FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2044167776> ?p ?o ?g. }

• W2044167776 endingPage "35254" @default.
• W2044167776 startingPage "35248" @default.
• W2044167776 abstract "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 (373) 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 (206) Google Scholar) and cytochrome c (3Colon W. Elove G.A. Wakem L.P. Sherman F. Roder H. Biochemistry. 1996; 35: 5538-5549Crossref PubMed Scopus (162) 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 (545) 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 (551) 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 (118) 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 (77) 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 (208) 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.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 (87) Google Scholar) by conventional t-butyloxycarbonyl chemistry (reviewed in Ref. 14Merrifield B. Methods Enzymol. 1997; 289: 3-13Crossref PubMed Scopus (86) 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 (19) 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 (Arg). A peptide resin core (1.0 g of MBHA resin containing 0.6 mmol of peptide chain was swelled and washed repeatedly with dichloromethane and N,N-dimethylformamide in a 25-ml polypropylene solid-phase extraction reservoir. Activation reagent O-benzotriazol-1-yl-1,1,3,3 tetramethyluronium hexafluorophosphate (0.45 m) was dissolved in N,N-dimethylformamide/dichloromethane/dimethyl sulfoxide (Me2SO) (85:10:5 v/v/v) and reacted with excess t-butyloxycarbonyl-protected amino acid (1.1 equivalent to mmol of polypeptide chain) and excess diisopropylethylamine (1.5 equivalent to mmol polypeptide chain bound to resin) for 5 min. The activated amino acid ester (4.0 equivalent excess as compared with mmol of polypeptide chain bound to resin) was then coupled onto the solid-phase support by agitation for 1 h. Excess unreacted amino acids were removed by three alternating washes of dichloromethane and N,N-dimethylformamide. Cleavage of the t-butyloxycarbonyl and side chain-protecting groups and the subsequent release of the completed peptides from the MBHA resin support was achieved with hydrogen fluoride containing scavengers, 10% (v/v) anisole, and 1% (v/v) 1,2 ethanedithiol, magnetically stirred for 60 min in a reaction vessel with the temperature controlled at (–4 °C) by immersion in a sodium chloride water bath. The peptide resin was then washed three times with cold diethyl ether to remove scavengers and amino acid-protecting groups. Subsequent resin extraction with glacial acetic acid and overnight lyopholization yielded the crude peptide. Crude peptides were purified by reversed-phase chromatography (reviewed in Ref. 16Mant C.T. Kondejewski L.H. Cachia P.J. Monera O.D. Hodges R.S. Methods Enzymol. 1997; 289: 426-469Crossref PubMed Scopus (43) Google Scholar) on a Zorbax semipreparative SB-C8 column (250 × 9.4 mm inner diameter, 5-μm particle size, 300-Å pore size) by linear AB gradient elution (0.2% increasing acetonitrile/min), where eluent A is 0.05% aqueous trifluoroacetic acid and eluent B is 0.05% trifluoroacetic acid in acetonitrile. The purification was carried out at room temperature with a constant flow rate of 2 ml/min. The purity and homogeneity of the peptide was verified by analytical reversed-phase chromatography on a Zorbax analytical 300-Å SB-C8 column (150 × 2.1 mm inner diameter, 5-μm particle size, 300-Å pore size), by quantitative amino acid analysis (Beckman Model 6300 amino acid analyzer), and by electrospray mass spectroscopy on a Fisons Quattro (Fisons, Pointe-Claire, Quebec, Canada). Formation of the disulfide-bridged two-stranded homodimeric coiled-coil was obtained by overnight stirring in a 100 mm NH4HCO3 buffer, pH 8.5, and the desired product was purified by reversed-phase chromatography (described above). Analytical Ultracentrifugation Equilibrium Experiments—Sedimentation equilibrium analysis was performed on a Beckman XLA analytical ultracentrifuge with absorbance optics at 274 nm for the detection of tyrosine. Samples were first dialyzed exhaustively against an aqueous solution of 100 mm KCl, 50 mm PO4, pH 7.0 (benign buffer) at 4 °C. A 