = 19 kcal/mol |
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What are the applications of wavelengths that correspond to x-rays (10^-12 - 10^-8 nm)?
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- Diffraction
- Scattering |
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What are the applications of wavelengths that correspond to UV (10^-8 nm) through Visible light (10^-7 nm)?
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Electronic Spectra
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What are the applications of wavelengths that correspond to IR (10^-7 - 10^-3 nm)?
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Vibrational Spectra
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What are the applications of wavelengths that correspond to microwaves (10^-3 - 10^1 nm)?
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EPR
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What are the applications of wavelengths that correspond to radio waves (10^1 - 10^8 nm)?
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NMR
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What type of electromagnetic waves are used for studying diffraction and scattering?
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X-rays
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What type of electromagnetic waves are used for studying electronic spectra?
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UV and Visible
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What type of electromagnetic waves are used for studying vibrational spectra?
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IR
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What type of electromagnetic waves are used for EPR?
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Microwave
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What type of electromagnetic waves are used for NMR?
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Radiofrequency
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Why does absorption occur by a molecule?
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The energy associated with a photon is used to bring an electron from a lower level orbital to a higher energy one
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What do S_0 and S_1 refer to?
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S_0 = ground state
S_1 = excited state |
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What do each S_0 and S_1 have?
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Vibrational sub-levels
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Why does absorption occur over a range of wavelengths of different energies?
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- Because of all of the vibrational sub-levels that the ground state and excited state have
- Also other things can affect it like interactions with solvent |
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Why are spectrums made of broad peaks and not sharp bands?
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Because the ground state and excited state have multiple vibrational sub-levels, absorption occurs over a range of wavelengths of different energies
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What does a spectrophotometer measure/compare?
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The intensity of a beam that went through the sample (I) and compares it to a beam that went through solvent only (Io)
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What equation relates the decrease in light intensity over an infinitesimal slab of sample?
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-dI/I = C*ξ' *dl
dI = change in light intensity I = intensity C = concentration of sample ξ = extinction coefficient dl = infinitesimal slab of sample |
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What is the decrease in light intensity over an infinitesimal slab of sample proportional to?
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C = concentration of sample
ξ' = extinction coefficient |
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By integrating and manipulating the equation relating the decrease in intensity over an infinitesimal slab of sample (-dI/I = C*ξ' *dl), what useful equation is produced?
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A(λ) = log (Io/I) = C*ξ*l
A = absorbance Io = Intensity through solvent I = Intensity through sample C = concentration of sample ξ = extinction coefficient = ξ' * 2.303 l = length of sample = Beer-Lambert Law |
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What are the units of absorption, A(λ)?
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A(λ) is dimension-less (optical density)
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What are the units of the extinction coefficient, ξ?
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ξ is expressed as 1/mol*cm
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What does the Beer-Lambert Law relate?
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The absorbance of a solution of a molecule is proportional to the molecule's concentration (C), the molar extinction coefficient of the molecule for that wavelength (ξ), and the length of the path (l)
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Why is the absorbance a more useful unit than transmittance (T = I/Io)?
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Absorbance in linearly proportional to the concentration (C)
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What is transmittance refer to?
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The proportion of the light not absorbed over the reference beam (T = I/Io)
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If you double the concentration of a sample, how will the absorbance be affected?
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The absorbance will also double
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What are some absorbing groups in proteins?
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- Peptide bond (backbone)
- Aromatic residues (Trp, Tyr, and Phe) |
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If a protein lacks aromatic groups, what structure can be used for detection during chromatography?
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Peptide bond - it absorbs (strong band ~190nm and weak band ~210-220nm)
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Around what wavelength do the aromatic groups of proteins absorb around?
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240-300nm (280nm)
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Which are the strongest absorbing residues?
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- Trp - E280 (5500 O.D.)
- Tyr - E280 (1450 O.D.) - Cys (in disulfide bonds) - E280 (150 O.D.) |
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At what wavelength does DNA absorb best?
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260nm
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What are the typical uses of UV/Vis spectrometry?
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- To check quantities (need to know extinction coefficient)
- To check purity (not widely applicable, but useful in some cases) - To monitor a reaction, a binding equilibrium, that produces a change of E at some wavelength |
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What does UV/Vis spectrometry require for accuracy in determining the quantity of something?
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Assumes that the sample is pure (or at least does not contain other contaminants that absorb in the same region)
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What do you need to know to determine the quantity of something based on UV/Vis spectrometry?
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Extinction coefficient
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What does the protein concentration equal?
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[Protein] = OD(280) / E(prot,280)
- OD(280) = optical density at 280 nm - E(prot,280) = extinction coefficient for that protein at 280 nm |
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How do you estimate the extinction coefficient of a protein?
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By summing up the contributions of the various groups:
E = #Trp*E(Trp) + #Tyr*E(Tyr) + #Disulf*E(disulf) - E(Trp) = 5500 - E(Tyr) = 1450 - E(disulf) = 150 |
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How much is the "calculation" for the extinction coefficient possibly off by? Why?
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Estimate could be off by ~10% because the actual extinction coefficient of the groups depends on the precise environment
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Why is it important to know the linear range of the spectrophotometer you are using?
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- The linear range is the range of input (solute concentration) in which the output (absorbance) is proportional to the input
- Above the linear range, the results are bogus |
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What is the typical linear range for the spectrophotometer?
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0 and 1.5-3.0 absorbance units
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Why is the linear range for the spectrophotometer so small?
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- Beer's law contains a "log"
- Small difference in absorbance equates to big differences in intensity |
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What is Beer's law?
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A(λ) = log(Io/I) = C*ξ*l
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What is a major factor for the loss of linearity for the spectrophotometer?
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- Stray light - the monochromator is unable to eliminate the totality of all other wavelengths
- A small fraction, typically 0.1% of all other wavelengths will contaminate the beam; some of them won't be absorbed and will contribute to the signal (making the reading incorrect) |
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What does it mean to "blank" a sample?
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It means subtracting the signal from another sample that contains everything but the substance we want to measure
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How do you blank the spectrophotometer?
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- Read the blank separately and subtract its absorbance from the sample
- Or by using a dual cuvette setup that subtracts as it measures |
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What are potential problems with blanking?
