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P16. Eriksson AE, Baase WA, Zhang X-J, Heinz DW, Blaber M, Baldwin EP, Matthews BW. (1992) “Response of a protein structure to cavity-creating mutations and its relation to the hydrophobic effect” Science 255:178-183
P17. Hughson,F.M., Wright, P.E., Baldwin, R.L. "Structural characterization of a partly folded apomyoglobin intermediate" Science 249:1544-1548 (1990)
Schulman, B., Kim, PS., Dobson, CM., Redfield, C. “A residue-specific NMR view of the non-cooperative unfolding of a molten globule” Nature Struct. Biol. 4:630-634. (1997)
P18. Chamberlain AK; Handel TM; Marqusee S. (1996) "Detection of rare partially folded molecules in equilibrium with the native conformation of RNaseH" NSB l 3:782-7.
Raschke TM, Marqusee S. (1997) “The kinetic folding intermediate of ribonuclease H resembles the acid molten globule and partially unfolded molecules detected under native conditions.” NSB 4:298-304.
P19. Carrion-Vazques,M., Oberhauser,A.F., Fowler,S.B., Marszalek,P.E., Broedel,S.E., Clarke,J., and Fernandez,J. “Mechanical and chemical unfolding of a single protein: a comparison.” PNAS 96:3694-99 (1999)
Brockwell, D.J., Paci,E., Zinober,R.C., Beddard, G.S., Olmsted,P.D., Smith,D.A., Perham,R.N. and Radford, SE. “Pulling geometry defines the mechanical resistance of a -sheet protein.” NSB 10:731-737 (2003)
P20. Kenniston, JA, Baker, TA, Fernandez, JM, & Sauer, RT (2003) “Linkage Between ATP Consumption and Mechanical Unfolding during the Protein Processing Reaction of an AAA+ Degradation Machine” Cell 114:511-520.
Discussion Papers
Measuring energetic contributions via ligand affinity, kcat/Km
significant differences from transfer free energies
transfer free energy
Chymotrypsin activity on diff substrates
⇒Protein binding site 2.2x more hydrophobic than octanol
Why?
How do we measure effects on protein stability?
what fraction of native protein is unfolded?
1. To measure Keq, need to increase [U] How?
How do we measure effects on protein stability?
what fraction of native protein is unfolded?
1. To measure Keq, need to increase [U] How?
2. Need to monitor either U or F to get Keq How?
Effects of denaturants on transfer free energy
7.1 cal/Å2 8.3 cal/Å2 co-solvent -> H20 transfer
18 cal/Å2 17 cal/Å2 octanol -> H20 transfer
Spectroscopy: an ideal method for monitoring protein foldingSpectroscopy – Studies the interaction of electromagnetic radiation and matter
Electromagnetic Spectrum High Energy Light Microwaves Radiowaves Low Energy γ rays X rays UV VIS IR NMR | | | | | | | | | 10-12 10-10 10-8 10-6 10-4 10-2 1 10 2 10 4
Wavelength (meters – log scale)
Ultraviolet Violet Blue Green Yellow Orange Red Infrared | | | | | | | | | 400 500 600 700 800 λ= nm 10-9 M (linear scale)
Types of transitions : Microwave –Rotational transitionsInfrared – Vibrational transitionsUV/Vis Electronic transitions in the outer shell UV/ X-ray – Inner shell transitions
What is UV/Vis spectroscopy good for?
1) Quantitative analysis – molecule identity, concentration
2) Non-invasive probe of macromolecular structure and dynamics
What happens when light interacts with matter?
This figure shows a section of the potential energy surfaces of the two lowest electronic states of a typical simple molecule. Superimposed on these are a series of vibrational levels that are subdivided into rotational levels. The energy spacing between the lowest states of S0 and S1
is ~ 80 kcal mol-1. This energy is much greater than kT (~0.5 kcal mol-1).
Vibrational level spacing is ~ 10 kcal mol-1.
Rotational level spacing is ~ 1 kcal mol-1 or less.
Franck-Condon Principle – Electronic transitions are vertical
wavelength
A molecule is perturbed by light because its distribution of electric charge is altered by the presence of the oscillating electric field of the incident wave. When light of the correct frequency is absorbed, the molecule can be excited to one of many vibration-rotation levels of the electronic state S1. In the absence of other effects, one should see a very complex spectrum, composed of many sharp spectral bands that are rich in information (Top). In practice, each of these bands is so broad that one generally observes a smooth envelope (Bottom).
Protein absorbance spectra
Phenylalanine 257.4 197
Tyrosine 274.6 1420
Tryptophan 279.8 5600
λ Max (nm) ε (M-1 cm-1)
Spectroscopic properties of Aromatic amino acids at Neutral pH
Absorption spectra of the aromatic amino acids at pH 6.
Measuring Protein Concentration
For a protein in 6.0 M guanidine HCl (pH 6.5), 0.02 M phosphate
ε280 = NTrp*5690 + NTyr* 1280 + N S-S*120
Wittung-Stafshede, Pernilla et al. (1999) Proc. Natl. Acad. Sci. USA 96, 6587-6590
Using Absorption spectroscopy to study folding
fract
ion
fold
ed
cytochrome c: foldingcoupled to Fe redox state
Fluorescence Spectroscopy: Jablonski Diagram• Excitation: Absorption of a photon of energy hνEX creates an excited electronic
singlet state S1’
• Exited-State Lifetime: The energy of S1’ is partially dissipated (conformational changes, interactions with environment); this yields a relaxed singlet excited state S1
• Fluorescence Emission: The excited molecule returns to the ground state S0 by emission of a photon with energy hνEM
Jablonski Diagram (cont.)
