Biomedical subjects

H R Bosshard

Publications and source records attributed to H R Bosshard.

At least 19 recordsLinked to original sources

PubMed2001

Energetics of coiled coil folding: the nature of the transition states.

Coiled coils are simple models for studying the association of two polypeptide chains to form a folded protein. Previous work has shown that the folding of a coiled coil can be described by a two-state transition between two unfolded monomeric peptide chains and a folded coiled coil dimer. Here we report the thermodynamic activation parameters for the folding and unfolding of two unrelated coiled coils: C62GCN4 and A(2). C62GCN4 corresponds to the 62 C-terminal residues of yeast transcription factor GCN4. The peptide forms a dimeric coiled coil through its 33 C-terminal residues. A(2) is a designed 30-residue dimeric coiled coil whose folding is induced by low pH [Dürr, E., Jelesarov, I., and Bosshard, H. R. (1999) Biochemistry 38, 870-880]. Folding and unfolding were assessed under identical native buffer conditions so that the microscopic reversibility applied and the transition state was the same for folding and unfolding. The time course of folding was followed from the self-quenching of a C-terminal fluorescent label (Texas Red). The overall folding of both peptides is enthalpy-driven and opposed by a loss of entropy. The main energetic changes occur after the system has passed the transition state. In the folding of C62GCN4, only 10-20% of the heat capacity change is attained between the monomeric state and the dimeric transition state. For coiled coil A(2), the fractional heat capacity change preceding the transition state is 30-40%. The results indicate that the activated states of folding of coiled coils are not well structured and differ considerably from the folded coiled coil conformation. These findings are in agreement with a rate-limiting transition state in which the coiled coil helices and the hydrophobic coiled coil interface are poorly developed.

Amino Acid Motifs↗

PubMed2001

Affinity capture of proteins from solution and their dissociation by contact printing.

Biological experiments at the solid/liquid interface, in general, require surfaces with a thin layer of purified molecules, which often represent precious material. Here, we have devised a method to extract proteins with high selectivity from crude biological sample solutions and place them on a surface in a functional, arbitrary pattern. This method, called affinity-contact printing (alphaCP), uses a structured elastomer derivatized with ligands against the target molecules. After the target molecules have been captured, they are printed from the elastomer onto a variety of surfaces. The ligand remains on the stamp for reuse. In contrast with conventional affinity chromatography, here dissociation and release of captured molecules to the substrate are achieved mechanically. We demonstrate this technique by extracting the cell adhesion molecule neuron-glia cell adhesion molecule (NgCAM) from tissue homogenates and cell culture lysates and patterning affinity-purified NgCAM on polystyrene to stimulate the attachment of neuronal cells and guide axon outgrowth.

Animals↗

PubMed2001

Molecular recognition by induced fit: how fit is the concept?

Induced fit explains why biomolecules can bind together even if they are not optimized for binding. However, induced fit can lead to a kinetic bottleneck and does not describe every interaction in the absence of prior complementarity. Preselection of a fitting conformer is an alternative to induced fit.

Antigen-Antibody Complex↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed2000

Compulsory order of substrate binding to herpes simplex virus type 1 thymidine kinase. A calorimetric study.

Isothermal titration calorimetry has been used to investigate the thermodynamic parameters of the binding of thymidine (dT) and ATP to herpes simplex virus type 1 thymidine kinase (HSV1 TK). Binding follows a sequential pathway in which dT binds first and ATP second. The free enzyme does not bind ATP, whose binding site becomes only accessible in the HSV1 TK.dT complex. At pH 7.5 and 25 degrees C, the binding constants are 1.9 x 10(5) m(-1) for dT and 3.9 x 10(6) m(-1) for ATP binding to the binary HSV1 TK.dT complex. Binding of both substrates is enthalpy-driven and opposed by a large negative entropy change. The heat capacity change (DeltaCp) obtained from DeltaH in the range of 10-25 degrees C is -360 cal K(-1) mol(-1) for dT binding and -140 cal K(-1) mol(-1) for ATP binding. These large DeltaCp values are incompatible with a rigid body binding model in which the dT and ATP binding sites pre-exist in the free enzyme. Values of DeltaCp and TDeltaS strongly indicate large scale conformational adaptation of the active site in sequential substrate binding. The conformational changes seem to be more pronounced in dT binding than in the subsequent ATP binding. Considering the crystal structure of the ternary HSV1 TK.dT.ATP complex, a large movement in the dT binding domain and a smaller but substantial movement in the LID domain are proposed to take place when the enzyme changes from the substrate-free, presumably more open and less ordered conformation to the closed and compact conformation of the ternary enzyme-substrate complex.

