and Hydrophobic-Induced pK, Shifts in Polypentapeptides - American

Page 1 ... pK, Shifts in Polypentapeptides: The Glutamic Acid. Residue. D. W. Urry,' S. ... 0.35 in the hydrophobic domain, salt decreases the pK, shi...
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J. Am. Chem. SOC. 1993,115, 7509-7510

Delineation of Electrostatic- and Hydrophobic-Induced pK, Shifts in Polypentapeptides: The Glutamic Acid Residue

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Polypentapeptides ofthe family ~ ~ ~ ~ V ; ( I P G V G ) ~ E ( I P ,lG E G ) ] where fv and fE are mole fractions with fv + fE = 1, were synthesized and characterized as to pKa in water and in 0.15 N NaCl with fEvaried from 1 to 0.06. As thevalueof fE is decreased from 1.0 to 0.7, in saline there is a slight increase in pKa from 4.35 to 4.40 and in water there is a decrease in pKa from 4.70 to 4.35 as the chargecharge repulsion is relieved due to dilution and polar moieties for hydration in a structurally-constrained, of charge. This is the chargecharge repulsion (electrostatic) and hence, water-limited, polymer system. domain. As the value of fE is further decreased from 0.7 toward The broader family of polypentapeptides, polyV;(VPGVG)fs 0 and the hydrophobicity increases as Val replaces Glu, in both (aPGPG)], where fv and fap are mole fractions with fv + fap = saline and water the pKa increases from the value near 4.4 to a 1, where a may be one of a few hydrophobic residues, e.g., Val, value of 6.6 in water and to a lower value of 6.1 in saline. The Ile, Phe, etc., and where @maybe any of the naturally occurring values OffE < 0.7 represent the hydrophobic domain in which the amino acid residues and chemical modifications thereof, exhibits hydrophobic-induced pKa shift dominates. These results unaman inverse temperature transition in which all members are soluble biguously delineate the domains for hydrophobic- and electroin water, if low enough temperatures can be reached, and on static-induced pKa shifts in these polypentapeptides, and they raising the temperature they hydrophobically fold and assemble suggest that in aqueous systems the latter is the dominant means in a phase separation as the more-structured water of hydrophobic of shifting pKa values. Nonetheless, for values of fE less than hydration becomes less-structured bulk water. The temperature 0.35 in the hydrophobic domain, salt decreases the pK, shift. Tt at which the inverse temperature transition occurs depends on Because of this, as discussed below, the decrease in affinity between the hydrophobicity of the a and @ residues, and a complete TIprotein subunits on addition of salt cannot be considered diagnostic based hydrophobicity scale has been obtained for all of the of the electrostatic domain. naturally occurring amino acids and certain chemical modifiTen polypentapeptides, poly[fv(1PGvG)f~(IPGEG)], with cations thereof.4 values offEvarying from 1 to 0.06, were synthesized as previously Increasing the degree of ionization of a Glu side chain strikingly The syntheses and compositions were verified by increases the value of Tt.5 Also from differential scanning amino acid analyses and carbon-1 3 nuclear magnetic resonance. calorimetry, the endothermic heat of the transition, interpreted The amino acid titration experiments were carried out at 37 OC, as the heat required to destructure the hydrophobic hydration, as previously de~cribed,~ using 45 min per data point (requiring is seen to decrease as the degree of ionization increases.6 This, approximately 24 h for each of three or more titrations per reported along with other data,5 indicates that the polar species, in the pKa value) and both in Type 1 (low-conductivity) water and in process of achieving their hydration shells, destructure the water 0.1 5 N NaC1. The plots of fE versus pKa are given in Figure 1 of hydrophobic hydration. Conversely, as the hydrophobicity for water and saline. increases, it becomes more difficult for an emerging carboxylate The plots in Figure 1 readily divide into two domains with anion to achieve its required hydration shell, as this necessitates respect to fE: fE > 0.65 and fE < 0.65. In water, the lowering destructuring of hydrophobic hydration. The consequence is the of the pKa on going from anfof 1.O to 0.7 is due to the removal hydrophobic-induced pKa The free energy for this of the chargecharge repulsion-induced pKa shift as the negatively competition for hydration, called an apolar-polar repulsive free charged carboxylate side chains of Glu become more dilute. In energy of hydration, can be obtained from the pKa shift, i.e., Ap salt, 0.15 N NaC1, the chargecharge repulsion is shielded by the = -2.3RT6pKa where Ap is the change in chemical potential of ions in solution. The fE > 0.65 values are clearly within the the proton indicated by the pKa shift, R is 1.987 cal/mol deg, and electrostatic domain. For the range of valuesfE < 0.65 in both Tis in Kelvins. With the largest hydrophobic-induced pKa shift water and physiological saline, as the mole fraction of Glubeing from 4.35 to 6.61, the Ap (the Gibbs free energy per mole) containing pentamers decreases below 0.65, the increases in pKa arising from the apolar-polar repulsive free energy of hydration become progressively greater as the mean hydrophobicity inis 3.2 kcal/mol. creases. This is the hydrophobic, or apolar-polar repulsive Waters of hydrophobic hydration, in general, cannot simulinteraction, domain. It is important to note that even in the taneously serve as waters of polar hydratioms Stated macrohydrophobic domain, for values of fE of 0.35 and less, the effect scopically, this means a lower effective dielectric constant of salt to reduce the pKa shift is the same as in the electrostatic surrounding the negatively charged moieties. Similar pKa shifts domain. This is discussed in terms of the competition of apolar

* To whom correspondence should be addressed.

