Clustering of Large Hydrophobes in the Hydrophobic Core of Two-stranded α-Helical Coiled-Coils Controls Protein Folding and Stability

The de novo design and biophysical characterization of two 60-residue peptides that dimerize to fold as parallel coiled-coils with different hydrophobic core clustering is described. Our goal was to investigate whether designing coiled-coils with identical hydrophobicity but with different hydrophobic clustering of non-polar core residues (each contained 6 Leu, 3 Ile, and 7 Ala residues in the hydrophobic core) would affect helical content and protein stability. The disulfide-bridged P3 and P2 differed dramatically in α-helical structure in benign conditions. P3 with three hydrophobic clusters was 98% α-helical, whereas P2 was only 65% α-helical. The stability profiles of these two analogs were compared, and the enthalpy and heat capacity changes upon denaturation were determined by measuring the temperature dependence by circular dichroism spectroscopy and confirmed by differential scanning calorimetry. The results showed that P3 assembled into a stable α-helical two-stranded coiled-coil and exhibited a native protein-like cooperative two-state transition in thermal melting, chemical denaturation, and calorimetry experiments. Although both peptides have identical inherent hydrophobicity (the hydrophobic burial of identical non-polar residues in equivalent heptad coiled-coil positions), we found that the context dependence of an additional hydrophobic cluster dramatically increased stability of P3 (ΔTm ≈ 18 °C and Δ[urea]½ ≈ 1.5 m) as compared with P2. These results suggested that hydrophobic clustering significantly stabilized the coiled-coil structure and may explain how long fibrous proteins like tropomyosin maintain chain integrity while accommodating polar or charged residues in regions of the protein hydrophobic core. The de novo design and biophysical characterization of two 60-residue peptides that dimerize to fold as parallel coiled-coils with different hydrophobic core clustering is described. Our goal was to investigate whether designing coiled-coils with identical hydrophobicity but with different hydrophobic clustering of non-polar core residues (each contained 6 Leu, 3 Ile, and 7 Ala residues in the hydrophobic core) would affect helical content and protein stability. The disulfide-bridged P3 and P2 differed dramatically in α-helical structure in benign conditions. P3 with three hydrophobic clusters was 98% α-helical, whereas P2 was only 65% α-helical. The stability profiles of these two analogs were compared, and the enthalpy and heat capacity changes upon denaturation were determined by measuring the temperature dependence by circular dichroism spectroscopy and confirmed by differential scanning calorimetry. The results showed that P3 assembled into a stable α-helical two-stranded coiled-coil and exhibited a native protein-like cooperative two-state transition in thermal melting, chemical denaturation, and calorimetry experiments. Although both peptides have identical inherent hydrophobicity (the hydrophobic burial of identical non-polar residues in equivalent heptad coiled-coil positions), we found that the context dependence of an additional hydrophobic cluster dramatically increased stability of P3 (ΔTm ≈ 18 °C and Δ[urea]½ ≈ 1.5 m) as compared with P2. These results suggested that hydrophobic clustering significantly stabilized the coiled-coil structure and may explain how long fibrous proteins like tropomyosin maintain chain integrity while accommodating polar or charged residues in regions of the protein hydrophobic core. Understanding protein folding remains a challenging problem: how does information encoded in the amino acid sequence translate into the three-dimensional structure necessary for protein function? Although hydrophobic interactions are generally accepted as the predominant source of free energy change that maintains the folded state, this non-specific stabilization does not describe how the “hydrophobic collapse” guides the formation of specific secondary structure (α-helices and β-sheets) in the final tertiary and quaternary structure in the native protein. The concomitant model suggests that the hydrophobic collapse restricts the conformation of the polypeptide chain into a “molten globule,” thus facilitating secondary structure folding in this limited conformational context (1Dill K.A. Protein Sci. 1999; 8: 1166-1180Crossref PubMed Scopus (384) Google Scholar). Examples of hydrophobic interactions participating in the early events of protein folding are observed via stopped flow fluorescence and nuclear magnetic resonance (NMR) studies in apomyogloblin (2Yao J. Chung J. Eliezer D. Wright P.E. Dyson H.J. Biochemistry. 