100-μl aliquot was loaded into the 12-mm Epon cell (charcoal-filled), and the initial loading concentrations of peptide stock solutions ranged from 50 to 500 μm in benign buffer. The samples were spun at 20 °C at 20,000, 25,000, and 35,000 rpm for 24 h to achieve equilibrium, as demonstrated by successive identical radial absorbance scans at 274 nm. The behavior of the peptide species at equilibrium is described by the following equation, Mbuoy=Mm(1-νρ)(Eq. 1) where M buoy is the measured buoyant weight, Mm is the molecular mass in daltons, ν is the partial specific volume of the sample, and ρ is the density of the buffer solution. The partial specific volume of the sample and density of the buffer were calculated using SednTerp (version 1.06, University of New Hampshire) using the weighted average of the amino acid content. The peptide oligomerization behavior was determined by fitting the sedimentation equilibrium data from different initial loading concentrations and rotor speeds to various monomer-oligomer equilibrium schemes using WIN NonLIN (version 1.035, University of Connecticut), a non-linear least squares algorithm for equilibrium ultracentrifugation analyses (17Johnson M.L. Correia J.J. Yphantis D.A. Halvorson H.R. Biophys J. 1981; 36: 575-588Abstract Full Text PDF PubMed Scopus (776) Google Scholar). Circular Dichroism Spectroscopy—Circular dichroism (CD) spectroscopy was performed on a Jasco-810 spectropolarimeter with constant N2 flushing (Jasco Inc., Easton, MD). A Lauda circulating water bath was used to control the temperature of the optic cell chamber, where rectangular cells of 1-mm path length were used. The concentration of peptide stock solutions was determined by absorbance at 275 nm in 6 m urea (extinction coefficient, ϵ = 1420 cm–1·m–1, 1 tyrosine per peptide chain). For wavelength scan analysis, a 5 mg/ml stock solution of each peptide in 100 mm KCl, 50 mm PO4, pH 7.0 (benign buffer) was diluted and scanned in the presence and absence of 50% trifluoroethanol (TFE). Mean residue molar ellipticity was calculated using the equation, [θ]=θobs·mrw/10lc(Eq. 2) where θobs is the observed ellipticity in millidegrees, mrw is the mean residue molecular weight, l is the optical path length of the CD cell (cm), and c is the peptide concentration (mg/ml). Each peptide spectrum was the average of eight wavelengths scans collected at 0.1-nm intervals from 195 to 250 nm. The uncertainty in the molar ellipticity values was ±300 degrees·cm2 · dmol–1. Protein stability measurements were monitored at wavelength 222 nm, indicative of the secondary structure of α-helices, by thermal and chemical (urea) denaturations (18Cooper T.M. Woody R.W. Biopolymers. 1990; 30: 657-676Crossref PubMed Scopus (217) Google Scholar). Temperature-induced Denaturation Monitored by Circular Dichroism—For thermal melting experiments, data points were taken at 1 °C intervals at a scan rate of 60 °C/h. The temperature dependence of the mean residue ellipticity θ was fitted to obtain fraction of the unfolded state, P U( t ), using a non-linear least-squares algorithm assuming a two-state unfolding reaction with pretransitional (folded state, θN( t )) and post-translational (unfolded state, θU( t )) baseline corrections (19Santoro M.M. Bolen D.W. Biochemistry. 1988; 21: 8063-8068Crossref Scopus (1589) Google Scholar, 20Lavigne P. Kondejewski L.H. Houston Jr., M.E. Sonnichsen F.D. Lix B. Sykes B.D. Hodges R.S. Kay C.M. J. Mol. Biol. 1995; 254: 505-520Crossref PubMed Scopus (97) Google Scholar), θ(t)=[(1-PU(t))·θN(t)]+[PU(t)·θU(t)](Eq. 3) where the pre- and post-transitional baselines are assumed to be linearly dependent on temperature, and with θN(0) and θU(0) as 0 °C intercepts, respectively, θN(t)=θN(0)-m·T(Eq. 4) and θU(t)=θU(0)-m·T(Eq. 5) The calculated fraction of the unfolded state, P U( t ), is given by, PU(t)=exp[(-ΔGU(t)/RT]/[1+exp(-ΔGU(t)/RT)](Eq. 6) where ΔG U( t ) is the apparent Gibbs free energy of folding described by the Gibbs-Helmholtz equation, ΔGU(t)=ΔH∘(1-T/tm)+ΔCp(T-Tm)-Tln(T/Tm)(Eq. 7) where tm is the temperature midpoint of the thermal transition, ΔH° is the apparent enthalpy of unfolding, and ΔCp is the change in heat capacity change associated with protein unfolding. Although ΔCp is temperature-dependent (21Makhatadze G.I. Privalov P.L. Adv. Protein. Chem. 1995; 47: 307-425Crossref PubMed Scopus (991) Google Scholar, 22Gomez J. Hilser V.J. Xie D. Freire E. Proteins. 