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- Improper blanking can cause baseline shifts, giving you incorrect optical density measurements
- The composition of the blank may be absorbing too high such that if the blank is "2", you are working with 1/100 of the light; this will lead to loss of linearity much earlier - Scattering - if there is particulate material, light can be scattered and diffused away from the detector, appearing as absorbance |
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When does scattering happen?
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Scattering occurs when the particles have a size comparable with the wavelength, and increases as the wavelength decreases
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Why are solutions with scattering difficult to analyze?
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Difficult to blank because the amount of scattering may be irreproducible between samples
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What must you pay attention to when using a spectrophotometer?
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- Baseline absorbance
- Noise - Scattering |
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What is purification a "fight against"?
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Entropy (opposite of mixing)
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What are the key questions to ask yourself when you want to purify something?
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- What are the physicochemical characteristics of the desired product(s) that set it apart from everything else?
- What techniques do I have available that can separate based on those characteristics? |
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What are the potential goals for a purification?
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- Desired outcome (purify one or a few components out of a mix or remove one or a few unwanted components)
- Purity needed (extreme to moderate) - Amount needed (nanograms to milligrams) - Recovery (all vs. some) |
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Why does purification require compromise?
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Obtaining a large amount of extremely pure sample with near 100% recovery would be VERY challenging
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What physical properties can be exploited for separation/purification?
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- Size
- Shape - Density - Solubility - Charge - Isoelectric point - Hydrophobicity - Solubility - Affinity - Stability |
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What are some techniques that can separate based on the physical properties of your product?
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- Chromatography
- Electrophoresis - Centrifugation - Dialysis - Extraction - Filtration |
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What is the first step of a protein purification procedure?
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Breaking up the cells (lysis)
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What are some techniques that can be used to lyse cells for protein purification?
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- Sonication
- French press - Grinding and mechanical disruption - Lytic enzymes and osmosis |
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When is sonication used? Why?
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- For lysing E. coli cells for the first step of protein purification
- Easy and rapid method |
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When is the French press method used? Why? How does it work?
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- Breaking up cells / lysis for protein purification
- Forces cell through a small orifice, the pressure differential breaks the cells - Used for E. coli and yeast cells - Good for smaller preparations, does not rescale well |
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When would the grinding and mechanical disruption method be used? Why?
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- Breaking up cells / lysis for protein purification
- Mechanical disruption works well with delicate mammalian cells - Yeast has a tough cell wall that can be broken by energetic stirring in the presence of glass beads |
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When would lytic enzymes and osmosis be used? Why?
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- Breaking up cells / lysis for protein purification
- Treatment with lysozyme and lipases can dissolve the cell wall and membrane - This is the least harsh system but the most expensive |
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What is the second step of protein purification?
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Bulk Purification
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What is the purpose of the bulk purification?
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- After the lysis, when possible some simple steps are performed to get rid of as much contaminant as possible before the chromatography
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What are some methods of bulk purification?
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- For proteins expressed in solution, the insoluble fraction is removed by centrifugation
- For proteins expressed as inclusion bodies, the soluble fraction is discarded before resolubilization (with urea or guanidinium) - For membrane proteins, the membrane fraction is pelleted and then extracted with detergents - If a thermophilic protein is overexpressed in E. coli, the lysate can be boiled; all proteins will unfold and precipitate except for the desired protein |
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How would you bulk purify a protein that is expressed in solution?
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- Centrifuge sample
- Remove insoluble fraction |
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How would you bulk purify a protein expressed as inclusion bodies?
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- Centrifuge sample
- Soluble fraction is discarded - Resolubilization (with urea or guanidinium) |
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How would you bulk purify a membrane protein?
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Membrane fraction is pelleted and then extracted with detergents
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How would you bulk purify a thermophilic protein that is over-expressed in E. coli?
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- Boil cell lysate
- Proteins will unfold and precipitate except for desired protein |
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How can you further bulk purify your protein?
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Using differential precipitation with salt (most commonly ammonium sulfate)
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What is the procedure for differential precipitation with ammonium sulfate?
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- Take solution containing protein and add saturated (NH4)2SO4
- Centrifuge - Some contaminant proteins will precipitate - Discard pellet - Save supernatant and add more (NH4)2SO4 - Centrifuge - Desired protein may precipitate - Save the pellet (discard supernantant) - Resolubilize - This takes practice to know when your protein is precipitated or is still in the solvent! |
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Why is ammonium sulfate used for differential precipitation?
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(NH4)2SO4 in high concentration reduces the activity of water, making the interactions between protein stronger, and thus inducing precipitation
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What is step #3 of the purification of proteins?
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Fine purification
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How is the vast majority of fine purification of protein done?
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Chromatography
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What is chromatography?
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A separation technique based on the partitioning of molecules between two phases, a stationary phase and a mobile phase
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During chromatography, what are the two phases the molecules/proteins can be in?
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- Solution - mobile phase
- Bound - stationary phase |
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Molecules that spend more time bound to the stationary phase are eluted when relatively?
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After a longer time because they travel much more slowly; stay in column for long time
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Are the molecules going through chromatography either all in the mobile phase or all in the stationary phase?
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- Equilibrium - spend some time in both phases
- If they spend more time in stationary phase they will take longer to come out - If they spend more time in mobile phase they will elute more quickly |
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How can you make the chromatography purification method more efficient?
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- Need a lot of surface contact between the phases
- This is achieved by reducing the size of the spheres (stationary phase) - increases surface area and decreases gaps |
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When larger spheres (stationary phase) are used what is the surface/volume ratio relative to when smaller spheres are used?
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- Larger spheres have larger gaps and a LOWER surface/volume ratio
- Smaller spheres have smaller gaps and a HIGHER surface/volume ratio |
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What is necessary as the size of the spheres (stationary phase of chromatography) decreases?
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- Pressure required for the flow increases
- High resolution chromatography (HPLC=high performance liquid chromatography) requires a high-pressure apparatus |
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What does traditional liquid chromatography use?
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- Uses gravity to drive the flow through the column
- Large, loosely packed column |
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What happens/is the procedure during chromatography?
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- Sample is applied ideally in the minimum volume to the column
- Bands separate, they become wider because of diffusion (which is proportional to the retention time) - The detector produces a chromatograph - Proteins elute in different fractions that can be tested for activity or by SDS-PAGE, Mass spec, etc. |
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What are some of the different types of chromatography?