2001; 12: 3561-3571Crossref Scopus (209) Google Scholar) and cytochrome c (3Colon W. Elove G.A. Wakem L.P. Sherman F. Roder H. Biochemistry. 1996; 35: 5538-5549Crossref PubMed Scopus (164) Google Scholar), illustrating the importance of the packing of non-polar residues in stabilizing helix-helix interactions. Recently, non-polar residues have also been observed to form non-native hydrophobic clustering in denatured proteins (4Shortle D. Ackerman M.S. Science. 2001; 293: 487-490Crossref PubMed Scopus (546) Google Scholar, 5Klein-Seetharaman J. Oikawa M. Grimshaw S.B. Wirmer J. Duchardt E. Ueda T. Imoto T. Smith L.J. Dobson C.M. Schwalbe H. Science. 2002; 295: 1719-1722Crossref PubMed Scopus (564) Google Scholar), and the authors postulated that non-native hydrophobic interactions can stabilize the long range order of the protein scaffold via an intermediate not observed in the folded state, thus indirectly guiding the extended polypeptide chain toward the correct native fold. Such an observation suggests that the amino acid sequence encodes for structural characteristics other than that of the native fold; in other words, the hydrophobic patterning in the sequence encodes the pathway that ultimately leads to the native functional state. Considering that hydrophobic interactions mediate protein folding both in the folded and the unfolded state, several questions arise: 1) How does a cluster of non-polar residues contribute to stability? 2) Is the free energy derived from the burial of hydrophobic residues simply a sum of the energy derived from the removal of non-polar surface area from aqueous medium? 3) Does hydrophobic clustering enhance stability via favorable enthalpic, geometric packing, and van der Waals interactions? The two-stranded α-helical coiled-coil is the simplest protein fold consisting of two amphipathic α-helices wound around one another forming a left-handed supercoil stabilized by hydrophobic burial (6Hodges R.S. Zhou N.E. Kay C.M. Semchuk P.D. Pept. Res. 1990; 3: 123-137PubMed Google Scholar, 7Zhou N.E. Zhu B.-Y. Kay C.M. Hodges R.S. Biopolymers. 1992; 32: 419-426Crossref PubMed Scopus (122) Google Scholar). All coiled-coils share a characteristic heptad (7-residue) repeat denoted as ( abcdefg) n in which non-polar residues occupy the a and d positions, forming an amphipathic surface where non-polar interactions allowed assembly of two-, three-, and higher oligomeric states (8Lupas A. Trends Biochem. Sci. 1996; 21: 375-382Abstract Full Text PDF PubMed Scopus (1008) Google Scholar). The quantitative contribution of 20 amino acids in positions a and d and their effects on protein stability and oligomerization state have been determined (9Wagschal K. Tripet B. Hodges R.S. J. Mol. Biol. 1999; 285: 785-803Crossref PubMed Scopus (78) Google Scholar, 10Wagschal K. Tripet B. Lavigne P. Mant C.T. Hodges R.S. Protein Sci. 1999; 11: 2312-2329Google Scholar, 11Tripet B. Wagschal K. Lavigne P. Mant C.T. Hodges R.S. J. Mol. Biol. 2000; 300: 377-402Crossref PubMed Scopus (211) Google Scholar). In addition, the secondary structure formation and hydrophobic collapse of coiled-coils are tightly coupled and cooperative since single-stranded amphipathic α-helices are unstable in aqueous medium. This hydrophobic surface where amphipathic α-helices interact via hydrophobic interactions provides an ideal model to test the effects of hydrophobic clustering. We postulated that hydrophobic clustering in the core of coiled-coils would have a significant influence on secondary structure formation and protein stability. Here we present the de novo design and characterization of two α-helical coiled-coils that have the same inherent hydrophobicity, i.e. the identical hydrophobic core residues (6 Leu, 3 Ile, and 7 Ala residues) but with different clustering of large and small hydrophobic core residues (see Fig. 1.). Their biophysical characteristics are compared by circular dichroism spectroscopy (CD), 1The abbreviations used are: CD, circular dichroism spectroscopy; DSC, differential scanning calorimetry; MBHA, copoly(styrene, 1% divinylbenzene)-4-methylbenzhydrylamine-HCl; TFE, trifluoroethanol. analytical ultracentrifugation, and differential scanning calorimetry (DSC). The results are discussed in the context of non-polar residue clustering enhancing protein stability. Peptide Synthesis and Purification—Peptides were synthesized by automated solid-phase methodology described previously (12Hodges R.S. Semchuk P.D. Taneja A.K. Kay C.M. Parker J.M. Mant C.T. Pept. Res. 1988; 1: 19-30PubMed Google Scholar, 13Sereda T.J. Mant C.T. Quinn A.M. Hodges R.S. J. Chromatogr. 1993; 646: 17-30Crossref PubMed Scopus (90) Google Scholar) by conventional t-butyloxycarbonyl chemistry (reviewed in Ref. 14Merrifield B. Methods Enzymol. 