1995; 22: 404-412Crossref PubMed Scopus (404) Google Scholar), but in the narrow temperature range of our experiments (5–60 °C), this term is generally insensitive to changes (23Betz S.F. Liebman P.A. DeGrado W.F. Biochemistry. 1997; 36: 2450-2458Crossref PubMed Scopus (87) Google Scholar). These thermodynamic parameters were fitted using the program Igor Pro (WaveMetrics, Inc.) with the protocol described in Ref. 20Lavigne P. Kondejewski L.H. Houston Jr., M.E. Sonnichsen F.D. Lix B. Sykes B.D. Hodges R.S. Kay C.M. J. Mol. Biol. 1995; 254: 505-520Crossref PubMed Scopus (97) Google Scholar. Chemical Denaturation Monitored by Circular Dichroism—For chemical denaturation experiments, the stock peptide solution was diluted with appropriate volumes of benign buffer and a stock solution of 10.0 m urea in benign buffer to give a series of data points in increasing denaturant concentration. Data points were left to equilibrate overnight before scanning, and to ensure accuracy, selected data points were rescanned to ascertain proper buffer equilibration. The data were fitted to a linear extrapolation method described previously in Ref. 15Kwok S.C. Mant C.T. Hodges R.S. Protein Sci. 2002; 11: 1519-1531Crossref PubMed Scopus (19) Google Scholar to determine denaturation midpoint, slope associated with the transition, and the change in free energy associated with the transition, ΔGU. A two-state unfolding model was used to derive peptide stability values from urea denaturation results, ff=([θ]obs-[θ]u)/([θ]f-[θ]u)(Eq. 8) where [θ] f and [θ] u represents the mean residue molar ellipticity for the fully folded and unfolded species, respectively, and the [θ]obs is the observed molar ellipticity at a given denaturant concentration. The free energy of unfolding was derived from the equation, ΔGU=RTlnKu(Eq. 9) where Ku is the equilibrium constant of the unfolding process. In the case of disulfide-bridged peptides, where the unfolding process is concentration-independent, Ku can be simplified as, Ku=(1-ff)/(ff)(Eq. 10) thus, ΔGU=RTln(1-ff)/(ff)(Eq. 11) Estimates of the free energy of unfolding in the absence of denaturant, ΔG U(water) and slope term m were obtained by linear extrapolation to zero, ΔGU=ΔGU(water)-m[denaturant](Eq. 12) where m is the slope associated with unfolding. Differential Scanning Calorimetry—Excess heat versus temperature for the peptides was determined using a Microcal differential scanning calorimeter (Microcal, Northampton, MA). Sample concentrations ranged from 105 to 140 μm coiled-coil dimer, and peptides were dissolved in 100 mm KCl, 50 mm PO4, pH 7.0, buffer. The sample solutions and buffer were filtered and degassed under vacuum and stored at 5 °C. Buffer scans were repeated until identical baselines were achieved. The heating rate was 60 °C/h, and the cooling rate was 90 °C/h with the excess heat monitored from 5 to 70 °C. Each sample was heated and cooled for three cycles to ensure folding reversibility. Data analyses were carried out in Microcal Origin software (Microcal DSC, version 1.2a) using a two-state model with change in heat capacity. Design of the α-Helical Coiled-coils with Different Hydrophobic Clustering—The peptides used in this study were modeled on heptad sequences that had strong α-helical potential and a heptad repeat ( gabcdef)n where non-polar residues at positions a and d facilitate coiled-coil formation. In the design of these hydrophobic clustered peptides, we took advantage of the features of the successful α-helical coiled-coil models in our laboratory (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 (118) Google Scholar, 9Wagschal K. Tripet B. Hodges R.S. J. Mol. Biol. 1999; 285: 785-803Crossref PubMed Scopus (77) 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 (208) Google Scholar), for example, complementary packing in the protein core (26Kellis Jr., J.T. Nyberg K. Fersht A.R. Biochemistry. 