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- Ionic exchange
- Gel filtration - Affinity chromatography - Immobilized metal affinity |
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How does ionic exchange chromatography separate molecules?
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Based on the charge of the molecules (charged stationary phase)
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How does gel filtration chromatography separate molecules?
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Based on size (porous stationary phase)
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How does affinity chromatography separate molecules?
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Based on binding of a specific ligand
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How does immobilized metal affinity chromatography separate molecules?
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Very common method for separating tagged proteins (His-tag)
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What is gel electrophoresis used for?
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- Analysis (what is it? how much do I have? how pure is it?)
- Preparation (purify a compound from a mixture; remove unwanted components of a mixture) |
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How does Polyacrylamide gel electrophoresis (PAGE) separate macromolecules?
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Based on size - smallest molecules come out first, largest molecules come out last
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Why is polyacrylamide used?
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It forms smaller pores which gives a better resolution
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Why is DNA easier to separate with electrophoresis?
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- Constant size to charge ratio
- Constant shape - Variable size - Protein has variable charge, variable shape, AND variable size |
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How can proteins be prepped so that they don't have a variable size to charge ratio and a variable shape?
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- Treat with SDS (sodium dodecyl sulfate)
- Treat with beta-mercaptoethanol or dithiothreitol (DTT) |
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What is SDS (sodium dodecyl sulfate) used for? Why?
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- Used to give proteins a constant charge to size ratio and constant shape (for electrophoresis)
- Anionic detergent that binds hydrophobic surfaces - Unfolds (denatures) proteins and coats them with negative charges |
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What is beta-mercaptoethanol used for?
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- Reducing agent that removes inter- and intra-chain disulfide bonds
- Important for protein electrophoresis |
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How does SDS bind to protein?
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- At a constant ratio (1 SDS molecule per 2 AA residues)
- Binds hydrophobic surfaces giving it a net negative charge and denatured shape |
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After proteins have been treated with SDS and beta-mercaptoethanol, how can they be separated?
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Electrophoresis separates proteins primarily by size
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What are the two gels in a polyacrylamide gel and how do they differ?
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- Stacking gel - low percentage, runs faster, "stacks" protein at the interface
- Resolving gel - high percentage, runs slower, separates by size |
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In what direction do the proteins run? Why?
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- From negative electrode to positive electrode
- SDS has added a surplus of negative charges to the proteins |
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How do you visualize the proteins after electrophoresis is completed? How does it work?
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Coomassie Blue treatment - binds (+)-charged and aromatic amino acids
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What is the procedure for setting up an SDS-PAGE gel?
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1. Assemble gel pouring apparatus (check for leaks with 75% ethanol)
2. Prepare resolving gel - avoid bubbles and work quickly 3. Pour resolving gel - gently layer water-saturated butanol on top and wait for gel to polymerize 4. Prepare stacking gel - avoid bubbles and work quickly 5. Pour stacking gel - insert comb, check for bubbles and wait for gel to polymerize 6. Prepare samples - thaw bacterial cell lysate, heat and spin 7. Load samples and markers 8. Run gel - 100V for ~15-20 min and 200V until dye front is within 1 cm of bottom 8. Stain gel and visualize 9. Photograph gel |
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If you want to use chromatography to purify your protein based on size, what technique should you use?
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Size Exclusion
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If you want to use chromatography to purify your protein based on charge, what technique should you use?
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Ionic Exchange
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If you want to use chromatography to purify your protein based on hydrophobicity, what technique should you use?
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Reverse Phase
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If you want to use chromatography to purify your protein based on Specific Interactions, what technique should you use?
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Affinity
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What kind of technique is used with a Ni-NTA column? How does this work?
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Affinity chromatography - His-tags specifically bind to Ni in resin
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What do proteins not like?
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- Heat
- Extreme pH (<6, >9) - Harsh detergents - Freeze/thaw cycles - Proteases - Oxidizers (Cu2+) |
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Which cell lysis technique did we use in lab?
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- Bugbuster - detergent based cell lysis
- Contains lysozyme for hydrolysis of cell wall peptidoglycans - Contains endonuclease for DNA and RNA degradation |
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Why did we have to centrifuge our samples after lysing the cells with BugBuster?
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- Unlysed cells and debris would clog the Ni-NTA column
- Lysate is cleared by centrifugation |
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What is contained in the soluble fraction after centrifuging out BugBusted cells? Do we want to keep this fraction?
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- Soluble proteins
- Small molecules - Our protein of interest! - YES - keep this fraction |
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What is contained in the insoluble fraction after centrifuging out BugBusted cells? Do we want to keep this fraction?
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- Cell walls/membranes
- Insoluble proteins - Unlysed cells - NO - don't keep this fraction |
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Why does the His-tag have a high affinity for the Ni-NTA resin?
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- NTA and Histidine with a lone pair on the nitrogen can chelate the Ni2+ atom
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Why did we remove the HCAII stop codon during the first lab when we did PCR?
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- We had to introduce the His-tag to the C-terminal
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What is the setup of the Ni-NTA column?
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- Frit
- Ni-NTA resin - Glass wool - Sample loaded on top |
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Why was imidazole so important for our Ni-NTA column?
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- Imidazole mimics the function group (side chain) of histidine
- Need a high enough concentration to compete with the locally high concentration 6-His tag |
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When else in lab (besides imidazole) did we mimic "biochemistry" to control our experimental system?
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We used IPTG, an allolactose mimic to activate transcription of the Lac operon
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Why did we use SDS-PAGE to analyze our fractions from the Ni-NTA column?
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- To figure out where our protein was and how much contamination was in each fraction
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Why did we have to dilute some of our fractions from the Ni-NTA column when determining the concentration with the spectrophotometer?
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To stay within the linear range!
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Why did we dialyze our most concentration elutions?
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The semipermeable membrane allows for small molecules to pass in and out but not protein; this removed salts and other small molecules (imidazole)
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Why did we do the Bradford assay if we already measured our protein concentration by UV absorbance?
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Limitations to UV quantification:
- Sensitivity depends on protein sequence and folding - Sensitive to contaminating molecules that also absorb at 280 nm - This method measures total protein, not just the protein of interest |
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What principle is the Bradford assay based on?