1997; 289: 3-13Crossref PubMed Scopus (91) Google Scholar). The peptides were synthesized on an Applied Biosystems model 430A peptide synthesizer as described previously (15Kwok S.C. Mant C.T. Hodges R.S. Protein Sci. 2002; 11: 1519-1531Crossref PubMed Scopus (24) Google Scholar). Briefly, the polypeptide chain was assembled on copoly(styrene, 1% divinylbenzene)-4-methylbenzhydrylamine-HCl (MBHA) resin, 100–200 mesh, substitution of 0.73 mmol amino groups/g (Novabiochem). The following side chain-protecting groups were used: benzyl (Thr, Ser), cyclohexyl (Asp), 4-methylbenzyl (Cys), trityl (Asn), and tosyl peptide core of mmol of peptide chain was and with and in a solid-phase m) was in and with amino acid equivalent to mmol of polypeptide and equivalent to mmol polypeptide chain to for The amino acid equivalent as compared with mmol of polypeptide chain to was coupled the solid-phase by for amino acids were by three of and of the t-butyloxycarbonyl and side chain-protecting groups and the of the peptides from the was with and 1% for in a with the temperature by in a The peptide was three with to and amino with acid and the peptides were by (reviewed in Ref. C.T. Hodges R.S. Methods Enzymol. 1997; 289: PubMed Scopus Google Scholar) on a by where is aqueous acid and is acid in The was temperature with a flow of The and of the peptide was by analytical on a analytical by quantitative amino acid amino acid and by spectroscopy on a of the disulfide-bridged two-stranded coiled-coil was by in a and the was by was on a analytical with for the of were an aqueous of was into the and the of peptide from to in benign The were 20 °C and for to as by identical The of the peptide is described by the following 1) where is the is the in is the specific of the and is the of the The specific of the and of the were of the of the amino acid The peptide oligomerization was determined by the from different and to of a for J. Full Text PDF PubMed Scopus Google Scholar). dichroism spectroscopy was on a with was used to the temperature of the where of were The of peptide was determined by in 6 peptide a of peptide in was and in the and of residue was the 2) where is the observed in is the residue is the of the and c is the peptide peptide was the of from to The in the was Protein stability were of the secondary structure of by thermal and chemical Biopolymers. 1990; PubMed Scopus Google Scholar). by thermal were °C a of The temperature dependence of the residue was to of the unfolded state, a a two-state with state, and state, Biochemistry. 1988; 21: Scopus Google Scholar, P. B. Hodges R.S. Kay C.M. J. Mol. Biol. PubMed Scopus Google Scholar), 3) where the and are to on and with and as °C and The of the unfolded state, is where is the free energy of folding described by the where is the temperature of the thermal is the enthalpy of and is the change in heat capacity change with protein Although is PubMed Scopus Google Scholar, J. D. E. PubMed Scopus Google Scholar), but in the temperature range of this is generally to changes Biochemistry. 1997; PubMed Scopus Google Scholar). These were the with the described in Ref. P. B. Hodges R.S. Kay C.M. J. Mol. Biol. PubMed Scopus Google by chemical denaturation the peptide was with of benign and a of in benign to a of in were to and to were to The were to a described previously in Ref. S.C. Mant C.T. Hodges R.S. Protein Sci. 2002; 11: 1519-1531Crossref PubMed Scopus (24) Google to denaturation with the and the change in free energy with the two-state model was used to peptide stability from denaturation where and the residue for the folded and unfolded and the is the observed a The free energy of was derived from the where is the of the In the of disulfide-bridged where the is can of the free energy of in the of and were by to where is the with heat temperature for the peptides was determined a differential scanning from to coiled-coil and peptides were in The and were and and were identical were The was and the was with the heat from to was and for three to folding were in DSC, a two-state model with change in heat of the with peptides used in this were on heptad that α-helical and a heptad repeat ( where non-polar residues positions a and d coiled-coil In the design of these hydrophobic we of the of the α-helical coiled-coil in (6Hodges R.S. Zhou N.E. Kay C.M. Semchuk P.D. Pept. Res. 1990; 3: 123-137PubMed Google Scholar, 7Zhou N.E. Zhu B.-Y. Kay C.M. Hodges R.S. Biopolymers. 