Electrostatic interactions in leucine zippers: thermodynamic analysis of the contributions of Glu and His residues and the effect of mutating salt bridges

Electrostatic interactions play a complex role in stabilizing proteins. Here, we present a rigorous thermodynamic analysis of the contribution of individual Glu and His residues to the relative pH-dependent stability of the designed disulfide-linked leucine zipper AB(SS). The contribution of an ionized side-chain to the pH-dependent stability is related to the shift of the pK(a) induced by folding of the coiled coil structure. pK(a)(F) values of ten Glu and two His side-chains in folded AB(SS) and the corresponding pK(a)(U) values in unfolded peptides with partial sequences of AB(SS) were determined by 1H NMR spectroscopy: of four Glu residues not involved in ion pairing, two are destabilizing (-5.6 kJ mol(-1)) and two are interacting with the positive alpha-helix dipoles and are thus stabilizing (+3.8 kJ mol(-1)) in charged form. The two His residues positioned in the C-terminal moiety of AB(SS) interact with the negative alpha-helix dipoles resulting in net stabilization of the coiled coil conformation carrying charged His (-2.6 kJ mol(-1)). Of the six Glu residues involved in inter-helical salt bridges, three are destabilizing and three are stabilizing in charged form, the net contribution of salt-bridged Glu side-chains being destabilizing (-1.1 kJ mol(-1)). The sum of the individual contributions of protonated Glu and His to the higher stability of AB(SS) at acidic pH (-5.4 kJ mol(-1)) agrees with the difference in stability determined by thermal unfolding at pH 8 and pH 2 (-5.3 kJ mol(-1)). To confirm salt bridge formation, the positive charge of the basic partner residue of one stabilizing and one destabilizing Glu was removed by isosteric mutations (Lys-->norleucine, Arg-->norvaline). Both mutations destabilize the coiled coil conformation at neutral pH and increase the pK(a) of the formerly ion-paired Glu side-chain, verifying the formation of a salt bridge even in the case where a charged side-chain is destabilizing. Because removing charges by a double mutation cycle mainly discloses the immediate charge-charge effect, mutational analysis tends to overestimate the overall energetic contribution of salt bridges to protein stability.     

Inverse electrostatic effect: electrostatic repulsion in the unfolded state stabilizes a leucine zipper

The pH-dependent stability of a protein is strongly affected by electrostatic interactions between ionizable residues in the folded as well as unfolded state. Here we characterize the individual contributions of charged Glu and His residues to stability and determine the NMR structure of the designed, heterodimeric leucine zipper AB consisting of an acidic A chain and a basic B chain. Thermodynamic parameters are compared with those of the homologous leucine zipper AB(SS) in which the A and B chains are disulfide-linked. NMR structures of AB based on (1)H NMR data collected at 600 MHz converge, and formation of the same six interchain salt bridges found previously in disulfide-linked AB(SS) [Marti, D. N., and Bosshard, H. R. (2003) J. Mol. Biol. 330, 621-637] is indicated. While the structures of AB and AB(SS) are very similar, their pH-dependent relative stabilities are strikingly different. The stability of AB peaks at pH approximately 4.5 and is higher at pH 8 than at pH 2. In contrast, AB(SS) is most stable at acidic pH where no interhelical salt bridges are formed. The different energetic contributions of charged Glu and His residues to stability of the two coiled coil structures were evaluated from pK(a) shifts induced by folding. The six charged Glu residues involved in salt bridges stabilize leucine zipper AB by 4.5 kJ/mol yet destabilize disulfide-linked AB(SS) by -1.1 kJ/mol. Two non-ion-paired Glu charges destabilize AB by only -1.8 kJ/mol but AB(SS) by -5.6 kJ/mol. The higher relative stability of AB at neutral pH is not caused by more favorable electrostatic interactions in the folded leucine zipper. It is due mainly to unfavorable electrostatic interactions in the unfolded A and B chains and may therefore be called an inverse electrostatic effect. This study illustrates the importance of residual interactions in the unfolded state and how the energetics of the unfolded state affect the stability of the folded protein.     