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Protein dye binding of an acidic solution of Coomassie Brilliant Blue G-250 to a protein solution which causes a measurable shift in absorbance maxima
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How does protein-dye binding (Bradford Assay) work?
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- Low pH of the solution (~1.1) causes protein to be disrupted and partially unfolded
- Dye donates a free e- to ionizable groups on the protein, exposing hydrophobic pockets for binding - Dye binds to exposed hydrophobic pockets (van der Waals) - Positive charges on the protein interact with the dye's negative sulfonates - These binding modes stabilize the blue form even at low pH |
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What amino acid residues are important for the detection of the protein-dye binding (Bradford assay) mechanism?
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Basic and aromatic side chains
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At what wavelength is maximum absorption from the coomassie blue / Bradford Assay?
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- 595 nm - occurs when it is in the presence of proteins
- 465 nm - no proteins |
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Why was BSA used in the Bradford Assay?
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- Used as a standard curve for quantification
- Cheap, stable, non-interfering protein - Use curve to determine concentration of unknowns |
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What are some possible explanations if you see your protein in the flow-through?
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- His-tag did not bind to Ni-NTA resin
- Buffer ran at wrong pH - Protein did not fold properly - HCAII is not tagged (cloning error) - Resin not added correctly - Resin not equilibrated before loading |
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What are some possible explanations if you see your protein in the wash?
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- Too much imidazole in wash buffer
- pH is too low - Used the wrong buffer |
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What are some possible explanations if your elution is too faint?
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- Not enough imidazole added to elute protein
- Incorrect elution volume added (not enough volume) - Used the wrong buffer - Column ran dry |
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What are some possible explanations if your elution is too messy?
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- Wash conditions not stringent enough (increase the amount of imidazole in wash)
- Washed with wrong buffer |
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How were the mutant sequence reads generated?
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Sanger sequencing reaction
- DNA template denatured - Primers annealed - Extend until a terminator is incorporated (no 3'-OH) - Fluorescent detector determines sequence |
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What is required for the typical set up for chromatography?
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- Reservoir
- Column - Resin - Detector - Pump - Fraction Collector |
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Why is ion exchange chromatography a very common technique?
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- High resolution
- High capacity - Relatively simple - Very applicable (all proteins have charges, and each one has a very distinct distribution) |
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What is the procedure of ion exchange chromatography?
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- A resin with charged groups is equilibrated with counter ions
- Protein is added with prevalent charge opposite that of charged groups on resin, which displaces the counter-ions - The protein can be released using excess salt (of the same charge as the protein) |
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Which charged groups for resins are ALWAYS charged, regardless of the pH?
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- Quaternary amino group (Q) =
-CH2-N+-(CH3)3 - Methyl sulfonate (S) = -CH2-SO3- |
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Which weaker charged groups for resins are charged only at some pH's?
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- Carboxymethyl (CM) (pH>4-5) =
-CH2-COO- - Diethyl-aminoethyl (tertiary amino) DEAE (pH <8-9) = -CH2-CH2-NH+-(CH2CH3)2 |
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What residues are negative?
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Asp + Glu >> Lys + Arg + His
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What is a proteins net charge when in acidic conditions?
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Positive
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What is a proteins net charge when in basic conditions?
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Negative
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What is a proteins charge at the pH corresponding to its isoelectric point?
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Zero
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Ion exchange chromatography is generally performed with what kind of elution?
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A salt gradient elution
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How does a salt gradient elution during ion exchange chromatography work?
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- As you increase the salt, the protein will start to partition and move
- At high enough salt concentration the protein does not interact with the resin anymore |
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What is the other name for size exclusion chromatography?
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Gel filtration chromatography
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What type of chromatography is often used in addition to ion exchange chromatography?
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Gel filtration / size exclusion chromatography
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How does gel filtration / size exclusion chromatography separate molecules?
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By size, or more correctly, by their hydrodynamic volume
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What occurs in the column during gel filtration / size exclusion chromatography?
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- Big particles are excluded by the gel and flow with the solvent
- The small particles can penetrate the gel and will be slowed down the most - Medium particles can penetrate some, they flow in between - Largest particles travel the fastest |
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What kind of gel is used in gel filtration / size exclusion chromatography?
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- Porous gel:
- Dextran, agarose, or acrylamide |
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What is the equation for the total volume of a column?
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V = h*pi*r^2
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What is the total volume of a column broken up into? What do those terms represent?
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- Excluded Volume (Ve) = volume around the particles
- Included Volume (Vi) = volume inside the particles + the excluded volume |
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As you add additional volume of buffer during a gel filtration / size exclusion chromatography experiment, with what amount of volume do the biggest particles elute with?
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- Biggest particles elute with Ve (excluded volume) = volume around the particles
- They come out first |
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As you add additional volume of buffer during a gel filtration / size exclusion chromatography experiment, with what amount of volume do the smallest particles elute with?
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- Smallest particles elute with Vi (included volume) = volume inside particles + excluded volume
- Come out last |
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|
If something elutes during a gel filtration / size exclusion chromatography experiment after you have added enough volume of buffer to surpass the Vi (included volume) what does that mean about those molecules?
|
They molecules must be binding to the resin (not good - you don't want your protein to bind to the resin)
|
|
|
Do you want your protein to bind to the resin during gel filtration / size exclusion chromatography?
|
No!
|
|
|
Does the sample stick to the top of the column during gel filtration / size exclusion chromatography?
|
No - unlike ionic exchange, the sample does not stick to the top of the column
|
|
|
During gel filtration / size exclusion chromatography, does the sample need to be very concentrated or dilute? Why?
|
The sample must be concentrated or the peaks will be very broad
|
|
|
What is affinity chromatography based on?
|
The selective binding affinity of a protein for a ligand; therefore very powerful and specific
|
|
|
What is in the column for affinity chromatography?
|
- The column contains an immobilized ligand attached to a resin
|
|
|
What is the procedure for affinity chromatography?
|
- Sample is loaded into a column containing an immobilized ligand on a resin
- The column is washed; only the protein with a specific affinity is bound to the resin - The protein is eluted with an excess of free ligand |
|
|
What is the downside to using affinity chromatography?
