Thermodynamic measurements

Transcription

1 Thermoynamic measurements When stuying protein structure an function, it is essential to measure various physical quantities, incluing: thermal an chemical stability bining affinity to a ligan solubility ynamics an flexibility reactivity One woul like to preict thermoynamic properties from structure alone When two interacting molecules come together in water solvent molecules are release from the bining surface solute molecules form bons (H-bons, van er Waals contact, etc) e.g. preict an tabulate all enthalpic an entropic changes

2 Stability One of the common protein engineering goals is to stabilize a protein while maintaining its function increase stability means longer shelf life, obviates special treatments such as refrigeration or free-rying, makes it possible to isseminate protein rugs cheaply an effectively ease of maintenance an hanling (no lyophilize human growth hormone that nees to be reconstitute before use) may require chemical moifications or site-irecte mutagenesis High solubility an resistance to (proteolytic) egraation are important Structural integrity can be monitore by spectroscopic (CD, fluorescence), hyroynamic an chromatographic methos

3 Thermal an chemical stability Measurement of thermal/chemical stability is commonly use uring protein engineering to monitor the effects of mutations an quality of esign Caveat: proteins with poorly efine core can have extremely high stability Bryson et al, Protein Sci 7, 1404 (1998)

4 Chemical enaturation Aition of enaturing reagents such as urea an guaniine hyrochlorie (GnHCl) can shift the equilibrium between fole an unfole states fole (native) Keq unfole (enature) G = RT log( K eq ) Both isrupt the hyrogen bon network within the protein as well as the protein-solvent interaction, thus estabilizing the fole structure mechanism of enaturation is not fully unerstoo see Bennion an Daggett, PNAS 100, 5142 (2003) In part ue to its net charge, GnHCl is about twice as effective as urea in inucing protein enaturation (i.e. will unfol protein at roughly half the concentration)

5 Tryptophan fluorescence Fluorescence is absorption of light at wavelength λ a an subsequent emission of light at λ e (λ e > λ a ) Trptophan fluorescence (λ a ~ 278 an λ e ~ 348 nm) is use to stuy protein conformation (i.e. tertiary structure) The exact λ e an intensity of fluorescence epens critically on the local environment aroun the inole ring RNase T1

6 Creighton, Protein Structure, 2 n eition, Ch 12 y n K f eq = = y y f f fn = 1 f = y n n n y y y y y y n yn y RT log Keq = RT log = G y y Require sufficient ata points in all three regions: pre, post an transition Nee to ensure equilibrium has been reache may take a long time (> hrs) slowest uring the transition reversing the irection of the experiment shoul yiel an ientical curve

7 Measuring Keq is feasible only within the range of G = G(H O) m[ D] 2 where [D] is the concentration of enaturant, e.g. urea, GnHCl, an m measures the epenence of G on [D] At the mipoint of the enaturation curve, [D] 1/2 = G(H 2 O)/m y [ ] + m D y [ ] n + mn D y n, m n G(H O), m y, m 2 y = ( yn + mn[ D]) + ( y + m [ D])*exp{ ( G(H2O) m[ D]) / RT} 1+ exp{ ( G(H O) m[ D]) / RT} 2

8 Thermal enaturation As for chemical enaturation, etermine Keq for the transition region G = RT log( K ) = at + b G = 0 T m eq b Tm = a G = 0 = H T S T T T m m m ( G) In general, ( H ) HT = T S m T = T = T a 0 m T ( T ) or H H S = C p = C p ( U ) C p ( F) log( Keq ) = (van't Hoff) T RT R (log( Keq )) H vh = from a T T G( T ) = HT 1 log m C p Tm T + T (1/ T ) R linear fit Tm Tm

9 Differential scanning calorimetry (DSC) DSC can measure heat capacity of a sample by computing the amount of heat require to raise the temperature by a fixe amount: C p Q = T When the protein enatures, its heat capacity increases, resulting in a positive change in Cp Cp Alternatively, if a calorimeter is not available, Cp can be estimate: C p N 164*(# of SS)

10 Bining affinity Characterizing bining affinity is essential for many biochemical processes DNA Protein DNA K A + B A B K [ A][ B] = = K [ A B] 1 a If [A] >> [B], then the fraction of B boun to A is f = [ A] [ A] + K log [A] K is the concentration of A where half of B is boun

11 Isothermal titration calorimetry (ITC) ITC can measure most of commonly use thermoynamic parameters, incluing H, S, C p Electronics monitors the amount of heat that was ae to keep the two cells at the same temperature (~ µw) When a secon component is ae to the sample cell, either more or less heat is require to maintain the sample cell at the same temperature, epening on whether the bining is enothermic or exothermic Can measure K between 1 nm 1mM Labury an Chowhry, Chem & Biology 3, 791 (1996)

12 For the reaction A+nB A nb, the heat input/output Q is relate to the enthalpy of bining ( H), association constant (K a ) an stoichiometry of bining (n) as 1 2 4B Q = nav H X X 2 na B 1 X = 1+ + na nk A a A is the total amount of sample #1 in the cell, B is the final amount of sample #2 ae, V is the volume of the liqui 1 2 B A

13 Surface plasmon resonance (Biacore) SPR measures the bining of nanoparticles (e.g. protein, DNA, small molecules, carbohyrates, etc) on a metal surface (silver or gol) by monitoring the change in the local inex of refraction When polarize light unergoes total internal reflection, the intensity of the reflecte light is reuce at a specific incient angle ue to the resonance energy transfer between evanescent wave an surface plasmons

14 Refractive inex varies with the molecular weight of the boun substrate bining of a ligan to the receptor immobilize on the surface changes the molecular weight of the complex, hence the angle of minimum intensity Kinetic information may be obtaine from SPR by measuring Kon an Koff Kon Koff A + B A B K = K K off on Reference: Silin an Plant, TIBiotech 15, 353 (1997)