1992; 32: 419-426Crossref PubMed Scopus (122) Google Scholar, K. Tripet B. Hodges R.S. J. Mol. Biol. 1999; 285: 785-803Crossref PubMed Scopus (78) Google Scholar, 10Wagschal K. Tripet B. Lavigne P. Mant C.T. Hodges R.S. Protein Sci. 1999; 11: 2312-2329Google Scholar, 11Tripet B. Wagschal K. Lavigne P. Mant C.T. Hodges R.S. J. Mol. Biol. 2000; 300: 377-402Crossref PubMed Scopus (211) Google Scholar), for packing in the protein core K. Biochemistry. PubMed Scopus Google Scholar), of charged residues the coiled-coil in heptad positions and Kay C.M. Hodges R.S. J. Mol. Biol. 1997; PubMed Scopus Google Scholar, J. Mol. Biol. 2001; PubMed Scopus Google Scholar), and a T. Science. PubMed Scopus Google Scholar). The coiled-coil of residues) on heptad and where positions hydrophobic core positions by Ile, or in positions a or d 1.). We a hydrophobic cluster as a of three large non-polar residues or in the core positions of the In non-polar residues Leu, Ile, in the d heptad positions a stabilizing hydrophobic Our was to design two proteins with identical inherent hydrophobicity, i.e. identical and of non-polar residues in equivalent coiled-coil core positions but with a different i.e. P3 three hydrophobic clusters and P2 two The hydrophobic cluster of P3 was by an of and Ala both heptad a positions, to P2 the two analogs have identical inherent hydrophobicity but different clustering The is that the hydrophobic clusters are that contribute to coiled-coil stability and folding the coiled-coil chain by of Ala residues this of large and small non-polar core residues the contribution of a hydrophobic cluster from inherent and interactions were by and positions and in stabilization to to or to and to or to coiled-coil a was to the formation of a parallel and and the residue for protein by The was from the hydrophobic cluster by dichroism is a of secondary structural and this was used to the in helical content the two P3 and P2 peptides were helical in benign but in the of Hodges R.S. Biochemistry. 1992; Scopus Google Scholar), significant helical structure was in both peptides Although the amino acid of these peptides have a helical stabilizing and in the state, is hydrophobic stabilization to the the of to form a folded been to enhance coiled-coil folding and stability by the that folding N.E. Zhu B.-Y. Kay C.M. Hodges R.S. Biopolymers. 1992; 32: 419-426Crossref PubMed Scopus (122) Google Scholar, N.E. Kay C.M. Hodges R.S. Biochemistry. 1993; 32: PubMed Scopus Google Scholar). In to the both the disulfide-bridged two-stranded coiled-coils P3 and P2 exhibited helical and the P3 coiled-coil with three hydrophobic clusters was folded temperature and The P2 coiled-coil with two hydrophobic clusters was only 65% folded 20 °C in benign was °C P3 coiled-coil showed a of a folded coiled-coil Taneja A.K. Hodges R.S. J. Biol. Scholar), whereas that of P2 coiled-coil is than to the of the single-stranded unfolded state the of the hydrophobic P2 not fold in benign In TFE, both the disulfide-bridged peptides showed helical and the for P3 and P2 and of the single-stranded α-helical conformation Taneja A.K. Hodges R.S. J. Biol. Scholar). The significant for P2 in TFE, an of showed the helical of this P2 unfolded in benign of hydrophobic stabilization in the hydrophobic core only two characterization of the and hydrophobic cluster are by the of stabilizing hydrophobic clusters in Fig. is the residue in a in the or of Peptide were and for P3 and is the residue in a of in the or of Peptide were and for P3 and was from on the benign by the by helical was by the observed by that was from on the benign by the by helical was by the observed by that were determined from of disulfide-bridged state was by the by the of the disulfide-bridged two-stranded peptide are by the of stabilizing hydrophobic clusters in Fig. is the residue in a in the or of Peptide were and for P3 and is the residue in a of in the or of Peptide were and for P3 and was from on the benign by the by The helical was by the observed by that were determined from of disulfide-bridged state was by the by the of the disulfide-bridged two-stranded peptide in a Fig. of the P3 and P2 temperature of the coiled-coils by circular dichroism spectroscopy were a temperature of In denaturation were 20 °C in a with of as Peptide P3 and peptide P2 of and disulfide-bridged two-stranded The of peptide P3 was as folded to the of The packing of hydrophobic core residues been to affect the oligomerization states of coiled-coils (9Wagschal K. Tripet B. Hodges R.S. J. Mol. Biol. 1999; 285: 785-803Crossref PubMed Scopus (78) Google Scholar, 10Wagschal K. Tripet B. Lavigne P. Mant C.T. Hodges R.S. Protein Sci. 1999; 11: 2312-2329Google Scholar). that P2 and P3 the same oligomerization state, were different protein to and we found that the oligomerization of coiled-coils P3 and P2 were to that of a with with a two-stranded coiled-coil and showed that the of hydrophobic residues and Ala that P2 and P3 not change the oligomerization state, i.e. these