|
- Not all proteins have a strong affinity for a ligand
- Resins are not commercially available for all ligands |
|
|
How do you get around the downsides of affinity chromatography (not all proteins have a strong affinity for a ligand and not all resins are commercially available for those that do have a strong affinity for a ligand)?
|
Express the protein as a fusion, with a commonly used affinity tag
|
|
|
What are some commonly used tags for affinity chromatography? Which are large protein tags?
|
- MBP Tag = maltose binding protein (large protein tag, 42 kDa)
- GST Tag = glutathione-S-transferase (large protein tag, 26 kDa) - His Tag = 6 histidine residues |
|
|
What is an MBP Tag (for affinity chromatography)? What does it bind to? How is it eluted?
|
- Maltose Binding Protein
- Binds to maltodextrin columns (poly-1,4-alpha-glucose) - Eluted with maltose (1,4-alpha-glucose disaccharide) |
|
|
What is an GST Tag (for affinity chromatography)? What does it bind to? How is it eluted?
|
- Glutathione-S-transferase
- Binds to glutathione columns - Eluted with free glutathione |
|
|
Why are the two large protein tags we learned about (MBP and GST) useful?
|
They often improve solubility of a recombinantly expressed protein
|
|
|
What does a protein that has a tag added have so that the tag can be removed later after affinity chromatography?
|
The construct often contains an engineered proteolytic site so that the tag can be removed
|
|
|
What does the His tag have a high affinity for?
|
High affinity for metals such as nickel or cobalt
|
|
|
Why is a His tag useful tag for affinity chromatography?
|
Most proteins tolerate (are functional and stable) the addition of an N-terminal or C-terminal His-tag
|
|
|
How are commercial plasmids that incorporate a His-tag made useful?
|
They are available with a His-tag in frame with the multiple cloning site (MCS) for easy addition
|
|
|
What is His-tag purification called?
|
Immobilized Metal Affinity Purification (IMAC)?
|
|
|
A protein with a His-tag is engineered to have a very high affinity for what phase of the column?
|
Stationary phase
|
|
|
Why might additional purification be necessary following IMAC (immobilized metal affinity chromatography - His-tag)?
|
There are a few natural proteins that have affinity for Ni; to remove these proteins another purification step is necessary
|
|
|
Which of the three chromatography techniques we learned about in more detail are known for having high resolution?
|
+++ Affinity chromatography
++ Ionic exchange chromatography (NOT gel filtration/size exclusion) |
|
|
Which of the three chromatography techniques we learned about in more detail are known for having a high capacity?
|
+++ Affinity chromatography
+++ Ionic exchange chromatography (NOT gel filtration/size exclusion) |
|
|
Which of the three chromatography techniques we learned about in more detail are known for having a high specificity?
|
+++ Affinity chromatography
+ Ionic exchange chromatography (NOT gel filtration/size exclusion) |
|
|
Which of the three chromatography techniques we learned about in more detail are known for being sensitive to oligomeric state?
|
+++ Gel filtration / size exclusion
(NOT Affinity chromatography or Ionic exchange chromatography) |
|
|
Which of the three chromatography techniques we learned about in more detail are known for being the most informative?
|
+++ Gel filtration / size exclusion
+ Ionic exchange chromatography (NOT Affinity chromatography) |
|
|
What are the perks of affinity chromatography?
|
Great for purification from complex mixes (high resolution, high capacity, high specificity) if you have a tagged protein; but not sensitive to oligomeric state or informative
|
|
|
What are the perks of ionic exchange chromatography?
|
Good for complex mixes (good resolution, high capacity, can be sort of specific, and slightly informative); but will require other steps to achieve complete purity
|
|
|
What are the perks of gel filtration/size exclusion chromatography?
|
An analytical technique and also very useful to purify the subset of proteins that are in the native oligomeric state (active state, normally) from a pure protein sample; does not have high resolution, high specificity or high capacity
|
|
|
Is it more difficult to purify a protein expressed in a bacterial host or a protein extracted from its native tissue?
|
- It is much more complicated to purify a protein from a natural source!
- Protein expressed in bacterial host using recombinant technology can have a tag added for ease |
|
|
Why is it important to study enzyme kinetics?
|
- Determine if purified protein is active
- Determine kinetic parameters (Vo, Vmax, kcat, Km, kcat/Km) - Check for alternate substrates - Investigate inhibitors and inhibitory modes - Determine the residues involved in catalysis, active site cavity, or substrate/product departure |
|
|
Why is it not realistic to test the enzyme function of the biologically relevant reaction of HCAII (CO2 + H2O --> HCO3- + H+)?
|
- This reaction is extremely fast and requires special electrodes to measure
|
|
|
Why can the esterase function of HCAII be useful for understanding its enzyme function?
|
- Natural reaction of CO2 to HCO3- is extremely fast and requires special electrodes to measure
- HCAII also catalyzes PNPA to PNP in the SAME ACTIVE SITE |
|
|
What is the Michaelis-Menton equation?
|
Vo = Vmas*[S] / Km + [S]
|
|
|
What does Km, the Michaelis-Menton constant, equal?
|
Km = (kcat + k-1) / k1
|
|
|
What does Vmax equal?
|
Vmax = kcat*[E]total
|
|
|
What are three important things to remember about Km?
|
1. It is NOT a dissociation constant (Kd = k-1/k1)
2. It is an apparent steady-state dissociation constant 3. If k-1 >>> kcat then Km = k-1/k1 = Kd, but for most enzymes this is not the case! |
|
|
How can you determine what Km is experimentally?
|
- Graph Vo vs. [S] by measuring the change in substrate concentration with time at different substrate concentrations
- Km is the concentration of substrate where V = 1/2 Vmax |
|
|
When [S] >>> Km, what does Vo equal?
|
Vo = Vmax when [S]>>>Km
|
|
|
When [S] = Km, what does Vo equal?
|
Vo = 1/2 Vmax
|
|
|
What is a Lineweaver-Burke plot, a plot of? Why is it used? What are its weaknesses?
|
1/V vs. 1/[S]
- Linearization of M-M plot - Data is condensed near the y-axis; at low substrate concentrations there is greater error |
|
|
How does the Michaelis-Menton equation change for a Lineweaver-Burke plot?