Interhelical ion pairing in coiled coils: solution structure of a heterodimeric leucine zipper and determination of pKa values of Glu side chains

Residues of opposite charge often populate heptad positions g (heptad i on chain 1) and e' (heptad i + 1 on chain 2) in dimeric coiled coils and may stabilize the dimer by formation of interchain ion pairs. To investigate the contribution to stability of such electrostatic interactions we have designed a disulfide-linked heterodimeric zipper (AB zipper) consisting of the acidic chain Ac-E-VAQLEKE-VAQAEAE-NYQLEQE-VAQLEHE-CG-NH(2) and the basic chain Ac-E-VQALKKR-VQALKAR-NYAAKQK-VQALRHK-CG-NH(2) in which all e and g positions are occupied by either E or K/R to form a maximum of seven interhelical salt bridges. Temperature-induced denaturation experiments monitored by circular dichroism reveal a stable coiled coil conformation below 50 degrees C and in the pH range 1.2-8.0. Stability is highest at pH approximately 4.0 [DeltaG(U) (37 degrees C) = 5.18 +/- 0.51 kcal mol(-)(1)]. The solution structure of the AB zipper at pH 5.65 has been elucidated on the basis of homonuclear (1)H NMR data collected at 800 MHz [heavy atom rmsd's for the ensemble of 50 calculated structures are 0.47 +/- 0.13 A (backbone) and 0.95 +/- 0.16 A (all)]. Both chains of the AB zipper are almost entirely in alpha-helical conformation and form a superhelix with a left-handed twist. Overhauser connectivities reveal close contacts between g position residues (heptad i on chain 1) and residues d/f (heptad i on chain 1), residues a/d (heptad i + 1 on chain 1), and residue a' (heptad i + 1 on chain 2). Residues in position e (heptad i on chain 1) are in contact with residues a/b/d/f (heptad i on chain 1) and residue d' (heptad i on chain 2). These connectivities hint at a relatively defined alignment of the side chains across the helix interface. Partial H-bond formation between the functional groups of residues g and e'(+1) is observed in the calculated structures. NMR pH titration experiments disclose pK(a) values for Glu delta-carboxylate groups: 4.14 +/- 0.02 (E(1)), 4.82 +/- 0.07 (E(6)), 4.52 +/- 0.01 (E(8)), 4.37 +/- 0.03 (E(13)), 4.11 +/- 0.02 (E(15)), 4.41 +/- 0.07 (E(20)), 4.82 +/- 0.03 (E(22)), 4.65 +/- 0.04 (E(27)), 4.63 +/- 0.03 (E(29)), 4.22 +/- 0.02 (E(1)(')). By comparison with pK(a) of Glu in unfolded peptides ( approximately 4. 3 +/- 0.1), our pK(a) data suggest marginal or even unfavorable contribution of charged Glu to the stability of the AB zipper. The electrostatic energy gained from interhelical ion pairs is likely to be surpassed by hydrophobic energy terms upon protonation of Glu, due to increased hydrophobicity of uncharged Glu and, thus, better packing against apolar residues at the chain interface.     

Electrostatic contribution to the thermodynamic and kinetic stability of the homotrimeric coiled coil Lpp-56: A computational study

The protein moiety of the Braun's E. coli outer membrane lipoprotein (Lpp-56) is an attractive object of biophysical investigation in several aspects. It is a homotrimeric, parallel coiled coil, a class of coiled coils whose stability and folding have been studied only occasionally. Lpp-56 possesses unique structural properties and exhibits extremely low rates of folding and unfolding. It is natural to ask how the specificity of the structure determines the extraordinary physical chemical properties of this protein. Recently, a seemingly controversial data on the stability and unfolding rate of Lpp-56 have been published (Dragan et al., Biochemistry 2004;43: 14891-14900; Bjelic et al., Biochemistry 2006;45:8931-8939). The unfolding rate constant measured using GdmCl as the denaturing agent, though extremely low, was substantially higher than that obtained on the basis of thermal unfolding. If this large difference arises from the effect of screening of electrostatic interactions induced by GdmCl, electrostatic interactions would appear to be an important factor determining the unusual properties of Lpp-56. We present here a computational analysis of the electrostatic properties of Lpp-56 combining molecular dynamics simulations and continuum pK calculations. The pH-dependence of the unfolding free energy is predicted in good agreement with the experimental data: the change in DeltaG between pH 3 and pH 7 is approximately 60 kJ mol(-1). The results suggest that the difference in the stability of the protein observed using different experimental methods is mainly because of the effect of the reduction of electrostatic interactions when the salt (GdmCl) concentration increases. We also find that the occupancy of the interhelical salt bridges is unusually high. We hypothesize that electrostatic interactions, and the interhelical salt bridges in particular, are an important factor determining the low unfolding rate of Lpp-56.     

