Journal of Molecular Biology
CommunicationPropensities of Aromatic Amino Acids versus Leucine and Proline to Induce Residual Structure in the Denatured-State Ensemble of Iso-1-cytochrome c
Introduction
There is strong evidence that nonrandom structure persists in disordered and denatured proteins.1, 2 Nonrandom structure is often local in nature,3 with persistent secondary structure being common.4, 5 However, recent work, especially with paramagnetic relaxation enhancement2, 6, 7, 8 and transverse relaxation NMR methods,9, 10, 11, 12 has shown that nonrandom structure can be stabilized by long-range tertiary interactions. Residual structure is most prominent in the absence of denaturing agents13, 14 and under weaker denaturing conditions.5, 6 In many instances, though, significant residual structure persists in the denatured state in the presence of 3 M guanidine hydrochloride (GdnHCl)5, 6, 15 or even 8 M urea.3, 11
There has been significant progress in the quantitative evaluation of the free energy associated with electrostatic interactions in the denatured-state ensemble (DSE).16, 17, 18, 19, 20, 21, 22 Electrostatic interactions have been shown to stabilize the DSE by 1–4 kcal mol− 1. Thus, electrostatic effects in the DSE can strongly influence the overall stability of a protein. Mutational studies on surface-exposed sites for several proteins show reverse hydrophobic effects indicating that hydrophobic interactions are important in the DSE of proteins as well.23, 24, 25, 26, 27, 28 Mutation of charged residues to hydrophobic residues has been shown to decrease the heat capacity change ΔCp for thermal unfolding, consistent with the formation of hydrophobic clusters in the DSE.29, 30 Simulation and experiment on the drkN SH3 domain, barnase, and hen egg white lysozyme have led to the conclusion that aromatic residues particularly promote hydrophobic clusters in the DSE.10, 11, 14, 31, 32, 33, 34, 35, 36, 37 However, simulation and experiment on chymotrypsin inhibitor 2 and α-lactalbumin show that larger aliphatic side chains are also sufficient to nucleate hydrophobic clusters in the DSE.9, 38 Various hydrophobicity scales rank the relative hydrophobicities of aliphatics and aromatics differently.39, 40, 41, 42 Thus, a quantitative assessment of the relative tendencies of these two types of amino acid side chains to promote residual structure under a particular set of denaturing conditions would be useful. Similarly, a comparison of the relative impact of electrostatic versus hydrophobic interactions on the energetics of the DSE would be valuable.
To quantify the relative free energies of hydrophobic residual structure interactions induced by aromatic and aliphatic residues in the DSE, we apply our denatured-state histidine–heme loop formation assay.23, 43, 44, 45 In particular, we substitute single aromatic or aliphatic residues into a 22-residue loop formed in the denatured state of yeast iso-1-cytochrome c (iso-1-Cytc) and measure the effect on loop stability. For comparison, we have also measured the effect of the insertion of a single proline into the loop to evaluate the relative importance of backbone versus side-chain effects on the DSE.
Section snippets
Design of variants
We use a host–guest approach to evaluate the effects of proline, aromatic amino acids, and the large aliphatic amino acid leucine on His–heme loop formation in the DSE of iso-1-Cytc. We have used leucine to represent large aliphatic residues because it is not β-substituted like valine and isoleucine; thus, its effect on main-chain sterics will be more similar to the effect of aromatic side chains on main-chain sterics.46 The host sequence that we have used to examine the effects of these amino
Theoretical treatment of loop formation
When a His–heme loop forms in the denatured state of a c-type cytochrome such as iso-1-Cytc, a proton is released (Fig. 2). Thus, an apparent pKa, pKa(obs), can be extracted from the midpoint of the pH-dependent loop formation equilibrium. Since this equilibrium is essentially a competition between the heme and a proton for binding to the histidine, a lower pKa(obs) indicates a more stable His–heme loop. This equilibrium can be treated as a stepwise process involving the deprotonation of
Stability of variants
Variants were prepared using the pBTR1 vector51, 52 carrying the NH5A variant as template for standard PCR-based mutagenesis. Proteins were expressed in BL21(DE3) Escherichia coli cells, as previously described.47, 48, 52 These variants all have a free N-terminal amino group that can bind to heme under denaturing conditions.53 However, histidines on the N-terminal side of the site of heme attachment have high-enough affinity for the heme47, 48, 54 that binding of the N-terminal amino group to
Equilibrium His–heme loop formation
The His–heme loop stabilities for each variant were measured under denaturing conditions (3 M GdnHCl) by increasing proton concentration in steps of ∼ 0.2 pH units from pH 7 to pH 2. Within this range, the unique histidine, a strong field ligand, is titrated off the heme group and replaced by water, a weak field ligand. This change is monitored at 398 nm, where the greatest change in absorbance occurs as the Fe3+–heme Soret band shifts due to the spin-state change of the heme (Fig. S1 in
Kinetics of His–heme loop formation
In previous work, we have shown that the kinetics of His–heme loop formation in the denatured state is consistent with a mechanism in which the histidine undergoes a rapid deprotonation equilibrium, followed by binding of the deprotonated histidine to the heme.57 For this kinetic mechanism, the pH dependence of the observed rate constant for loop formation and breakage kobs is given by Eq. (4):where kb is the rate constant for loop breakage, kf is the rate
Importance of aromatic residues for residual structure in the DSE
We have used a host–guest approach to compare the tendencies of aromatic versus aliphatic residues to promote hydrophobic residual structure in the DSE. Both kinetic and thermodynamic data (ΔΔGloop(His) in Table 2 and ΔΔGb‡ in Table 3) show that leucine has a weaker tendency to induce residual structure in 3 M GdnHCl compared to the aromatic amino acids. Thus, our data indicate that aromatics are more effective than aliphatics at inducing residual structure in the DSE. Given that the transfer
Relative importance of hydrophobic versus electrostatic residual structure for the DSE
Electrostatic interactions have been shown to stabilize the DSE by 1–4 kcal mol− 1.16, 17, 18, 19, 20, 21 The stabilization of a His–heme loop by 0.3–0.5 kcal mol− 1 (which we observe here for an aromatic guest residue in a 22-residue loop), while significant, is modest by comparison. However, electrostatic interactions in the DSE likely involve multiple residues; in our case, we measure the impact of introducing only a single aromatic residue relative to alanine. Introduction of multiple
Importance of the present work for protein folding
The present work adds to a growing body of evidence that the DSE is not a thermodynamically featureless part of the energy landscape for protein folding.16, 19 More importantly, it shows that “hydrophobic” amino acids, particularly aromatics, can contribute significantly to stabilizing interactions that bias the DSE. In terms of the mechanism of protein folding, the role of small loops is often emphasized.69, 70 However, our work on denatured-state His–heme loop formation suggests that
Summary
Using a host–guest approach, we have shown that aromatic residues have a stronger effect on loop stability in comparison to the aliphatic residue leucine. Previous work on the factors that stabilize native-state structure in proteins suggests that the stronger stabilizing effect of aromatic residues in the DSE likely arises from the weakly polar interactions available to aromatic, but not aliphatic, residues. The residual structure induced by aromatic amino acids primarily affects the
Acknowledgements
This work was supported by award number GM074750 (B.E.B.) and American Recovery and Reinvestment Act supplement GM074750-04S1 from the National Institute of General Medical Sciences. M.L.F. acknowledges support from The University of Montana and MT NSF EPSCoR grant EPS-0701906.
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Characterization of the denatured state
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2020, Proceedings of the National Academy of Sciences of the United States of America