|
MM: Vo = Vmax*[S] / Km + [S]
LB: 1/Vo = (Km/Vmax)*(1/[S]) + (1/Vmax) |
|
|
What is the slope of the Lineweaver-Burke plot? What is the y-intercept? What is the x-intercept?
|
- Slope = Km/Vmax
- y-intercept = 1/Vmax - x-intercept = -1/Km |
|
|
What is a Eadie-Hofstee plot, a plot of? Why is it used? What are its weaknesses?
|
Vo vs. Vo/[S]
- Helps correct for error at different substrate concentrations - Dependent variable on both axes |
|
|
How does the Michaelis-Menton equation change for a Eadie-Hofstee plot?
|
MM: Vo = Vmax*[S] / Km + [S]
EH: Vo = -Km*(Vo/[S]) + Vmax |
|
|
What is the slope of the Eadie-Hofstee plot? What is the y-intercept?
|
- Slope = -Km
- y-intercept = Vmax |
|
|
What are the three types of inhibition?
|
- Competitive
- Uncompetitive - Non-competitive |
|
|
What is a competitive inhibitor?
|
Inhibitor that directly competes with substrate binding
|
|
|
What is an uncompetitive inhibitor?
|
Inhibitor that binds to the ES complex
|
|
|
What is a non-competitive inhibitor?
|
Inhibitor does not change the binding of substrate or ES complex; most commonly allosteric
|
|
|
How does a competitive inhibitor affect substrate binding? Vmax? Km?
|
- Inhibitor directly competes with substrate binding
- Vmax is unchanged - Apparent Km increased resulting from a distribution of enzyme between "full affinity" and "no affinity" |
|
|
What is a Dixon plot, a plot of?
|
- 1/Vo vs. [I]
- Multiple lines with varying substrate concentrations held constant for each line |
|
|
How do you determine Ki for a competitive inhibitor
|
With a Dixon plot; inhibitor concentration where lines intersect reflects -Ki
|
|
|
On a Dixon plot, as substrate is increased, how do the slopes of the lines change?
|
- As substrate concentration increases, slope decreases
- Slope = Km / (Vmax*Ki*[S]) |
|
|
How does an uncompetitive inhibitor affect substrate binding? Vmax? Km?
|
- Inhibitor binds to ES complex preventing formation of E+P
- Vmax will be reduced - Apparent Km will be reduced |
|
|
How does a Dixon plot change between a competitive inhibitor and an uncompetitive inhibitor?
|
- Competitive inhibitor - lines intersect, where lines intersect indicates -Ki
- Uncompetitive inhibitor - lines are parallel to each other; as substrate concentration increases, reach a certain point where line with lowest y-intercept (the x-intercept corresponds to -Ki) |
|
|
How does a non-competitive inhibitor affect substrate binding? Vmax? Km?
|
- Inhibitor binds to both enzyme and enzyme-substrate complex
- Reduces Vmax - No effect on Km |
|
|
How do you determine Ki for a non-competitive inhibitor
|
- Generate a Dixon plot
- Lines from different substrate concentrations will intersect at the x-axis; this inhibitor concentration equals -Ki |
|
|
Why does absorption of a photon occur?
|
The energy associated with a photon is used to bring an electron from a lower level orbital to a higher energy one
|
|
|
What happens to the energy that is accumulated when an electron transitions to a higher energy state due to absorption of a photon?
|
- Normally the energy is given back to the system very quickly (10^-12 sec) and converted to heat
- In some cases the energy is released in the form of another photon |
|
|
When energy accumulates due to absorption of photons, what happens to the energy when it is released as a photon?
|
- Some energy is released in the form of a photon
- Not all of the energy absorbed with the first photon is converted back to the second photon |
|
|
What is the process of absorption and emission of photons?
|
1. Energy is absorbed (hv') bringing the electron from a certain sub-level of So to a sub-level of S1
2. The excited state rapidly decays to its lowest sublevel (relaxation) 3. The electron returns to a sub-level of the ground state So and a photon is emitted (hv'') |
|
|
Why is the energy emitted by an electron usually lower than the energy absorbed?
|
- Because of relaxation in the excited state (S1) vibrational sub-levels
- Also, interactions with the solvent may stabilize the excited state (S1) |
|
|
Lower energy affects the frequency and wavelength how? What equation relates these?
|
- Lower energy corresponds to a lower frequency (v) and a higher wavelength (λ)
- λ = c/v (c = speed of light) |
|
|
What is a fluorophore?
|
A molecule that has fluorescent properties
|
|
|
What kind of functional groups / orbital structures do fluorophores typically have?
|
- Generally aromatic compounds
- Large delocalized orbitals |
|
|
What does the quantum yield refer to? What is the equation for quantum yield, Q?
|
- A property of the fluorophore that defines how efficient the molecule is as a fluorophore
- Measure of how many photons are emitted compared to how many are absorbed - Q = Γrad / (Γrad + Γnon-rad) - Γrad is a rate constant of decay from the excited state with emission of radiation - Γnon-rad is a rate constant of non-radiative decay |
|
|
How is fluorescence measured? What are the components of this machine?
|
Spectrofluorometer
- Light source reflects off of mirror through monochromator - Beam passes through sample cell - Light that is emitted at 90* angle from sample cell hits another mirror and passes through another monochromator - Light hits detector |
|
|
Why is a second monochromator necessary for a spectrofluorometer?
|
The second monochromator is necessary because the fluorescence includes a range of wavelengths
|
|
|
What unit do we use for fluorescence measurements?
|
Fluorescence Intensity - relative number proportional to the number of photons captured by the detector
|
|
|
Why is absorption units not used for fluorescence?
|
- The light emitted from fluorescence is in all directions, but only part of it is collected
- So unless the system is very carefully calibrated, it is not generally used for quantitation |
|
|
What kind of unit is "absorption"?
|
A = log (Io/I)
- Very quantitative because we compare the intensity without the sample to the reduced intensity with the sample |
|
|
What is used to measure fluorescence in vivo? In vitro?
|
- In vivo = microscopic imaging techniques
- In vitro = using a spectrofluorometer |
|
|
What are some common examples of fluorescence in biochemistry?