Conformational specificity of the lac repressor coiled-coil tetramerization domain

Predictive understanding of how the folded, functional shape of a native protein is encoded in the linear sequence of its amino acid residues remains an unsolved challenge in modern structural biology. Antiparallel four-stranded coiled coils are relatively simple protein structures that embody a heptad sequence repeat and rich diversity for tertiary packing of alpha-helices. To explore specific sequence determinants of the lac repressor coiled-coil tetramerization domain, we have engineered a set of buried nonpolar side chains at the a-, d-, and e-positions into the hydrophobic interior of the dimeric GCN4 leucine zipper. Circular dichroism and equilibrium ultracentrifugation studies show that this core variant (GCN4-pAeLV) forms a stable tetrameric structure with a reversible and highly cooperative thermal unfolding transition. The X-ray crystal structure at 1.9 A reveals that GCN4-pAeLV is an antiparallel four-stranded coiled coil of the lac repressor type in which the a, d, and e side chains associate by means of combined knobs-against-knobs and knobs-into-holes packing with a characteristic interhelical offset of 0.25 heptad. Comparison of the side chain shape and packing in the antiparallel tetramers shows that the burial of alanine residues at the e positions between the neighboring helices of GCN4-pAeLV dictates both the antiparallel orientation and helix offset. This study fills in a gap in our knowledge of the determinants of structural specificity in antiparallel coiled coils and improves our understanding of how specific side chain packing forms the teritiary structure of a functional protein.     

A calorimetric characterization of the salt dependence of the stability of the GCN4 leucine zipper.

The effects of different salts (LiCl, NaCl, ChoCl, KF, KCl, and KBr) on the structural stability of a 33-residue peptide corresponding to the leucine zipper region of GCN4 have been studied by high-sensitivity differential scanning calorimetry. These experiments have allowed an estimation of the salt dependence of the thermodynamic parameters that define the stability of the coiled coil. Independent of the nature of the salt, a destabilization of the coiled coil is always observed upon increasing salt concentration up to a maximum of approximately 0.5 M, depending on the specific cation or anion. At higher salt concentrations, this effect is reversed and a stabilization of the leucine zipper is observed. The effect of salt concentration is primarily entropic, judging from the lack of a significant salt dependence of the transition enthalpy. The salt dependence of the stability of the peptide is complex, suggesting the presence of specific salt effects at high salt concentrations in addition to the nonspecific electrostatic effects that are prevalent at lower salt concentrations. The data is consistent with the existence of specific interactions between anions and peptide with an affinity that follows a reverse size order (F- > Cl- > Br-). Under all conditions studied, the coiled coil undergoes reversible thermal unfolding that can be well represented by a reaction of the form N2<==>2U, indicating that the unfolding is a two-state process in which the helices are only stable when they are in the coiled coil conformation.     

Effect of side chain length on intrahelical interactions between carboxylate- and guanidinium-containing amino acids

The charge-containing hydrophilic functionalities of encoded charged amino acids are linked to the backbone via different numbers of hydrophobic methylenes, despite the apparent electrostatic nature of protein ion pairing interactions. To investigate the effect of side chain length of guanidinium- and carboxylate-containing residues on ion pairing interactions, α-helical peptides containing Zbb–Xaa (i, i + 3), (i, i + 4) and (i, i + 5) (Zbb = carboxylate-containing residues Aad, Glu, Asp in decreasing length; Xaa = guanidinium residues Agh, Arg, Agb, Agp in decreasing length) sequence patterns were studied by circular dichroism spectroscopy (CD). The helicity of Aad- and Glu-containing peptides was similar and mostly pH independent, whereas the helicity of Asp-containing peptides was mostly pH dependent. Furthermore, the Arg-containing peptides consistently exhibited higher helicity compared to the corresponding Agp-, Agb-, and Agh-containing peptides. Side chain conformational analysis by molecular mechanics calculations showed that the Zbb–Xaa (i, i + 3) and (i, i + 4) interactions mainly involved the χ ₁ dihedral combinations (g+, g+) and (g?, g+), respectively. These low energy conformations were also observed in intrahelical Asp–Arg and Glu–Arg salt bridges of natural proteins. Accordingly, Asp and Glu provides variation in helix characteristics associated with Arg, but Aad does not provide features beyond those already delivered by Glu. Importantly, nature may have chosen the side chain length of Arg to support helical conformations through inherent high helix propensity coupled with stabilizing intrahelical ion pairing interactions with the carboxylate-containing residues.     