1989; 28: 4914-4922Crossref PubMed Scopus (330) Google Scholar), balance of charged residues across the coiled-coil interface in heptad positions e and g (27Kohn W.D. Kay C.M. Hodges R.S. J. Mol. Biol. 1997; 267: 1039-1052Crossref PubMed Scopus (113) Google Scholar, 28McClain D.L. Binfet J.P. Oakley M.G. J. Mol. Biol. 2001; 313: 371-383Crossref PubMed Scopus (43) Google Scholar), and a flexible disulfide bridge linkage (29O'Shea E.K. Klemm J.D. Kim P.S. Alber T. Science. 1991; 254: 539-544Crossref PubMed Scopus (1268) Google Scholar). The coiled-coil sequences consisted of 8 heptads (56 residues) based on 2 repeating heptad sequences: EXEAXKA and KXEAXEG where positions X represent hydrophobic core positions occupied by Ala, Ile, or Leu in positions a or d (Fig. 1.). We defined a hydrophobic cluster as a consecutive string of three large non-polar residues (Ile or Leu) in the core positions of the coiled-coil. In our coiled-coils, non-polar residues Leu, Ile, Leu in the consecutive d, a, d heptad positions defined a stabilizing hydrophobic cluster. Our approach was to design two proteins with identical inherent hydrophobicity, i.e. identical number and character of non-polar residues in equivalent coiled-coil core positions but with a different arrangement, i.e. P3 having three hydrophobic clusters and P2 having two (Fig. 1, rectangular boxes). The N-terminal hydrophobic cluster of P3 was disrupted by an interchange of Ile at position 9 and Ala at position 16, both at heptad a positions, to give P2 (Fig. 1, bottom). Therefore, the two analogs have identical inherent hydrophobicity but different clustering patterns. The hypothesis is that the hydrophobic clusters are independent units that contribute to coiled-coil stability and folding when separated along the coiled-coil chain by consecutive strings of Ala residues (Fig. 1, open circles). Thus, this pattern of large and small non-polar core residues helped distinguish the contribution of a hydrophobic cluster from inherent hydrophobicity. Interchain and intrachain ionic interactions were engineered by placing Lys and Glu at positions b, e , and g , resulting in ionic stabilization due to interchain electrostatic attractions (i to i′ + 5 or g to e ′) and intrachain ionic attractions (i to i +3 or i to i + 4). To promote coiled-coil formation, a C-terminal disulfide bridge, Gly-Gly-Cys-Tyr linker was introduced to facilitate the formation of a parallel and in-register coiled-coil, and the single Tyr residue allows for protein concentration determination by UV spectroscopy. The disulfide bridge was distant from the N-terminal hydrophobic cluster under investigation (Fig. 1). Secondary Structure Characterization by Circular Dichroism—Circular dichroism is a sensitive probe of secondary structural features, and this technique was used to detect the difference in helical content between the two peptides. Reduced P3 and P2 peptides were helical in benign condition (∼50% α-helical), but in the helix-promoting environment of 50% TFE (30Sonnichsen F.D. Van Eyk J.E. Hodges R.S. Sykes B.D. Biochemistry. 1992; 37: 8790-8798Crossref Scopus (610) Google Scholar), significant helical structure was induced in both peptides (Table I). Although the amino acid sequences of these peptides have a strong underlying helical propensity, potential stabilizing ionic interactions, and amphipathicity, in the reduced state, there is insufficient hydrophobic stabilization to overcome the monomer-dimer equilibrium, at the concentration of ∼200 μm (Table I), to form a fully folded coiled-coil. An interchain disulfide bridge has been shown to enhance coiled-coil folding and stability by eliminating the concentration-dependent monomer-dimer equilibrium that prevented folding (7Zhou N.E. Zhu B.-Y. Kay C.M. Hodges R.S. Biopolymers. 1992; 32: 419-426Crossref PubMed Scopus (118) Google Scholar, 31Zhou N.E. Kay C.M. Sykes B.D. Hodges R.S. Biochemistry. 