|
- Intrinsic fluorescence of tryptophan to measure changes around its environment
- DNA probes - fluoresce when intercalated between DNA bases - FRET - Fluorescence Resonance Energy Transfer |
|
|
Why can Trp, Tyr and Phe be used as intrinsic fluorophores?
|
- These aromatic amino acids all have fluorescent properties (Trp used most often due to good quantum yield)
- Since they are relatively rare in proteins (<1% of all AA) they can be used - Need no addition of chemical labels |
|
|
Why is Trp usually the only of the three aromatic amino acids used for intrinsic fluorescence measurements?
|
- Phe has a low quantum yield
- Tyr has a normal quantum yield but its emission maxima is not as significantly different from its absorption maxima as it is for Trp - Trp has a normal quantum yield and its emission maxima is further from the absorption maxima |
|
|
What can the intrinsic fluorescence of Trp be used to monitor?
|
Used to monitor protein folding, by monitoring emission changes at 350 nm
|
|
|
If you have a folded protein with a buried Trp residue, what happens to the fluorescence intensity at 350 nm when it is denatured?
|
- Low intensity initially
- Once denatured, water exposed Trp increases the fluorescence intensity |
|
|
When are DNA probes not very fluorescent? When are they fluorescent?
|
- Not very fluorescent in water
- Acquire fluorescent properties when they intercalate between the DNA bases |
|
|
What is the most well known example of a DNA probe?
|
- Ethidium bromide - fluorescence is increased by 30 fold when it binds to DNA
|
|
|
What technique is important for studying molecular interaction?
|
FRET - fluorescence resonance energy transfer
|
|
|
When does FRET occur?
|
- When two fluorescent molecules are at close distance, the energy can be passed from an excited molecule ("donor") to the second fluorophore ("acceptor")
- The acceptor will then emit light at its characteristic wavelength |
|
|
What is necessary for energy transfer during FRET to occur efficiently?
|
- The fluorophores' (acceptors and donors) spectra need to overlap in a certain way and their distance needs to be below a certain range
- Ideally the emission spectra of the donor and the absorption spectra of the acceptor should overlap - There should be a wavelength in which the donor is excited but the acceptor isn't - There should be a wavelength in which the acceptor emits but the donor does not |
|
|
FRET occurs only at a short range and its efficiency decays how?
|
Very quickly with an r^6 power with distance
|
|
|
What is the equation for FRET efficiency, E?
|
E = Ro^6 / (Ro^6 + r^6)
Ro = Forster distance of the FRET pair r = distance |
|
|
What is the Forster distance, Ro? On what order is this value usually?
|
The distance at which FRET is 50%; typically in the order of ~50Å
|
|
|
What happens to the fluorescence in a molecular association application of FRET?
|
- Donor emission is reduced upon association (the energy is transferred)
- Acceptor emission is increased |
|
|
Which molecules were important for FRET in our experiment?
|
- HCAII which has 7 Trp residues
- Fluorescent ligand (dansyl amide) |
|
|
What is the most remarkable about the GFPs (Green Fluorescence Proteins)?
|
The reaction is self-catalyzed, which means that the proteins can be expressed in a foreign host by themselves and will acquire fluorescence without the need of specific enzymes
|
|
|
What is Kd?
|
The rate of dissociation of a ligand from the enzyme
|
|
|
What does Trp fluorescence depend upon? How so?
|
- Depends on the environment of each individual Trp residue
- Hydrophobic environments tend to increase fluorescence - Aqueous environments quench fluorescence |
|
|
In our experiment, at what wavelengths were the FRET experiments excited at? At what emission wavelengths were noted?
|
- Excitation at 295 nm (corresponding to absorption by Trp)
- Emission at 340 nm (corresponding to emission by Trp) - Emission at 470 nm = FRET (corresponding to emission by DNSA) |
|
|
How do you determine the Kd of DNSA binding to HCAII?
|
- Run FRET experiment
- Plot r vs [L] (determine r by calculating Fadjusted, Fmax, and Fmin) - Fit Langmuir Isotherm equation to obtain Kd(DNSA) |
|
|
What is necessary to determine "r" for the Langmuir Isotherm equation?
|
- r = (Fadj = Fmin) / (Fmax - Fmin)
- Fadjusted = (F470)(Final volume) / (initial volume) - Fmin = Fadjusted in absence of DNSA - Fmax = Fadjusted at saturating DNSA |
|
|
What is the Langmuir Isotherm equation?
|
r = [Lf] / (KdDNSA + [Lf])
|
|
|
What do enzymes do? What don't they do?
|
- Catalyze reactions
- They do not change the difference in energy between the substrate and the products - Enzymes do not affect the direction or equilibrium of the reaction - Enzymes do lower the transition barrier (ΔGϮ) and thus speed up the rate of the reaction |
|
|
Enzyme catalysis involves the formation of what?
|
Formation of complexes between the enzyme and the substrate (and product:
E + S <=> ES <=> EP <=> E + P |
|
|
Why must the energy of the unbound product be lower than for the ES and EP complexes?
|
Otherwise the enzyme would have a slow turn-over rate
|
|
|
Enzymes are optimized to bind what?
|
Transition state product
|
|
|
What are some examples of important enzymes that are drug targets?
|
- Reverse transcriptase - inhibitors block the replication of HIV
- Cyclooxygenase (COX) - inhibitors are potent anti-inflammatory drugs - Statins - inhibitors of HMG-CoA reductase used to lower blood cholesterol |
|
|
What are some of the uses of enzyme assays?
|
1. To detect the presence of an enzyme in a natural source (or in the expression medium if using recombinant technology)
2. Estimate total amount of enzyme present 3. Aid in the purification of an enzyme 4. To corroborate the purity of an enzyme, the specific activity should approach constant values as the prep approaches homogeneity |
|
|
What is a "unit"?
|
μmol product / min = UNIT
|
|
|
What is the "volumetric activity"?
|
- The amount of "activity" per a certain volume
- Volumetric activity = Unit / mL = μmol product / min*mL |
|
|
How do you estimate the total amount of enzyme present?
|
- Total activity = Volumetric Activity x Volume = UNITS
|
|
|
How can enzyme assays aid in the purification of an enzyme?