Contribution of arginine-glutamate salt bridges to helix stability

Peptide side chain interactions were studied by molecular dynamics simulation using explicit solvent on a peptide with the sequence AAARAAAAEAAEAAAARA. Three different protonation states of the glutamic acid side chains were simulated for four 20 ns runs each, a total simulation time of 240 ns. Two different salt bridge geometries were observed and the preferred geometry was found to depend on Glu — Arg residue spacing. Stable charge clusters were also observed, particularly in the fully charged peptide. Salt bridges were selectively interrupted upon protonation, with concomitant changes in secondary structure. The fully charged peptide was highly helical between residues 9 and 13, although protonation increased helicity near the N-terminus. The contribution of salt bridges to helix stability therefore depends on both position and relative position of charged residues within a sequence.     

Kinetics and thermodynamics of the unfolding and refolding of the three-stranded alpha-helical coiled coil, Lpp-56

Temperature-induced reversible unfolding and refolding of the three-stranded alpha-helical coiled coil, Lpp-56, were studied by kinetic and thermodynamic methods, using CD spectroscopy, dynamic light scattering, and scanning calorimetry. It was found that both unfolding and refolding reactions of this protein in neutral solution in the presence of 100 mM NaCl are characterized by unusually slow kinetics, which permits detailed investigation of the mechanism of these reactions. Kinetic analyses show that the unfolding of this coiled coil represents a single-stage first-order reaction, while the refolding represents a single-stage third-order reaction. The activation enthalpy and entropy for unfolding do not depend noticeably on temperature and are both significantly greater than those for the folding reaction, which show a significant dependence on temperature. The activation heat capacity change for the unfolding reaction is close to zero, while it is quite significant for the folding reaction. The correlation between the activation and structural parameters obtained for the Lpp-56 coiled coil suggests that interhelical van der Waals interactions are disrupted in the transition state, which is nevertheless still compact, and water has not yet penetrated into the interface; the transition from the transient state to the unfolded state results in hydration of exposed apolar groups of the interface and the disruption of helices. The low propensity for the Lpp-56 strands to fold and associate is caused by the high number of charged groups at neutral pH. On one hand, these charges give rise to considerable repulsive forces destabilizing the helical conformation of the strands. On the other hand, they align the folded helices in parallel and in register so that the apolar sides face each other, and the oppositely charged groups may form salt links, which are important for the formation of the trimeric coiled coil. A decrease in pH, which eliminates the salt links, dramatically decreases the stability of Lpp-56; its structure becomes less rigid and unfolds much faster.     

Tertiary structure in 7.9 M guanidinium chloride--the role of Glu53 and Asp287 in Pyrococcus furiosus endo-beta-1,3-glucanase

The thermodynamic stability of family 16 endo-beta-1,3-glucanase(EC 3.2.1.39) from the hyperthermophilic archaeon Pyrococcus furiosus is decreased upon single (D287A, E53A) and double (E53A/D287A) mutation of Asp287 and Glu53. In accordance with the homology model prediction,both carboxylic acids are involved in the composition of a calcium binding site, as shown by titration of the wild-type and the variant proteins with a chromophoric chelator. The present study shows that, in P. furiosus, endo-beta-1,3-glucanase residues Glu53 and Asp287 also make up a calcium binding site in 7.9 M guanidinium chloride. The persistence of tertiary structure in 7.9 M guanidinium chloride, a feature of the wild-type enzyme,is observed also for the three variant proteins. The DeltaG(H2O) values relative to the guanidinium chloride-induced equilibrium unfolding of the three variants are approximately 50% lower than that of the wild-type. The destabilizing effect of the combined mutations of the double mutant is non-additive, with an energy of interaction of 24.2 kJ x mol(-1), suggesting a communication between the two mutated residues. The decrease in the thermodynamic stability of D287A, E53A and E53A/D287A is contained almost exclusively in the m-values, a parameter which reflects the solvent exposed surface area upon unfolding. The decrease in m-value suggests that the substitution with alanine of two evenly charged repulsive side chains induces a stabilization of the non-native state in 7.9 M guanidinium chloride comparable to that induced by the presence of calcium on the wildtype. These results suggest that the stabilization of a compact non-native state may be a strategy for P. furiosus endo-beta-1,3-glucanase to thrive under adverse environmental conditions.     