1993; 32: 6190-6197Crossref PubMed Scopus (78) Google Scholar). In contrast to the reduced peptides, both the disulfide-bridged two-stranded coiled-coils P3 and P2 exhibited more helical structure, and the P3 coiled-coil with three hydrophobic clusters was fully folded (98% α-helical) at room temperature (Fig. 2 and Table I). The P2 coiled-coil with two hydrophobic clusters was only 65% folded at 20 °C in benign buffer, although more helicity was induced at 5 °C (Fig. 3A). P3 coiled-coil showed a [θ]222/208 = 1.02, indicative of a fully folded coiled-coil (32Lau S.Y.M. Taneja A.K. Hodges R.S. J. Biol. Chem. 1984; 317: 129-140Google Scholar), whereas that of P2 coiled-coil is less than 1 (0.74) due to the presence of the single-stranded unfolded state (Fig. 2.). Thus, without the presence of the third hydrophobic cluster, P2 did not fully fold in benign condition. In 50% TFE, both the disulfide-bridged peptides showed nearly 100% helical content, and the [θ]222/208 ratios for P3 and P2 were, respectively, 0.91 and 0.89, indicative of the single-stranded α-helical conformation (32Lau S.Y.M. Taneja A.K. Hodges R.S. J. Biol. Chem. 1984; 317: 129-140Google Scholar). The significant helix induction for P2 in TFE, an increase of 35% helicity, showed the underlying high helical propensity of this sequence. However, P2 remained partially unfolded in benign condition because of insufficient hydrophobic stabilization in the hydrophobic core (having only two clusters).Table IBiophysical characterization of the oxidized and reduced hydrophobic cluster peptidesPeptide nameaPeptides are named by the number of stabilizing hydrophobic clusters shown in Fig. 1.[θ]222 (reduced)b[θ]222 (reduced) is the mean residue molar ellipticity measured at 222 nm in a reducing buffer 2 mM dithioerythritol (DTE), 50 mM PO4 (K2HPO4/KH2PO4), 100 mm KCl buffer, pH 7.0, in the absence (Benign) or presence of 50% trifluoroethanol (TFE, v/v) at 20°C. Peptide concentrations were 206 um and 220 um (of monomer) for P3 and P2, respectively.Benign (20° C)[θ]222 (oxidized)c[θ]222 (oxidized) is the mean residue molar ellipticity measured at 222 nm in a non-reducing buffer of 50 mm PO4 (K2HPO4/KH2 PO4), 100 mm KCl buffer, pH 7.0, in the absence (Benign) or presence of 50% trifluoroethanol (TFE, v/v) at 20°C). Peptide concentrations were 103 um and 110 um (of dimer) for P3 and P2, respectively.Benign (20° C)Sedimentation equilibriumBenign (20° C)50% TFE% helix (reduced)d% Helix was calculated from [θ]222 based on the benign value divided by the value at 50% TFE multiplied by 100.[θ]222/208eThe helical ratio [θ]222/208 was calculated by dividing the observed molar ellipticity value at 222 nm by that at 208 nm.Benign (20° C)50% TFE% helix (oxidized)d% Helix was calculated from [θ]222 based on the benign value divided by the value at 50% TFE multiplied by 100.[θ]222/208eThe helical ratio [θ]222/208 was calculated by dividing the observed molar ellipticity value at 222 nm by that at 208 nm.Apparent MWfApparent molecular weights (MW) were determined from sedimentation equilibrium analyses of disulfide-bridged peptides.Oligomer ratiogOligomeric state was calculated by dividing the apparent molecular weight by the mass of the disulfide-bridged two-stranded peptide (theoretical MW, 12,334).P3-18,800-35,10053.60.74-34,200-34,90098.01.0214,8001.2P2-16,500-34,60047.70.69-22,800-35,20064.80.7413,7001.1a Peptides are named by the number of stabilizing hydrophobic clusters shown in Fig. 1.b [θ]222 (reduced) is the mean residue molar ellipticity measured at 222 nm in a reducing buffer 2 mM dithioerythritol (DTE), 50 mM PO4 (K2HPO4/KH2PO4), 100 mm KCl buffer, pH 7.0, in the absence (Benign) or presence of 50% trifluoroethanol (TFE, v/v) at 20°C. Peptide concentrations were 206 um and 220 um (of monomer) for P3 and P2, respectively.c [θ]222 (oxidized) is the mean residue molar ellipticity measured at 222 nm in a non-reducing buffer of 50 mm PO4 (K2HPO4/KH2 PO4), 100 mm KCl bu" @default.
• W2044167776 created "2016-06-24" @default.
• W2044167776 creator A5023948822 @default.
• W2044167776 creator A5052295449 @default.
• W2044167776 date "2003-09-01" @default.
• W2044167776 modified "2023-09-28" @default.
• W2044167776 title "Clustering of Large Hydrophobes in the Hydrophobic Core of Two-stranded α-Helical Coiled-Coils Controls Protein Folding and Stability" @default.
• W2044167776 cites W1481404660 @default.
• W2044167776 cites W1495475575 @default.
• W2044167776 cites W1533569014 @default.
• W2044167776 cites W1677326748 @default.
• W2044167776 cites W1687147049 @default.
• W2044167776 cites W1964610252 @default.
• W2044167776 cites W1966135653 @default.
• W2044167776 cites W1967802967 @default.
• W2044167776 cites W1971003485 @default.