Adenosine Triphosphate↗

PubMed2000

Folding of a three-stranded coiled coil.

Coiled coils consist of two or more amphipathic a-helices wrapped around each other to form a superhelical structure stabilized at the interhelical interface by hydrophobic residues spaced in a repeating 3-4 sequence pattern. Dimeric coiled coils have been shown to often form in a single step reaction in which association and folding of peptide chains are tightly coupled. Here, we ask whether such a simple folding mechanism may also apply to the formation of a three-stranded coiled coil. The designed 29-residue peptide LZ16A was shown previously to be in a concentration-dependent equilibrium between unfolded monomer (M), folded dimer (D), and folded trimer (T). We show by time-resolved fluorescence change experiments that folding of LZ16A to D and T can be described by 2M (k1)<==>(k(-1)) D and M + D (k2)<==>(k(-2)) T. The following rate constants were determined (25 degrees C, pH 7): k1 = 7.8 x 10(4) M(-1) s(-1), k(-1) = 0.015 s(-1), k2 = 6.5 x 10(5) M(-1) s(-1), and k(-2) = 1.1 s(-1). In a separate experiment, equilibrium binding constants were determined from the change with concentration of the far-ultraviolet circular dichroism spectrum of LZ16A and were in good agreement with the kinetic rate constants according to K(D) = k1/2k(-1) and K(T) = k2/k(-2). Furthermore, pulsed hydrogen-exchange experiments indicated that only unfolded M and folded D and T were significantly populated during folding. The results are compatible with a two-step reaction in which a subpopulation of association competent (e.g., partly helical) monomers associate to dimeric and trimeric coiled coils.

Amides↗

PubMed1999

Antigen recognition by conformational selection.

Conformational adaptation between antigen and antibody can modulate the antibody specificity. The phenomenon has often been proposed to result from an 'induced fit', which implies that the binding reaction induces a conformational change in the antigen and the antibody. Thus, an 'induced fit' requires initial complex formation followed by a conformational change in the complex. However, an antibody may select those antigen molecules that happen to be in a fitting conformational state. This leads to the same end result as an induced fit. Here, we demonstrate conformational selection by a single chain antibody fragment, raised against a random coil variant of the leucine zipper domain of transcription factor GCN4, when it cross-reacts with the wild-type dimeric leucine zipper. Kinetic and equilibrium data show that the single chain antibody fragment fragment selects monomeric peptides from the population in equilibrium with the leucine zipper dimer.

Amino Acid Sequence↗

PubMed1999

Extremely fast folding of a very stable leucine zipper with a strengthened hydrophobic core and lacking electrostatic interactions between helices.

