Science Science 249: Nature Struct. Biol. NSB l 3: NSB PNAS 96: NSB

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1 Discussion Papers 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: P17. Hughson,F.M., Wright, P.E., Baldwin, R.L. "Structural characterization of a partly folded apomyoglobin intermediate" Science 249: (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: (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: 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: 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: (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: (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:

2 water structure plays a critical role in folding: the hydrophobic effect slope ~ 25cal/Å 2

3 Measuring energetic contributions via ligand affinity, k cat /K m significant differences from transfer free energies transfer free energy Chymotrypsin activity on diff substrates Protein binding site 2.2x more hydrophobic than octanol Why?

4 How do we measure effects on protein stability? what fraction of native protein is unfolded?

5 How do we measure effects on protein stability? what fraction of native protein is unfolded? 1. To measure K eq, need to increase [U] How?

6 How do we measure effects on protein stability? what fraction of native protein is unfolded? 1. To measure K eq, need to increase [U] How? 2. Need to monitor either U or F to get K eq How?

7 Effects of denaturants on transfer free energy 7.1 cal/å cal/å 2 co-solvent -> H 2 0 transfer 18 cal/å 2 17 cal/å 2 octanol -> H 2 0 transfer

8 Unfolding demonstrated on Urea gradient gel

9 Spectroscopy: an ideal method for monitoring protein folding Spectroscopy 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 Wavelength (meters log scale) Ultraviolet Violet Blue Green Yellow Orange Red Infrared λ= nm 10-9 M (linear scale) Types of transitions : Microwave Rotational transitions Infrared Vibrational transitions UV/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

10 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 S 0 and S 1 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

11 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 S 1. 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). wavelength

12 Protein absorbance spectra Absorption spectra of the aromatic amino acids at ph 6. Spectroscopic properties of Aromatic amino acids at Neutral ph λ Max (nm) ε (M -1 cm -1 ) Phenylalanine Tyrosine Tryptophan

13 Measuring Protein Concentration For a protein in 6.0 M guanidine HCl (ph 6.5), 0.02 M phosphate ε 280 = N Trp * N Tyr * N S-S *120

14 Wittung-Stafshede, Pernilla et al. (1999) Proc. Natl. Acad. Sci. USA 96, Using Absorption spectroscopy to study folding cytochrome c: folding coupled to Fe redox state fraction folded

15 Fluorescence Spectroscopy: Jablonski Diagram Excitation: Absorption of a photon of energy hν EX creates an excited electronic singlet state S 1 Exited-State Lifetime: The energy of S 1 is partially dissipated (conformational changes, interactions with environment); this yields a relaxed singlet excited state S 1 Fluorescence Emission: The excited molecule returns to the ground state S 0 by emission of a photon with energy hν EM

16 Jablonski Diagram (cont.) S 0 : ground state S 1 : first excited state Vibrational energy levels s: transitions s: vibrational relaxation 10-8 s: excited state lifetime Fluorescence emission generally results from lowest vibrational level of S 1 Return to the ground state typically occurs to a higher vibrational ground-state level

17 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)

18 Consequences of vibrational relaxation 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.

19 Fluorescence Instrumentation 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 Light source Excitation Monochromator Sample photomultiplier Emission Monochromator

20 Protein fluorescence spectra absorbance fluorescence 100µM 6µM 1µM Spectroscopic properties of Aromatic amino acids at Neutral ph λ Max (nm) ε (M -1 cm -1 ) Fluorescence quantum yield Phenylalanine Tyrosine Tryptophan

21 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 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

22 Measurement of protein stability Emission spectra upon excitation at 278 nm of native and unfolded RNAse T1 Unfolding conditions: 8M urea Tryptophan fluorescence: generally see increased fluorescence intensity in the native state & blue-shift

23 Measuring stability for several barnase mutants Example from Fersht

24 The light illustrated is right- circularly polarized Circular Dichroism A solution of randomly oriented molecules will be optically active if the molecules are 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

25 Circular Dichroism - Proteins Short wavelength CD probes backbone amide 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 disulfide bonds at low wavelengths.

26 Circular Dichroism - Proteins 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 10 o C 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.

27 Circular Dichroism - Proteins Pro Gly Thr Example of the use of CD to monitor protein stability. Minor & Kim Nature (1994)

28 Measuring stability for several barnase mutants Example from Fersht Extrapolate to 0M urea to get stability Slope (m-value) proportional to Δ hydrophobic surface area ΔG ~ 50 cal/å 2 why not 25cal /Å 2??

29 Vast array of mutant studies on T4 lysozyme by Brian Matthews lab

30 Genetic screen for ts mutants in T4 lysozyme Mapped onto plot of B factors Not all residues contribute equivalently to stability

31 Doing structures of mutants is impt for understanding effects

32 Huge variations in ΔG for similar mutations Why?

33 Discussion Paper P16 Paper 16 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:

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35 Determined structures of all mutants Different mutants had quite diff cavity volumes

36 Probing configurational entropy contribution How about effects of Gly, Pro mutations?

37 Most simple mutations lead to small changes in m-value (understand via loss of interactions in N) But not in stapholococal nuclease

38 Two classes of SN-mutants remember: m-value slope of denaturation curves related to Δ hydrophobic surface area ΔG denat = ΔG o - m[denat] ΔG denat = ΔG o - k[denat](a D - A N ) ΔG denat = ΔG o - k[denat] ΔA Rationalizable by effects on unfolded state

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40 Protein stability vs temperature Proteins are both cold and heat sensitive (cs mutants) Note strong entropy/enthalpy compensation

41 How do proteins fold: 2 models

42 Separating out configurational entropy provides more informative view of energy landscape 2-state more complex single intermediate Chan & Dill Models of Energy Landscape

43 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?

44 (2): Detection of partially folded proteins 8-Anilino-1-naphthalene sulfonate ANS + molten globule ANS + buffer ANS fluorescence is strongly quenched in aqueous solution, but displays a large fluorescence enhancement in a nonpolar environment

45 Intermediate Denatured Native ANS

46 hydrogen exchange is a powerful tool to study protein folding rate of exchange related to H 2 O accessibility. Thus greatly slowed down in protein interior.

47 Hydrogen exchange measured in crystal of trypsin by neutron diffraction crystal soaked in D 2 O for ~ 1yr note non-exchanged H!

48 Hydrogen exchange in BPTI protection factor = k intrinsic /k Native typically

49 αlp residues having Protection Factors > unique suppression of Native state fluctuations => impt or function

50 Significant structure in The unfolded state