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

water structure plays a critical role in folding:the hydrophobic effect

slope ~ 25cal/Å2

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?

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

Unfolding demonstrated on Urea gradient gel

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

Measuring stability for several barnase mutants

Example from Fersht

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??

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

Genetic screen for ts mutants in T4 lysozymeMapped onto plot of B factors

Not all residues contribute equivalently to stability

Doing structures of mutants is impt for understanding effects

Huge variations in ΔG for similar mutations

Why?

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

Determined structures of all mutants

Different mutants had quite diff cavity volumes

Probing configurational entropy contribution

How about effects of Gly, Pro mutations?

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

How do proteins fold: 2 models

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

Intermediate

NativeDenatured

ANS

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!

Hydrogen exchange in BPTI

protection factor = kintrinsic/kNativetypically 104-106

αLP residues having Protection Factors > 1010

unique suppression of Native state fluctuations => impt or function

Significant structure inThe unfolded state