(1) The singleletter codes for the amino acid residues are Gly(G), Val(V),

Ile(I), Pro(P), and Glu(E). (2) Zhang, H.; Prasad, K. U.; Urry, D. W. J . Protein Chem. 1989, 8, 173-182.

( 3 ) Urry, D. W.; Peng, S.Q.; Parker, T. M. Biopolymers 1992,32,373379.

OOQ2-7863/93/1515-7509$04.0Q/0

(4) Urry, D. W.; Gowda, D. C.; Parker, T. M.; Luan, C.-H.; Reid, M. C.; Harris, C. M.; Pattanaik, A.; Harris, R. D. Biopolymers 1992, 32, 12431250. ( 5 ) Urry, D. W. Angew. Chem., Int. Ed. Engl. 1993, 32, 819-841. (6) Urry, D. W.; Luan, C.-H.; Harris, R. D.; Prasad, K. U. Polym. Prepr., Am. Chem. SOC.Diu. Polym. Chem. 1990, 31, 188-189. (7) Urry, D. W.; Peng, S. Q.; Hayes, L.; Jaggard, J.; Harris, R. D. Biopolymers 1990, 30, 215-218.

0 1993 American Chemical Society

7510 J . Am. Chem. Soc., Vol. 115, No. 16, 1993 are seen for positively charged species, where increased hydrophobicity results in a decrease in the pK, of a lysyl side-chain ammonium moiety (D. W. Urry, S.Peng, D. C. Gowda, T. Parker, and R. D. Harris, unpublished results). Again, the effective dielectric constant surrounding the ammonium moiety could be considered to decrease with increased hydrophobicity. Therefore, if two protein subunits had interfacial lysine and glutamic acid residues with hydrophobically shifted pKa values, the Coulombic attraction between the two would be increased. That attraction, due to the hydrophobic domains in which the Lys and Glu residues were located, would be decreased by the addition of salts. Analogously, the addition of salt to the medium of a protein with a hydrophobically shifted pKa of a Glu residue would, by providing counterions, relax the apolar-polar repulsive free energy of hydration and decrease the magnitude of the pK, shift, as seen in Figure 1 for fE for less than 0.35. In Figure 1 the electrostatic-induced pKa shift is less than the hydrophobic-induced pKa shift. The charge density can, of course, be increased in polypeptides or proteins to more than occurs in these polypentapeptides, that is, to more than one carboxylate per five residues which is a charge density of one carboxylate per 15 backbone atoms. The charge density could not be made as great as in poly(methacry1ic acid) where there can be one carboxylate for every two backbone atoms. With the very large charge-charge repulsion in poly(methacry1ic acid), the pKa is raised only to 7.3.8 On the other hand, the hydrophobicity can also be greatly increased. With an fB of 0.17 and with five Phe residues per tricosamer, a pK, of 8.1 has been observed.9 Even larger shifts could conceivably occur for Glu residues in globular proteins with sufficiently hydrophobic domains or in membrane (8) Katc:halsky, A. J. Polym. Sei. 1951, 7, 393412. (9) Urry, D. W.; Gowda, D. C.; Peng, S.Q.;Parker, T. M.; Harris, R. D. J . Am. Chem. Soc. 1992, 114, 8716-8717.

Communications to the Editor

proteins. Thus it is not unreasonable to expect that the larger pKa shifts are due dominantly to the apolar-polar repulsive free energy of hydration. Amino acid residues with shifted pK, values have been increasingly identified as key to protein function whether observed in globular proteins in solution1@15or in proteins in membranes.16-17 Accordingly, as the apolar-polar repulsive free energy of hydration is the most effective means of shifting pK, values, it should be considered to be significant in general in protein mechanisms. More specifically, the apolar-polar repulsive free energies of hydration have been used to achieve free energy transductions involving the intensive variables of mechanical force, temperature, pressure, chemical potential, electrochemical potential, and electromagnetic r a d i a t i ~ n . ~Furthermore, J~ chemomechanical transduction has been demonstrated, when using the apolarpolar repulsive free energy of hydration mechanism, to be 1 order of magnitude more efficient than when using the charg-harge repulsion mechanism.5.18

Acknowledgment. This work was supported in part by contract NOOO14-89-5-1970 (to D.W.U.) from the Department of the Navy, Office of Naval Research. (IO) McGrath, M. E.; Vasquez, J. R.; Craik, C. S.;Yang, A. S.;Honig, B.; Fletterick, R. J. Biochemistry 1992. 31. 3059-3064. (1 1) Langetsmo, K.; Fuchs, J:A.; W'oodward, C. Biochemistry 1991,30, 7603-7609. (12) Langetsmo,K.; Fuchs, J. A.; Woodward, C.;Sharp,K. A. Biochemistry 1991, 30, 7609-7614. (13) Dao-pin, S.;Anderson, D. E.; Baase, W. A,; Dahlquist, F. W.; Matthews, 8. W. Biochemistry 1991, 30, 11521-11529. (14) Inoue,M.;Yamada,H.;Yasukochi,T.;Kuroki,R.;Miki,T.;Horiuchi, T.; Imoto, T. Biochemistry 1992, 31, 5545-5553. (15) Auzat, I.; Garel, J.-R. Protein Sci. 1992, 1, 254258. (16) Khorana, H . G . Proc.Nutl. Acud.Sci. U.S.A. 1993,90, 1166-1171. (17) Okamura, M. Y.; Feher, G. Annu. Reu. Biochem. 1992,61,861-896. (18) Urry, D. W. Prog. Biophys. Molec. Biol. 1992, 57, 23-57,