coiled-coils are two-stranded and not into order oligomerization we the in to a in stability to the of an additional stabilizing hydrophobic cluster in peptide of by and P3 coiled-coil with three hydrophobic clusters exhibited a to that of native proteins In P2 coiled-coil was only folded with stability. We determined the stability of these two analogs thermal and denaturation and found that P3 was significantly stable than with an of thermal of than 18 °C and a in denaturation of 3 and the free energy these two the was used to the chemical denaturation is to and from thermal in In addition, a would to the free energy change from chemical denaturation the around the denaturation transition is K. Biochemistry. PubMed Scopus Google Scholar). The free energy contribution of the hydrophobic cluster in the P3 coiled-coil to stability was to and this in stability can explain P2 coiled-coil not fold. The with the transition for P3 was significantly higher than that of P2 and describe the of the secondary structure of P2. The generally with the of changes in the non-polar surface area the folded and the unfolded state J.M. Sci. PubMed Scopus Google of peptides and stability contribution of hydrophobic are by the of stabilizing hydrophobic clusters in Fig. is the temperature which is a in compared to the peptide with three clusters as determined by circular dichroism is the denaturation of the two-state of an α-helical coiled-coil to a i.e. the to a in with a coiled-coil as is the by the and is the free energy of denaturation in the of whereas is the free energy of a is the by the and is the free energy of denaturation in the of whereas is the free energy of a is the free energy by a stabilizing clusters are by the of stabilizing hydrophobic clusters in Fig. is the temperature which is a in compared to the peptide with three clusters as determined by circular dichroism is the temperature with the transition differential scanning is the change in enthalpy derived from the thermal denaturation is the change in enthalpy derived from the calorimetry is the change in heat capacity with the the denaturation transition observed in the differential scanning calorimetry are by the of stabilizing hydrophobic clusters in Fig. is the temperature which is a in compared to the peptide with three clusters as determined by circular dichroism is the denaturation of the two-state of an α-helical coiled-coil to a i.e. the to a in with a coiled-coil as is the by the and is the free energy of denaturation in the of whereas is the free energy of a is the free energy by a stabilizing clusters is the temperature with the transition differential scanning is the change in enthalpy derived from the thermal denaturation is the change in enthalpy derived from the calorimetry is the change in heat capacity with the the denaturation transition observed in the differential scanning calorimetry in a of to the was also used to the contribution of hydrophobic clusters to coiled-coil stability. This is one of the for protein and of enthalpy and heat capacity changes of Methods Enzymol. PubMed Scopus Google Scholar). the showed a cooperative transition with a and but the not transition not thus illustrating the and of the coiled-coil to the of a hydrophobic the that these two coiled-coils have the to identical hydrophobic surface results suggested that the two-state of peptide a of three hydrophobic clusters to form a folded protein ≈ We compared the and for P3 from thermal and and found to in The was which to This was a of the in non-polar surface area the folded and unfolded states Methods Enzymol. PubMed Scopus Google Scholar, D. E. J. Mol. Biol. PubMed Scopus Google Scholar) and with of of small proteins J. Mol. Biol. 1990; PubMed Scopus Google Scholar), in the range of interactions contribute significantly to protein stability the burial of non-polar surface area is favorable in aqueous and this that the hydrophobic stabilization is In the coiled-coil the additional cluster of three large non-polar residues in P3 the folding of secondary structure and protein stability compared with where this cluster is the P3 and the P2 peptides have the same inherent hydrophobicity (6 Leu, 3 Ile, and 7 Ala residues in the hydrophobic their folding and stability P3 with three hydrophobic clusters is a native protein-like two-stranded coiled-coil with a In P2 with two hydrophobic clusters is only folded and significantly stable compared with P3 coiled-coil showed a transition in DSC, whereas the transition was not with P2 identical conditions. the of the hydrophobic cluster in P2 the enthalpy of hydrophobic which would affect the packing of the two The of the and the of i.e. to Ala is and E. 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