Primary structure effects on peptide group hydrogen exchange

Abstract

The rate of exchange of peptide group NH hydrogens with the hydrogens of aqueous solvent is sensitive to neighboring side chains. To evaluate the effects of protein side chains, all 20 naturally occurring amino acids were studied using dipeptide models. Both inductive and steric blocking effects are apparent. The additivity of nearest-neighbor blocking and inductive effects was tested in oligo- and polypeptides and, surprisingly, confirmed. Reference rates for alanine-containing peptides were determined and effects of temperature considered. These results provide the information necessary to evaluate measured protein NH to ND exchange rates by comparing them with rates to be expected for the same amino acid sequence is unstructured oligo- and polypeptides. The application of this approach to protein studies is discussed.

Fig. 1

H to D exchange of peptide NHs in the alanine dipeptide reference molecule (a–c) and for oligo- and polypeptide reference molecules (d) at 5°C in 0.5 M KCl. a: NMR spectra after various times of exchange at pD_read 4.05; b: exponential decay of the NH resonances; c: dependence of H-D exchange rates on pD_read; d: H-D exchange behavior of alanine oligo- and polypeptide reference molecules. The data in c and d are fit with rate constants for acid, base, and water catalysis (solid curves) to obtain the alanine reference rate constants listed in Table I. Results for acid catalysis of PDLA exchange in low salt concentration (○) were treated separately (rates 20% slower); results for base catalysis at high (0.5 M KCl) and low salt concentrations agree within 7% and were merged.

Fig. 2

a–d: H-D exchange behavior of neutral polar amino acids in dipeptide models. The leftward shift relative to the alanine dipeptide reference curve (dashed line) results from an inductive effect in which electron density is withdrawn from the peptide group, increasing the base-catalyzed rate and decreasing the acid-catalyzed rate. The side chain parameters in Table II quantify the changes in acid and base rate constants for the L and R peptide NHs in these residues.

Fig. 3

H-D exchange behavior of titratable amino acids in dipeptide models. The dashed curves show the alanine dipeptide reference rates. a,b: Lys and arg were fit by the usual three parameters for acid, base, and water catalysis. c,d: His, Asp, and Glu require additional rate constants for both the protonated and unprotonated forms. Values for pK_a,read in D₂O at 5°C are: His, 7.02; Glu, 4.53; Asp, 4.08. To shield coulombic effects these solutions contained 0.50 M KCl, as did all other solutions used here.

Fig. 4

a–d: H-D exchange curves of aliphatic and aromatic amino acids in dipeptide models. The downward shifts relative to the alanine curve (dashed line) reflect side chain-dependent steric hindrance effects that slow both acid and base rates. The aromatics show both steric and inductive effects.

Fig. 5

H-D exchange curves for the peptide NHs of Asn and Gln dipeptide models (a,b) and for the R peptide of the proline dipeptide model in cis and trans forms (c). The dashed curve is the alanine reference.

Fig. 6

H-D exchange curves for NHs in some test oligopeptides. The dashed curve shows the exchange behavior of an internal NH in the reference trialanine peptide (from Fig. 1d). For each residue NH underlined, the solid line is the curve predicted using Eq. (2) with the reference rates in Table I and the side chain-specific factors in Table II. The success of the predictions demonstrates the additivity of inductive and blocking effects. The unblocked oligopeptides (free amino and carboxyl termini) are: Lys-Val-Ile-Leu-Phe (KVILF); Lys-Ala-Ile-Val-Asn-Lys (KAIVNK); Gly-Ala-Val-Ser-Thr-Ala (GAVSTA). Exchange behavior of the last NH of each oligopeptide was affected by titration of the terminal carboxyl group in the pD region measured and is not shown.

Fig. 7

Water-catalyzed HX rate: a: Water rates measured for the dipeptide NHs are correlated with the relative basicity of the peptide group, expressed in terms of the shift in pD_min relative to the alanine dipeptide. Data are shown for the 17 dipeptides (including Cys₂) with well-determined water rates (Pro and the titratable residues—Asp, Glu, and His—are excluded), b: To test predictive schemes for the water rate, water rates predicted as in the last term in Eq. (2a) (using only the base factors) are compared with water rates measured by fitting the oligopeptide data in Figure 6. The data points represent the eight moderately well-determined NHs in Figure 6 (left-most panels excluded). The diagonal at unit slope indicates the position for a perfect prediction of the rates.

Fig. 8

Overall H-D exchange behavior of the NHs in random chain oxidized ribonuclease near the pD_min (center) and where dominated by acid (left) and base (right) catalysis (5°C). The solid line is the H-D exchange curve obtained by summing the predicted exchange behavior of all the individual NHs in the polymer. The dashed line shows the reference HX behavior of PDLA drawn with an amplitude of 119 NHs, the number of NHs in ribonuclease.

Fig. 9

H-D exchange curves for some side chain NHs in dipeptide models in D₂O at 5°C. Rate constants are listed in Table IV.