Importance of potential interhelical salt-bridges involving interior residues for coiled-coil stability and quaternary structure

Coiled coils are formed by two or more alpha-helices that align in a parallel or an antiparallel relative orientation. Polar interactions involving residues at the interior a and d positions are important for determining the quaternary structure of coiled coils. In the model heterodimeric coiled-coil Acid-a1-Base-a1, a buried a-d' Asn-Asn interaction is sufficient to specify both a dimeric structure and an antiparallel relative helix orientation. Although the equivalent a-a' interaction is found in parallel coiled coils, there is no example of an a-d' Asn-Asn interaction in structurally characterized, naturally occurring antiparallel coiled coils. Instead, interior charged residues form interhelical salt-bridges with residues at the adjacent e or g positions. Using a model coiled-coil heterodimer, we have explored the role of a potential interhelical interaction between an Arg at an interior d position and a Glu at the adjacent g' position. Our results demonstrate that this potentially attractive interhelical Coulombic interaction has little or no influence on helix orientation. Instead, we show that burying a single Arg residue at an interior position is sufficient to specify a dimeric state at a significantly lower thermodynamic cost than burial of two interacting Asn residues.     

Removing an interhelical salt bridge abolishes coiled-coil formation in a de novo designed peptide

Alpha-helical coiled coils represent a common protein oligomerization motif that are mainly stabilized by hydrophobic interactions occurring along their coiled-coil interface, the so-called hydrophobic seam. We have recently de novo designed and optimized a series of two-heptad repeat long coiled-coil peptides which are further stabilized by a complex network of inter- and intrahelical salt bridges. Here we have extended the de novo design of such two heptad-repeat long peptides by removing the central and most important g-e' Arg to Glu (g-e'RE) ionic interhelical interaction and replacing these residues by alanine residues. The effect of the missing interhelical ionic interaction on coiled-coil formation and stability has been analyzed by CD spectroscopy, analytical ultracentrifugation, and X-ray crystallography. We show that the peptide, while being highly alpha-helical, is no longer able to form a parallel coiled-coil structure but rather assumes an octameric globular helical assembly devoid of any coiled-coil interactions.     

Hydrogen bonding is the prime determinant of carboxyl pKa values at the N-termini of alpha-helices

Experimentally determined mean pK(a) values of carboxyl residues located at the N-termini of alpha-helices are lower than their overall mean values. Here, we perform three types of analyses to account for this phenomenon. We estimate the magnitude of the helix macrodipole to determine its potential role in lowering carboxyl pK(a) values at the N-termini. No correlation between the magnitude of the macrodipole and the pK(a) values is observed. Using the pK(a) program propKa we compare the molecular surroundings of 18 N-termini carboxyl residues versus 233 protein carboxyl groups from a previously studied database. Although pK(a) lowering interactions at the N-termini are similar in nature to those encountered in other protein regions, pK(a) lowering backbone and side-chain hydrogen bonds appear in greater number at the N-termini. For both Asp and Glu, there are about 0.5 more hydrogen bonds per residue at the N-termini than in other protein regions, which can be used to explain their lower than average pK(a) values. Using a QM-based pK(a) prediction model, we investigate the chemical environment of the two lowest Asp and the two lowest Glu pK(a) values at the N-termini so as to quantify the effect of various pK(a) determinants. We show that local interactions suffice to account for the acidity of carboxyl residues at the N-termini. The effect of the helix dipole on carboxyl pK(a) values, if any, is marginal. Backbone amide hydrogen bonds constitute the single biggest contributor to the lowest carboxyl pK(a) values at the N-termini. Their estimated pK(a) lowering effects range from about 1.0 to 1.9 pK(a) units.     