• W2044167776 cites W1971018890 @default.
• W2044167776 cites W1974774710 @default.
• W2044167776 cites W1975061641 @default.
• W2044167776 cites W1983452280 @default.
• W2044167776 cites W1985374711 @default.
• W2044167776 cites W1989417429 @default.
• W2044167776 cites W1996518756 @default.
• W2044167776 cites W1999515733 @default.
• W2044167776 cites W2005034833 @default.
• W2044167776 cites W2010622646 @default.
• W2044167776 cites W2011222301 @default.
• W2044167776 cites W2018525943 @default.
• W2044167776 cites W2018531588 @default.
• W2044167776 cites W2021015773 @default.
• W2044167776 cites W2032561032 @default.
• W2044167776 cites W2032930889 @default.
• W2044167776 cites W2038263678 @default.
• W2044167776 cites W2043829693 @default.
• W2044167776 cites W2044997557 @default.
• W2044167776 cites W2046488637 @default.
• W2044167776 cites W2047030183 @default.
• W2044167776 cites W2050519187 @default.
• W2044167776 cites W2055290772 @default.
• W2044167776 cites W2059229862 @default.
• W2044167776 cites W2067798279 @default.
• W2044167776 cites W2080302102 @default.
• W2044167776 cites W2080757193 @default.
• W2044167776 cites W2084035996 @default.
• W2044167776 cites W2084140416 @default.
• W2044167776 cites W2086288291 @default.
• W2044167776 cites W2086666214 @default.
• W2044167776 cites W2089869461 @default.
• W2044167776 cites W2096272977 @default.
• W2044167776 cites W2116076986 @default.
• W2044167776 cites W2117747873 @default.
• W2044167776 cites W2165890129 @default.
• W2044167776 cites W28039676 @default.
• W2044167776 cites W4243228481 @default.
• W2044167776 doi "https://doi.org/10.1074/jbc.m305306200" @default.
• W2044167776 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12842878" @default.
• W2044167776 hasPublicationYear "2003" @default.
• W2044167776 type Work @default.
• W2044167776 sameAs 2044167776 @default.
• W2044167776 citedByCount "39" @default.
• W2044167776 countsByYear W20441677762012 @default.
• W2044167776 countsByYear W20441677762013 @default.
• W2044167776 countsByYear W20441677762014 @default.
• W2044167776 countsByYear W20441677762017 @default.
• W2044167776 countsByYear W20441677762018 @default.
• W2044167776 countsByYear W20441677762020 @default.
• W2044167776 countsByYear W20441677762021 @default.
• W2044167776 crossrefType "journal-article" @default.
• W2044167776 hasAuthorship W2044167776A5023948822 @default.
• W2044167776 hasAuthorship W2044167776A5052295449 @default.
• W2044167776 hasBestOaLocation W20441677761 @default.
• W2044167776 hasConcept C119599485 @default.
• W2044167776 hasConcept C12554922 @default.
• W2044167776 hasConcept C127413603 @default.
• W2044167776 hasConcept C154945302 @default.
• W2044167776 hasConcept C185592680 @default.
• W2044167776 hasConcept C204328495 @default.
• W2044167776 hasConcept C2164484 @default.
• W2044167776 hasConcept C2776545253 @default.
• W2044167776 hasConcept C41008148 @default.
• W2044167776 hasConcept C55493867 @default.
• W2044167776 hasConcept C73555534 @default.
• W2044167776 hasConcept C76155785 @default.
• W2044167776 hasConcept C8010536 @default.
• W2044167776 hasConcept C86803240 @default.
• W2044167776 hasConceptScore W2044167776C119599485 @default.
• W2044167776 hasConceptScore W2044167776C12554922 @default.
• W2044167776 hasConceptScore W2044167776C127413603 @default.
• W2044167776 hasConceptScore W2044167776C154945302 @default.
• W2044167776 hasConceptScore W2044167776C185592680 @default.
• W2044167776 hasConceptScore W2044167776C204328495 @default.
• W2044167776 hasConceptScore W2044167776C2164484 @default.
• W2044167776 hasConceptScore W2044167776C2776545253 @default.
• W2044167776 hasConceptScore W2044167776C41008148 @default.
• W2044167776 hasConceptScore W2044167776C55493867 @default.
• W2044167776 hasConceptScore W2044167776C73555534 @default.
• W2044167776 hasConceptScore W2044167776C76155785 @default.
• W2044167776 hasConceptScore W2044167776C8010536 @default.

• next