The dimer interface of a leucine zipper involves hydrophobic as well as electrostatic interactions between the component helices. Here we ask how hydrophobic effects and electrostatic repulsion balance the rate of folding and thermodynamic stability of a designed dimeric leucine zipper formed by the acidic peptide A that contains four repeating sequence units, (abcdefg)4. The aliphatic a and d residues of peptide A were the same as in the GCN4 leucine zipper but the e and g positions were occupied by Glu, which prevented folding above pH 6 because of electrostatic repulsion. Leucine zipper A2 was formed by protonation of the e and g side chains with a sharp transition midpoint at pH 5.2. Folding could be described by a two-state transition from two unfolded random coil monomers to a coiled coil dimer. There was a linear relationship between the logarithm of the rate constants and the number of repulsive charges on the folded leucine zipper dimer. The same linear relationship applied to the free energy of unfolding and the number of repulsive charges at thermodynamic equilibrium. Fully protonated peptide A folded at a near diffusion-limited rate (kon = 3 x 10(8) M-1 s-1), and the free energy of folding was -55 kJ mol-1 at 25 degrees C. The present work shows that protonation of Glu in positions e and g increases both the folding rate and the stability of the leucine zipper in the absence of any interhelical electrostatic interactions. Protonated Glu is proposed to act like a nonpolar residue and to strengthen the hydrophobic core by folding back toward the core residues in the a and d positions. This effect adds more to the free energy of unfolding and to the rate of folding than maximizing the number of salt bridges across the helix interface in an electrostatically stabilized heterodimeric leucine zipper [Wendt, H., Leder, L., Härmä, H., Jelesarov, I., Baici, A., and Bosshard, H. R. (1997) Biochemistry 36, 204-213].

Amino Acid Sequence↗

PubMed1998

Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries.

Ribosome display was applied for affinity selection of antibody single-chain fragments (scFv) from a diverse library generated from mice immunized with a variant peptide of the transcription factor GCN4 dimerization domain. After three rounds of ribosome display, positive scFvs were isolated and characterized. Several different scFvs were selected, but those in the largest group were closely related to each other and differed in 0 to 5 amino acid residues with respect to their consensus sequence, the likely common progenitor. The best scFv had a dissociation constant of (4 +/- 1) x 10(-11) M, measured in solution. One amino acid residue in complementarity determining region L1 was found to be responsible for a 65-fold higher affinity than the likely progenitor. It appears that this high-affinity scFv was selected from the mutations occurring during ribosome display in vitro, and that this constitutes an affinity maturation inherent in this method. The in vitro-selected scFvs could be functionally expressed in the Escherichia coli periplasm with good yields or prepared by in vitro refolding. Thus, ribosome display can be a powerful methodology for in vitro library screening and simultaneous sequence evolution.

Amino Acid Sequence↗

PubMed1998

Thermodynamics and kinetics of the reaction of a single-chain antibody fragment (scFv) with the leucine zipper domain of transcription factor GCN4.

Single-chain Fv (scFv) fragments of antibodies have become important analytical and therapeutic tools in biology and medicine. The reaction of scFv fragments has not been well-characterized with respect to the energetics and kinetics of antigen binding. This paper describes the thermodynamic and kinetic behavior of the high-affinity scFv fragment SW1 directed against the dimeric leucine zipper domain of the yeast transcription factor GCN4. The scFv fragment was selected by the phage display technique from the immune repertoire of a mouse that had been immunized with the leucine zipper domain of GCN4. The scFv fragment was produced in high yield in Escherichia coli inclusion bodies and refolded from the denatured state. Differential scanning calorimetry showed that SW1 was stable up to about 50 degreesC, but the subsequent thermal denaturation was irreversible (Tm approximately 68 degreesC). The scFv fragment specifically recognized the dimeric leucine zipper conformation. Two scFv fragments bound to the GCN4 dimer to form the complex (scFv)2-GCN4. Because of its repetitive structure, the rod-shaped GCN4 leucine zipper may present two similar epitopes for the scFv fragment. Surprisingly, the binding reaction was highly cooperative, that is, the species (scFv)2-GCN4 dominated over scFv-GCN4 even in the presence of a large excess of the antigen GCN4. It is speculated that cooperativity resulted from direct interaction between the two GCN4-bound scFv fragments. At 25 degreesC, the average binding enthalpy for a scFv fragment was favorable (-61 kJ mol-1), the entropy change was unfavorable, and the change in heat capacity was -1.27 +/- 0.14 kJ mol-1 K-1. As a result of enthalpy-entropy compensation, the free binding energy was virtually independent of temperature in the physiological temperature range. Antigen binding in solution could be described by a single-exponential reaction with an apparent rate constant of 1 x 10(6) M-1 s-1. Binding followed in a biosensor with the dimeric GCN4 coupled to the surface of the metal oxide sensor chip was 20 times slower.