Optimizing pKa computation in proteins with pH adapted conformations

pK(A) in proteins are determined by electrostatic energy computations using a small number of optimized protein conformations derived from crystal structures. In these protein conformations hydrogen positions and geometries of salt bridges on the protein surface were determined self-consistently with the protonation pattern at three pHs (low, ambient, and high). Considering salt bridges at protein surfaces is most relevant, since they open at low and high pH. In the absence of these conformational changes, computed pK(A)(comp) of acidic (basic) groups in salt bridges underestimate (overestimate) experimental pK(A)(exp), dramatically. The pK(A)(comp) for 15 different proteins with 185 known pK(A)(exp) yield an RMSD of 1.12, comparable with two other methods. One of these methods is fully empirical with many adjustable parameters. The other is also based on electrostatic energy computations using many non-optimized side chain conformers but employs larger dielectric constants at short distances of charge pairs that diminish their electrostatic interactions. These empirical corrections that account implicitly for additional conformational flexibility were needed to describe the energetics of salt bridges appropriately. This is not needed in the present approach. The RMSD of the present approach improves if one considers only strongly shifted pK(A)(exp) in contrast to the other methods under these conditions. Our method allows interpreting pK(A)(comp) in terms of pH dependent hydrogen bonding pattern and salt bridge geometries. A web service is provided to perform pK(A) computations.     

Dissociation and unfolding of GCN4 leucine zipper in the presence of sodium dodecyl sulfate.

The dissociation and unfolding behavior of the GCN4 leucine zipper has been studied using SDS titration. Circular dichroism (CD) spectra showed that the alpha-helix content of the leucine zipper (20 microM) decreased during the sodium dodecyl sulfate (SDS) titration. However, the alpha-helix content of the leucine zipper still remained significant in the presence of 1 mM SDS, with little change detected when the SDS concentration further increased to 2 mM. The dimer dissociation of the leucine zipper is also a co-operative process during SDS titration; with no dimer remaining when SDS concentration reached 1 mM, as shown by electrophoresis and the the theta(222)/theta(208) ratio. Our results indicate that SDS efficiently induces leucine zipper dimer dissociation with the monomers still partially folded. The experimental results provide important evidence for the previous model that partial helix formation precedes dimerization in coiled coil folding.     

Complementation of buried lysine and surface polar residues in a designed heterodimeric coiled coil

The coiled coil is an attractive target for protein design. The helices of coiled coils are characterized by a heptad repeat of residues denoted a to g. Residues at positions a and d form the interhelical interface and are usually hydrophobic. An established strategy to confer structural uniqueness to two-stranded coiled coils is the use of buried polar Asn residues at position a, which imparts dimerization and conformational specificity at the expense of stability. Here we show that polar interactions involving buried position-a Lys residues that can interact favorably only with surface e' or g' Glu residues also impart structural uniqueness to a designed heterodimeric coiled coil with the nativelike properties of sigmoidal thermal and urea-induced unfolding transitions, slow hydrogen exchange and lack of ANS binding. The position-a Lys residues do not, however, confer a single preference for helix orientation, likely reflecting the ability of Lys at position a to from favorable interactions with g' or e' Glu residues in the parallel and antiparallel orientations, respectively. The Lys-Glu polar interaction is less destabilizing than the Asn-Asn a-->a' interaction, presumably reflecting a higher desolvation penalty associated with the completely buried polar position-a groups. Our results extend the range of approaches for two-stranded coiled-coil design and illustrate the role of complementing polar groups associated with buried and surface positions of proteins in protein folding and design.     

Hierarchical Cascades of Instability Govern the Mechanics of Coiled Coils: Helix Unfolding Precedes Coil Unzipping

Coiled coils are a fundamental emergent motif in proteins found in structural biomaterials, consisting of α-helical secondary structures wrapped in a supercoil. A fundamental question regarding the thermal and mechanical stability of coiled coils in extreme environments is the sequence of events leading to the disassembly of individual oligomers from the universal coiled-coil motifs. To shed light on this phenomenon, here we report atomistic simulations of a trimeric coiled coil in an explicit water solvent and investigate the mechanisms underlying helix unfolding and coil unzipping in the assembly. We employ advanced sampling techniques involving steered molecular dynamics and metadynamics simulations to obtain the free-energy landscapes of single-strand unfolding and unzipping in a three-stranded assembly. Our comparative analysis of the free-energy landscapes of instability pathways shows that coil unzipping is a sequential process involving multiple intermediates. At each intermediate state, one heptad repeat of the coiled coil first unfolds and then unzips due to the loss of contacts with the hydrophobic core. This observation suggests that helix unfolding facilitates the initiation of coiled-coil disassembly, which is confirmed by our 2D metadynamics simulations showing that unzipping of one strand requires less energy in the unfolded state compared with the folded state. Our results explain recent experimental findings and lay the groundwork for studying the hierarchical molecular mechanisms that underpin the thermomechanical stability/instability of coiled coils and similar protein assemblies.     