|
- During purification, enzyme assays can be used to identify the fractions that contain the enzyme
- Comparison with the estimation of the total amount of enzyme provides info about the percentage recovery |
|
|
What is the specific activity?
|
Specific Activity = total activity / total protein = Units/mg
|
|
|
Why is the unit "units" used as a measure of enzyme quantity?
|
It is a convenient way to express activity (what is needed for practical purposes) and it can be used even when amount, molecular weight, and purity are unknown
|
|
|
Why are colorimetric enzyme assays done when available?
|
- Very convenient
- Simple - Inexpensive - Reliable - Often very sensitive |
|
|
Why is the product of the esterase activity (PNP) useful for?
|
- The electronic structure of the delocalized pi orbital of the aromatic compound changes significantly when it is ionized
- PNPA (substrate) can not be ionized |
|
|
At what pKa does PNP become negatively charged / ionized?
|
7.1 (higher pH)
|
|
|
Why is it important to consider the pH at which the esterase activity assay was run?
|
- The pH at which we ran the reaction does not completely shift the PNP towards the absorbing form
- Therefore we need to obtain an extinction coefficient for our specific buffer conditions that takes into account the fact that ~80% of all the reagent is in the deprotonated highly absorbing form and 20% in the low absorbing form |
|
|
What are the characteristics of studying pre-steady-state kinetics?
|
- Require a complex apparatus
- Very powerful - Can observe a reaction milliseconds after it is started - Can detect transient enzyme bound species - Necessary to interrogate a mechanism |
|
|
What are the characteristics of studying steady-state kinetics?
|
- Simpler, although less powerful
- Measure enzymes in a state (steady state) - Can access variables such as Km, Kcat, Vmax |
|
|
What is Km?
|
Apparent dissociation constant of the ES complex
Km = Kcat + k-1 / k1 |
|
|
What is Kcat?
|
Catalytic constant ES-->P
|
|
|
What is Vmax?
|
Maximum speed of catalysis of saturation
|
|
|
What are some important assumptions for Michaelis-Menten kinetics?
|
- [E]o <<< [S] (much less enzyme than substrate)
- Vo is the initial rate (before more than the first few % of substrate have been depleted) - Vmax is the max theoretical velocity when all the enzyme is bound to substrate |
|
|
What is wrong with only fitting the lower end of the concentration range of substrate for MM plot or EH plot?
|
- Very hard to know where Vo will saturate (poor Vmax estimation)
- If you don't know Vmax it is hard to know Km - Poor estimation of both parameters - It is best to have points across the entire range of substrate concentrations |
|
|
What is wrong with only fitting the higher end of the concentration range of substrate for MM plot or EH plot?
|
- Vmax will be well estimated
- There is a lot of uncertainty about Km - It is best to have points across the entire range of substrate concentrations |
|
|
What is wrong with only fitting the middle concentration range of substrate for MM plot or EH plot?
|
- Still some inaccuracy of determining Km and Vmax
- It is best to have points across the entire range of substrate concentrations |
|
|
How does gel filtration / size exclusion chromatography separate molecules?
|
By size, or more correctly, by their hydrodynamic volume
|
|
|
What occurs in the column during gel filtration / size exclusion chromatography?
|
- Big particles are excluded by the gel and flow with the solvent
- The small particles can penetrate the gel and will be slowed down the most - Medium particles can penetrate some, they flow in between - Largest particles travel the fastest |
|
|
What kind of gel is used in gel filtration / size exclusion chromatography?
|
- Porous gel:
- Dextran, agarose, or acrylamide |
|
|
What is the equation for the total volume of a column?
|
V = h*pi*r^2
|
|
|
What is the total volume of a column broken up into? What do those terms represent?
|
- Excluded Volume (Ve) = volume around the particles
- Included Volume (Vi) = volume inside the particles + the excluded volume |
|
|
How is an NMR machine set up?
|
- Coil wound like spool of thread; current runs through it generating a magnetic field
- Coil is submerged in liquid helium (very cold) - Liquid helium surrounded by liquid nitrogen (also very cold) - Surrounded by vacuum chamber - Sample loaded in from top until it rests on top of probe |
|
|
What kind of nuclei can be "seen" by the NMR?
|
Only nuclei that possess a property called "spin"
|
|
|
What does it mean for a nuclei to have a spin?
|
If a nucleus can have more than one energy state in a magnetic field, the quantum spin number (l) is not 0, and energy transitions for this nucleus are possible
|
|
|
What does the quantum spin number (l) depend upon?
|
- Number of protons (Z)
- Number of neutrons (n) |
|
|
What is the Zeeman Effect?
|
When inserted in a magnetic field (B), nuclei that possess "spin" align themselves according to their energy states (either parallel or antiparallel)
|
|
|
Spins are split into what two populations?
|
Parallel: +1/2 (lower energy state than when no magnetic field)
Anti-parallel: -1/2 (higher energy state than when no magnetic field) |
|
|
What is a magnetic moment and what is it produced by?
|
The spinning, charged nuclei generate a magnetic field and possess a magnetic moment (μ) which is partially aligned along the magnetic field (Bo) axis
|
|
|
Why is it said that the magnetic moment of the spinning nuclei are "partially aligned along the magnetic field"?
|
The magnetic moment vector "precesses" around the z-axis like a spinning top
|
|
|
Why is there a tendency for more spins to "precess" (or spin like a top) around the component aligned with the +z (+1/2 parallel) direction?
|
Boltzmann! There are more spins in the lower energy state
|
|
|
What is the effect of the tendency for more spins to precess (spin like a top) with the +z (+1/2 parallel) direction?
|
This results in a net magnetization (Mo) vector in the +z direction
|
|
|
How do they knock the processing nuclei over 90* (and subsequently measure its relaxation)?
|
They use a radio frequency pulse which is specific for the different nuclei (H, C, N, F, etc)
|
|
|
What is FID?
|
Free Induction Decay - sinusoidal, exponential function modulated by a decay function; this is collected after the radio frequency pulse tips the net magnetization of the spinning nuclei
|
|
|
What kind of information does a 1D spectrum give us?
|
- Chemical shift (δ) - chemical environment
- Scalar coupling (J) - provides conformational info - Integration - area under curve provides ratio of atoms in chemical environment - NOE - nuclear Overhauser effect - |
