Biophysik der Moleküle 4. Vorlesung Rädler WS 2010 Protein folding - Afinsen hypothesis - hydrophobic interaction 28. Oct. 2010
Protein Unfolding: Sushi Restaurant 1. Distinguish salmon roe from imitation salmon roe by dropping into hot tea. 2. Mackerel is pickled in vinegar for preservation. When foods with proteins are exposed to heat and certain chemicals (such as vinegar), they turn white. Gaub/SS 2005 BPM 1.3 2
Gaub/SS 2005 BPM 1.3 3
The Thermodynamic Hypothesis (Afinsen 1973) the native state is thermodynamically stable C. Afinsen 1916-1995 => the sequence alone determines 3D structure! Nobel Prize for Chemistry in 1972
Afinsen s model protein: ribonuclease A alpha-helix beta-sheet loop (usually exposed on surface) ribonuclease A
Ribonuclease kann durch Oxidation (Spaltung der S-S Bindung) denaturiert werden o) Nofürtiche Ribonuclea se b)0enoturierte Ribonu cleose 95 H H 65 Abb. 3.12: Die zwei Zuslände der Ribonuklease: links: KomDakt! Funktionsfom rechts: Oenatudert! Form Das Enzym hat 8 s-s Bindungen. Im Prinzip könnten 56 verschiedene Zustände (Isomere) gebildet werden. Es gibt aber offenbar nur einen Zustand niedrigster Energie.
Folding of RNAse A in the test tube denaturation Incubate protein in guanidine hydrochloride (GuHCl) or urea (aggregation) renaturation 100-fold dilution of protein into physiological buffer - the amino acid sequence of a polypeptide is sufficient to specify its three-dimensional conformation Thus: protein folding is a spontaneous process that does not require the assistance of extraneous factors Anfinsen, CB (1973) Principles that govern the folding of protein chains. Science 181, 223-230. 7
However: Folding of proteins in vivo is promoted by chaperones this bears only on the rate of folding
What drives protein folding? Minimization of G=E-TS+PV Minimize the solvation energy. Decrease the conformational entropy. 9
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GFP Fluoreszenz Siehe Biophysik F-Praktikum
Other techniques to probe unfolding High-resolution techniques (local): FTIR Fluorescence NMR UV absorption Low-resolution techniques: SAXS DLS
Which forces are dominant in protein folding? Local vs. non-local interactions Nonlocal interactions drive collapse transition, whereas local interactions drive helix transitions.
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Early model in which protein folding was proposed to be driven by ion-paired hydrogen bonding among side chains (Mirsky& Pauling, 1936; Eyring & Stearn, 1939) disproven by Jacobsen and Linderstrom-Lang
Electrostatic Contributions! i =(z i e/4"# o )(1/r 2 ) coulomb potential Sensitive to ph and ion concentrations ph determines total charge (pi) Ionic strength determines effective range of interactions Ion pairs contribute 1-3 kcal/mol (on surface) Ion pairs generally destabilizing if buried (cost up to 19 kcal/mol/ion to completely bury Ion pairs contribute ~5-15 kcal/mol per 150 aa s
The Kauzmann Hypothesis hydrophobic interactions determine the thermal stability of the native state Key arguments * non-polar solvents denature proteins * unusual temperature dependence: (stability decreases at high AND low temperatures) * protein stability follows same salt dependence as lyotropic (Hofmeister) series
Determination of protein stability. This can be measured with a variety of tools including, microcalorimetry, spectroscopy, and enzyme function. The transition can be accomplished with heat or denaturants. The area under the curve gives $H which agrees with measurements based on the van't Hoff equation 20
Denaturants Temperature ph Change the ionization state of critical residues Detergents Bind strongly to the unfolded protein High concentrations of water soluble organic substances Aliphatic alcohols. These disrupt the water structure Ionic or polar denaturants including urea and guanidinium 21
Denaturants: The Hofmeister Series The ability of an ion to stabilize a protein follows the Hofmeister series Anions! SO 4 2->H 2 PO 4 ->CH 3 COO - >Cl - >Br - >I' - >ClO 4 ->SCN - Cations NH 4 +,Cs +,K +,Na + >Li + >Mg 2+ >Ca 2+ >Ba 2+!!! >guanidinium>urea 22
Thermal stability of RNase A as a function of salt This illustrates the effect on protein stability for many commonly used salts. Potassium phosphate and ammonium sulfate stabilize proteins which accounts for their frequent use in protein purification. From Voet and Voet second edition 23
The hydrophobic effect water forms cluster with coordination number 4 proteins are surrounded by a shell of structured water
Solubility and partition function chemical potential,!, and partition coefficient,x of oil molecule in water (w) and oil (K) 0 µ K,W = µ K,W + RT ln x K,W at equilibrium: ( ) µ W = µ K ln x * W = µ 0 0 K " µ W RT µ 0 K " µ 0 W = ( H 0 0 K " H ) W " T( S 0 0 K " S ) W enthalpic entropic The entropic change (cost of inducing water order) dominates over the enthalpy change (gain in intermolecular interaction), which is also negative. "µ = µ W 0 # µ K 0 = 2.44 + 0.88n C [kcal/mol] for alcanes "µ = #4.2 + 0.825n C for alcoholes
!"#$%&'()*%+,-"#./*0#).1&).2&"3#)4*,,#$,0&5#).+).6*,,#$.7#+.89:.; Stoff µ W 0! µ K 0 H W 0! H K 0 in kcal/mol in kcal/mol C 2 H 6 3.9-2.5-21 C 3 H 8 4.9-1.7-22 C 4 H 10 5.9-0.8-23 S W 0! S K 0 in cal/mol K H W 0! H K 0 S W 0! S K 0!"#$%&'$()*$+,-$.&%$/%&$.%-$0/%-+,1-2'3$%&'%"$(45"$.%"$67$84'$9$'):1$;$+-%&;%-.%'.%$ 7<-=%>$?)$.&%"%-$7%-#$'%3)@8$&"#A$&"#$.&%$0/%-+,1-2'3$%&'$%B4#1%-=%-$C-4D%*>$?&%$ E)'F.%-F7))5"FG'D&%12'3$D;&":1%'$.%=$67F(45%9,5$2'.$7)""%-$&"#$/%#-<:1#5&:1>$ $!"#$'%3)@8A$.)"$/%.%2#%#A$%"$H'.%#$%&'%$G/')1=%$.%-$I)-@%55%'$J'#-4I&%$/%&$.%- 0/%-+,1-2'3$.%"$67$84'$9$'):1$;$"#)K>$LJ&"/%-3"#-29#2-$.%"$7)""%-"M %=I&-&":1N!µ = µ W 0 " µ K 0 = 2.44 + 0.88n C ' : NO)15$.%-$6415%'"#4P)#4=%
Hydrophobic Effect At normal temp s the hydrophobic effect is entropic water molecules form ordered structures around nonpolar compounds Hydrophobic residues collapse in to exclude water Additional forces can then stabilize (vdw, h-bond,intrinsic properties) Hydrophobic effect is dependent on temperature (unstable at high AND low temp).