Hierarchical Cascades of Instability Govern the Mechanics of Coiled Coils: Helix Unfolding Precedes Coil Unzipping

Coiled coils are a fundamental emergent motif in proteins found in structural biomaterials, consisting of α-helical secondary structures wrapped in a supercoil. A fundamental question regarding the thermal and mechanical stability of coiled coils in extreme environments is the sequence of events leading to the disassembly of individual oligomers from the universal coiled-coil motifs. To shed light on this phenomenon, here we report atomistic simulations of a trimeric coiled coil in an explicit water solvent and investigate the mechanisms underlying helix unfolding and coil unzipping in the assembly. We employ advanced sampling techniques involving steered molecular dynamics and metadynamics simulations to obtain the free-energy landscapes of single-strand unfolding and unzipping in a three-stranded assembly. Our comparative analysis of the free-energy landscapes of instability pathways shows that coil unzipping is a sequential process involving multiple intermediates. At each intermediate state, one heptad repeat of the coiled coil first unfolds and then unzips due to the loss of contacts with the hydrophobic core. This observation suggests that helix unfolding facilitates the initiation of coiled-coil disassembly, which is confirmed by our 2D metadynamics simulations showing that unzipping of one strand requires less energy in the unfolded state compared with the folded state. Our results explain recent experimental findings and lay the groundwork for studying the hierarchical molecular mechanisms that underpin the thermomechanical stability/instability of coiled coils and similar protein assemblies.     

Guanidinium chloride- and urea-induced unfolding of FprA, a mycobacterium NADPH-ferredoxin reductase: stabilization of an apo-protein by GdmCl

The guanidinium chloride- and urea-induced unfolding of FprA, a mycobacterium NADPH-ferredoxin reductase, was examined in detail using multiple spectroscopic techniques, enzyme activity measurements and size exclusion chromatography. The equilibrium unfolding of FprA by urea is a cooperative process where no stabilization of any partially folded intermediate of protein is observed. In comparison, the unfolding of FprA by guanidinium chloride proceeds through intermediates that are stabilized by interaction of protein with guanidinium chloride. In the presence of low concentrations of guanidinium chloride the protein undergoes compaction of the native conformation; this is due to optimization of charge in the native protein caused by electrostatic shielding by the guanidinium cation of charges on the polar groups located on the protein side chains. At a guanidinium chloride concentration of about 0.8 m, stabilization of apo-protein was observed. The stabilization of apo-FprA by guanidinium chloride is probably the result of direct binding of the Gdm+ cation to protein. The results presented here suggest that the difference between the urea- and guanidinium chloride-induced unfolding of FprA could be due to electrostatic interactions stabilizating the native conformation of this protein.     

Stabilities of leucine zipper dimers estimated by an empirical free energy method.

The leucine zipper motif is a characteristic amino acid sequence found in dimeric DNA-binding proteins. Computer-generated models for leucine zippers were constructed as alpha-helical coiled dimers with leucine repeated every seventh residue. An empirical Gibbs free energy, delta G, function which incorporates hydrophobic force, electrostatic interactions, and conformational entropy loss as the major intermolecular interactions was used to estimate the delta G of dimer formation in fos, jun, and GCN4 zipper sequences. The calculations showed that complexes known to form stable homo- or heterodimers have favorable (negative) delta G, while other less stable complexes have unfavorable (positive) delta G. Leucines in position d of the coiled coil contribute large hydrophobic stabilization energies while residues in the a position contribute less to dimer stability. Hydrophobic contributions show little sequence specificity, however, and do not contribute significantly to homo/heterodimer preference. Charged residues in the e and g positions, on the other hand, determine homo/heterodimer specificity. In GCN4 homodimers, residues GLU el, Glu b2, Lys g2, and Lys e4 greatly contribute to dimer stability. The preferential stability of fos-jun heterodimer over the jun-jun and fos-fos homodimers is primarily due to the side chains Asp b1, Glu g1, Asp b2, Glu e2, Glu g2, Glu g3, and Lys a5 of the fos helix, and Arg c1, Lys g1, Lys b2, Lys e2, Arg e4, and Glu g4 of the jun helix.