Amino Acid Sequence↗

PubMed1998

Salt effects on hydrophobic interaction and charge screening in the folding of a negatively charged peptide to a coiled coil (leucine zipper).

The stability of a coiled coil or leucine zipper is controlled by hydrophobic interactions and electrostatic forces between the constituent helices. We have designed a 30-residue peptide with the repeating seven-residue pattern of a coiled coil, (abcdefg)n, and with Glu in positions e and g of each heptad. The glutamate side chains prevented folding at pH values above 6 because of electrostatic repulsion across the helix dimer interface as well as within the individual helices. Protonation of the carboxylates changed the conformation from a random coil monomer to a coiled coil dimer. Folding at alkaline pH where the peptide had a net charge of -7e was promoted by the addition of salts. The nature of the charge screening cation was less important than that of the anion. The high salt concentrations (>1 M) necessary to induce folding indicated that the salt-induced folding resulted from alterations in the protein-water interaction. Folding was promoted by the kosmotropic anions sulfate and fluoride and to a lesser extent by the weak kosmotrope formate, whereas chloride and the strong chaotrope perchlorate were ineffective. Kosmotropes are excluded from the protein surface, which is preferentially hydrated, and this promotes folding by strengthening hydrophobic interactions at the coiled coil interface. Although charge neutralization also contributed to folding, it was effective only when the screening cation was partnered by a good kosmotropic anion. Folding conformed to a two-state transition from random coil monomer to coiled coil dimer and was enthalpy driven and characterized by a change in the heat capacity of unfolding of 3.9 +/- 1.2 kJ mol-1 K-1. The rate of folding was analyzed by fluorescence stopped-flow measurements. Folding occurred in a biphasic reaction in which the rapid formation of an initial dimer (kf = 2 x 10(7) M-1 s-1) was followed by an equally rapid concentration-independent rearrangement to the folded dimer (k > 100 s-1).

Amino Acid Sequence↗

PubMed1998

Diffusion-controlled DNA recognition by an unfolded, monomeric bZIP transcription factor.

Basic leucine zipper (bZIP) transcription factors are dimers that recognize mainly palindromic DNA sites. It has been assumed that bZIP factors have to form a dimer in order to bind to their target DNA. We find that DNA binding of both monomeric and dimeric bZIP transcription factor GCN4 is diffusion-limited and that, therefore, the rate of dimerization of the bZIP domain does not affect the rate of DNA recognition and GCN4 need not dimerize in order to bind to its specific DNA site. The results have implications for the mechanism by which bZIP transcription factors find their target sites for transcriptional regulation.

Amino Acid Sequence↗

PubMed1997

A monoclonal antibody induces opening of a coiled coil. Global protection of amide protons from H/D exchange decreases by up to 1000-fold in antibody-bound triple-stranded coiled coil.

Antibody specificity is limited, and different antigens may cross-react to varying degrees with the same antibody. An example is monoclonal antibody 42PF elicited against a 29-residue random-coil peptide. 42PF cross-reacts with a triple-stranded coiled coil of related amino acid sequence, and there was circumstantial evidence for an antibody-induced opening of the coiled coil [Leder, L., Berger, C., Bornhauser, S., Wendt, H., Ackermann, F., Jelesarov, I. & Bosshard, H. R. (1995) Biochemistry 34, 16,509-16,518]. To reveal in a direct way that antibody 42PF induces the opening of the coiled coil, we have compared the rates of H/D exchange of amide protons in the free and in the antibody-bound coiled-coil peptide using electrospray-ionization mass spectrometry for the analysis of deuterium incorporation. Complete H/D exchange lasted several days in the tightly folded coiled-coil conformation while in the presence of antibody 42PF the overall exchange took only minutes, corresponding to a thousand-fold decrease of protection of the most slowly exchanging amide hydrogens of the folded coiled coil. This demonstrates that the antibody induced a considerable opening of the coiled coil.

Amides↗

PubMed1997

Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.