Thermodynamic considerations Protein stability is composed of two components. % % $G = $H-T$S There is a complex temperature dependence for $H and T$S which means that the contribution of the enthalpic and entropic terms changes with temperature. This temperature dependence arises from the anomalously high change in heat capacity on transferring hydrophobic compounds into water. This is the hall-mark of the hydrophobic effect and arises from the water-ordering.
Heat Capacity The heat capacity influences both the temperature dependence of the enthalpy and entropy Cp = "H "T = T"S "T It is proportional to the buried non-polar surface area as are all of the thermodynamic parameters. The large heat capacity is indicative of a well ordered water structure around non-polar molecules in water as is evident from their partial specific volumes when dissolved in water
Temperature dependence of $G Thermodynamics of transfer of a hydrocarbon from liquid to aqueous solution. The temperature dependence is the result of different heat capacities of the two phases. The large changes in $H and T$S compensate so that $G is fairly constant with temperature 31
Temperature dependence of $H and T$S continued $H becomes more favorable at lower temperatures, whereas the entropic term becomes less favorable. This is consistent with an increase in the order in the water surrounding the non-polar molecule. The water-ordering increases the interaction between solvent and solute and thus "enhances" the solubility that would occur in its absence. Even so, the interactions between solute and water eliminate hydrogen bonds within the water that cannot be compensated for by the ordering of the water. Significantly the van der Waals interactions are greater in the pure water and solute than in the dissolved solute. It is the loss of hydrogen bonds and van der Waals interactions that is the cause of the hydrophobic interaction. $H is ~0 at room temperature Terms counterbalance 32
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Buried hydrophobic surface area The buried hydrophobic surface area for a protein correlates with the protein stability. Although it is difficult to predict the overall stability of a protein, it is possible to predict the worst case scenario that a mutation might produce based on changes in buried surface area. Occlusion of 1Å 2 of hydrophobic surface area provides ~25 cal mol-1 of stability. 34
$C p vs $A np for proteins There is a linear correlation between the heat capacity change for protein unfolding and the buried non-polar surface area. This relationship is identical to that seen for the transfer of hydrocarbons from aqueous solution to the pure liquid phase From Livingstone JR, Spolar RS, Record MT Jr. Biochemistry. 1991 Apr 30;30(17):4237-44 35
Protein Unfolding: Pressure? 1895 Royer discovered that high hydrostatic pressure kills bacteria. 1899 Hite uses pressure for milk preservation. 1914 Bridgman notices that egg white looks cooked after pressure treatment. Though it isn t intuitive, proteins also unfold with pressure. 36
High-Pressure SAXS Study SAXS: shine X-ray on sample, look at scattering intensity vs. scattering angle. Guinier approximation: I~I o exp(-r g 2/3) Detect global size changes. -> for pressure studies, this may give the most relevant information. 37
Minimization of Volume atmospheric P hydrophobic packing? unfolding? More efficient packing is accomplished when small water molecules penetrate the hydrophobic core. (10 basket balls and 1000 golf balls pack the basket balls clustered or separated. Which takes up less space?)
Faltungsproblem Konformation eines Proteins als Random Walk: Mother nature has no folding problem, but we do! Gitter-Modell: Kleines Protein mit 100 Aminosäuren => Mögliche Konformationen: 3 100 " 10 30 Interne Dynamik typ ns &Zeit, um alle möglichen Kombinationen durchzuspielen " 10 21 s Vergleiche: Alter des Universums " 10 20 s!
How many conformations are there in the Native state? the HP model molten globules have many configurations the number of sequences that have N native states decreases strongly
The reason that only one native structure is encoded in the amino acid sequence may be largely attributable to the hydrophobic interaction; there are only a small number of ways to configure a chain to maximize the number of nonpolar contacts. These forces are of a nature such that proteins should be tolerant of amino acid substitution, a given native structure should be encodable in many different sequences, and a large fraction of all possible sequences should fold to compact structured native states.
in vitro Levinthal paradox in vivo denatured protein: random coil 10 30 possible conformations folding folding Native protein 1 stable conformation t = seconds or much less t = seconds 42
Up To Date No Unified Folding Theory