S0: ground stateS1: first excited stateVibrational energy levels10-15s: transitions10-12s: vibrational relaxation10-8s: excited state lifetime
• Fluorescence emission generally results from lowest vibrational level of S1
• Return to the ground state typically occurs to a higher vibrational ground-state level
Consequences of vibrational relaxation
1.Stokes shift:
The energy of the emitted photon is lower (therefore longer wavelength) than the excitation photon:
Stokes Shift = (hνEX - hνEM)
The Stokes shift is fundamental to the sensitivity of fluorescence techniques:Emission photons can be isolated from excitation photons!(Contrast to absorption: requires measurement of transmitted light relative to high incident light levels at the same wavelength)
2. Under the same conditions, the fluorescence emission spectrum is independent of the excitation wavelength.
3. The emission intensity is proportional to the amplitude of the fluorescence excitation spectrum at the excitation wavelength.
Consequences of vibrational relaxation
Fluorescence Instrumentation
Light source
Excitation Monochromator
Sample
Emission Monochromator
photomultiplier
• Spectrofluorometers / microplate readers: average properties of bulk samples
• Fluorescence microscopes: resolve fluorescence as a function of spatial coordinates
• Fluorescence scanners/microarray readers• Flow cytometers: measure fluorescence per cell in a flowing stream
Protein fluorescence spectra
Phenylalanine 257.4 197 .04
Tyrosine 274.6 1420 .21
Tryptophan 279.8 5600 .20
λ Max (nm) ε (M-1 cm-1) Fluorescence quantum yield
Spectroscopic properties of Aromatic amino acids at Neutral pH
absorbance fluorescence100µM 6µM 1µM
Sensitivity of fluorescence to the environment
is due the to relatively long time a molecule stays in the excited state (absorption and CD are over in 10-15 sec!)
During this time, a number of processes can occur:• Protonation/deprotonation• Solvent-cage relaxation• Local conformational changes• Processes coupled to rotational/translational motion
Fluorescence is therefore dependent on:• Solvent polarity• Proximity and concentration of quenching species• pH of the aqueous medium
Measurement of protein stability
Emission spectra upon excitation at 278 nm of native and unfolded RNAse T1Unfolding conditions: 8M urea
Tryptophan fluorescence:
generally see increased fluorescence intensity in the native state & blue-shift
Circular DichroismA solution of randomly oriented molecules will be optically active if the moleculesare asymmetric. Differential absorbance of right versus left hand circularly polarized light is known as circular dichroism.
Example of elliptically polarized light emerging towards the observer form a circularly dichroic sample
The light illustrated is right- circularly polarized
α-helices show a strong characteristic double minima at 208 and 222 nm. This can be very diagnostic. β-sheets show a weaker CD signal with a broad minimum around 218 nm. It is not so clean or easy to deconvolve the CD spectra of an average protein into its components because of possible significant and unpredictable contributions from aromatic residues and disulf ide bonds at low wavelengths.
Circular Dichroism - Proteins
Short wavelength CD probesbackbone amide
Aromatic sidechains as well as disulfide bonds display CD bands in the far UV. The magnitude of the contributions cannot be readily computed.
A) Aromatic CD spectra of RNase at 10oC in the folded (solid line) and unfolded (dashed line) states. (Protein conc 78 mM, 1 cm cell. pH 6.0, 0.1M sodium cacodylate (6M GdmHCl)) B) Far UV CD spectra of RNase under the same solution conditions as (A), protein concentration 28 mM in a 0.1cm cell.
Circular Dichroism - Proteins
Pro
Gly
Thr
Circular Dichroism - Proteins
Example of the use of CD to monitor protein stability.
Minor & Kim Nature 371 264-267 (1994)
Measuring stability for several barnase mutants
Extrapolate to 0M urea to get stability
Example from Fersht
Slope (m-value) proportional to Δ hydrophobic surface areaΔG ~ 50 cal/Å2 why not 25cal /Å2??
Genetic screen for ts mutants in T4 lysozymeMapped onto plot of B factors
Not all residues contribute equivalently to stability
31
Eriksson AE, Baase WA, Zhang X-J, Heinz DW, Blaber M, Baldwin EP, Matthews BW. (1992) “Response of a protein structure to cavity-creating mutations and its relation to the hydrophobic effect” Science 255:178-183
Discussion Paper P16
Paper 16
Most simple mutations lead to small changes in m-value (understand via loss of interactions in N)
But not in stapholococal nuclease
Two classes of SN-mutants
Rationalizable by effects on “unfolded state”
remember:m-value slope of denaturation curves related to Δ hydrophobic surface areaΔGdenat = ΔGo - m[denat]ΔGdenat = ΔGo - k[denat](AD - AN)ΔGdenat = ΔGo - k[denat] ΔA
Protein stability vs temperature
Proteins are both cold and heat sensitive (cs mutants)Note strong entropy/enthalpy compensation
2-state
Separating out configurational entropy providesmore informative view of energy landscape
Chan & Dill Models of Energy Landscape
single “intermediate”
more complex
Searching for intermediates
i) biphasic transitions
ii) unusual solvent conditions (low pH, etc) a) expanded but compact b) 2° structure c) little or no 3° structure d) ANS binding => Molten Globule
iii) what do MG's look like?
(2): Detection of partially folded proteins
ANS fluorescence is strongly quenched in aqueous solution, but displays a large fluorescence enhancement in a nonpolar environment
8-Anilino-1-naphthalene sulfonate
ANS + buffer
ANS + molten globule
hydrogen exchange is a powerful tool to study protein folding
rate of exchange related to H2O accessibility. Thus greatly slowed down in protein interior.
Hydrogen exchange “measured” in crystal of trypsin by neutron diffraction
crystal soaked in D2O for ~ 1yrnote non-exchanged H!
αLP residues having Protection Factors > 1010
unique suppression of Native state fluctuations => impt or function
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