A prerequisite for the use of recombinant antibody technologies starting from hybridomas or immune repertoires is the reliable cloning of functional immunoglobulin genes. For this purpose, a standard phage display system was optimized for robustness, vector stability, tight control of scFv-delta geneIII expression, primer usage for PCR amplification of variable region genes, scFv assembly strategy and subsequent directional cloning using a single rare cutting restriction enzyme. This integrated cloning, screening and selection system allowed us to rapidly obtain antigen binding scFvs derived from spleen-cell repertoires of mice immunized with ampicillin as well as from all hybridoma cell lines tested to date. As representative examples, cloning of monoclonal antibodies against a his tag, leucine zippers, the tumor marker EGP-2 and the insecticide DDT is presented. Several hybridomas whose genes could not be cloned in previous experimental setups, but were successfully obtained with the present system, expressed high amounts of aberrant heavy and light chain mRNAs, which were amplified by PCR and greatly exceeded the amount of binding antibody sequences. These contaminating variable region genes were successfully eliminated by employing the optimized phage display system, thus avoiding time consuming sequencing of non-binding scFv genes. To maximize soluble expression of functional scFvs subsequent to cloning, a compatible vector series to simplify modification, detection, multimerization and rapid purification of recombinant antibody fragments was constructed.

Amino Acid Sequence↗

PubMed1997

Very rapid, ionic strength-dependent association and folding of a heterodimeric leucine zipper.

Leucine zippers (coiled coils) are dimerization motifs found in several DNA-binding transcription factors. A parallel leucine zipper composed of the acidic chain X1-EYQALEKEVAQLEAENX2-ALEKEVAQLEHEG-amide and the basic chain X1-EYQALKKKVAQLKAKNX2ALKKKVAQLKHKG-amide was designed to study the kinetics of folding of a heterodimeric leucine zipper and to investigate the role of electrostatic attraction between oppositely charged peptide chains to the folding reaction. Each peptide alone did not form a leucine zipper at ionic strength (mu) < 1 M because of electrostatic repulsion between like charges in a homodimer. Therefore, the formation of the heterodimeric leucine zipper could be investigated by simple mixing of acidic and basic chains. To monitor folding, a fluorescent label was located either at the N-terminus (X1 = fluorescein-GGG, X2 = Q) or in the center of the coiled coil (X1 = acetyl, X2 = W). Folding could be described by a simple two-state reaction involving the disordered monomers and the folded heterodimer. The same bimolecular rate constant (k(on)) was observed independent of the location of the fluorescent label, indicating that both fluorescence probes monitored the same reaction. Lowering of the ionic strength increased k(on) from 4 x 10(6) M-1 s-1 (mu = 525 mM) to about 5 x 10(7) M-1 s-1 (mu = 74 mM). When extrapolated to mu = O, k(on) was approximately 10(9) M-1 s-1, which is near the diffusion limit. In contrast, the rate of dissociation depended very weakly on ionic strength; k(off) decreased only by about 2-fold when mu was lowered from 525 to 74 mM. Equilibrium association constants (Ka) of the heterodimeric zippers measured directly and calculated from kinetic constants (Ka = k(on)/k(off) were in good agreement. The observed two-state mechanism, the strong dependence on ionic strength of k(on) but not of k(off), and the nearly diffusion-limited association rate at very low ionic strength point to a folding pathway in which the formation of an electrostatically stabilized dimeric intermediate may be rate-limiting and the subsequent folding to the final dimer is very rapid and follows a "down-hill" free energy landscape. The small increase of k(off) at increasing ionic strength indicates a minor contribution of electrostatics to the stability of the folded leucine zipper.

Amino Acid Sequence↗

PubMed1997

Recombinant wild-type and mutant complexes of ferredoxin and ferredoxin:NADP+ reductase studied by isothermal titration calorimetry.

The interaction of spinach ferredoxin:NADP+ reductase (FNR) with ferredoxin (Fd) is driven by a favorable change of entropy and shows almost no change in enthalpy. The change in heat capacity between the free proteins and the complex is -0.47 +/- 0.1 kJ mol(-1) K(-1), a value indicating a relatively small surface area buried in the complex. A single proton is taken up from the environment when the ferredoxin:FNR complex forms. In the complex, the protonated residue(s) is (are) probably located in the vicinity of E92 of Fd because charge reversal in Fd(E92K) quenches protonation. Substitution of K88 by Q in FNR(K88Q) destabilizes the complex by a 7 kJ mol(-1) reduction in binding entropy, which indicates that dehydration of the complex interface contributes to stability.

Calorimetry↗

PubMed1996

Coupled folding and site-specific binding of the GCN4-bZIP transcription factor to the AP-1 and ATF/CREB DNA sites studied by microcalorimetry.

The site-specific interaction of the basic leucine zipper protein C62GCN4, which corresponds to the C-terminal sequence 220-281 of the yeast transcription factor GCN4, with the AP-1 and ATF/ CREB DNA recognition sites was analyzed by isothermal titration microcalorimetry. Free C62GCN4 is a dimer composed of a C-terminal leucine zipper and a basic, mainly unstructured DNA binding domain. Upon association with the target DNA, C62GCN4 folds to a fully alpha-helical dimer [Ellenberger et al. (1992) Cell 71, 1223-1237; König and Richmond (1993) J. Mol. Biol. 233, 139-154]. The protein-bound AP-1 site is straight, and the protein-bound ATF/CREB site is bent by 20 degrees toward the leucine zipper domain. The coupling between protein folding and DNA association resulting in two conformationally different complexes with C62GCN4 poses interesting thermodynamic problems. The association was strongly exothermic for both DNA target sites. The free energies of binding were indistinguishable in buffers of low salt concentration, and no change of the protonation state of C62GCN4 and/or the DNA target site occurred on formation of the complexes. Both complexes exhibited large and negative heat capacity changes. The empirical correlation between buried nonpolar and polar surfaces and the reduction in heat capacity concomitant to complexation did hold for the reaction with the AP-1 site at low salt concentration. However, in the case of the ATF/CREB site, the change in heat capacity was larger than could be accounted for by the burial of solvent-accessible surface. Potential sources of the extra decrement in the heat capacity could be restrictions in the vibrational modes of polar groups and of bound water molecules at the protein-DNA interface, thought to result from the bending of the ATF/CREB site. In the presence of high concentrations of glutamate and NaCl, the complex with the ATF/CREB site was significantly weaker than the complex with the AP-1 site.

Activating Transcription Factors↗

PubMed1996

Thermodynamic characterization of the coupled folding and association of heterodimeric coiled coils (leucine zippers).

Folding thermodynamics of nine heterodimeric, parallel coiled coils were studied by isothermal titration calorimetry (ITC) and thermal unfolding circular dichroism measurements. The heterodimers were composed of an acidic and a basic 30-residue peptide, which when in isolation were monomeric and essentially unstructured. The reaction followed a two-state mechanism indicating that folding and association were coupled. delta Hfold, delta Sfold and delta Cp normalized per mol of residue were of the same magnitude as for monomeric globular proteins, hence the energetics of folding and association of the heterodimeric coiled coils was balanced similarly to the folding of a single polypeptide chain. Cavity creating Leu/Ala substitutions revealed strong and position-dependent energetic coupling between leucine residues in the hydrophobic core of the coiled coil. delta Gunfold (equivalent to -delta Gfold in the two-state reaction) was determined from thermal unfolding. Global stability curves were calculated according to the Gibbs-Helmholtz equation and using the combined free energy data from ITC and thermal unfolding. Maximum stabilities were between 15 and 37 degrees C and cold denaturation could be demonstrated by direct calorimetry. The stability curves were based on free energies of folding measured between 10 and 85 degrees C and under identical solvent conditions. This represents a novel experimental approach which circumvents the use of varying solvent conditions as is typically required to measure protein stability curves. Discrepancies were noticed between van't Hoff enthalpies deduced from thermal unfolding and measured by direct calorimetry. The discrepancies are thought to be due to residual ordered structure in the denatured single chains around room temperature but not near the transition midpoint temperature Tm. This demonstrates that over an extended temperature range the assumption of a common denatured state implicit in the van't Hoff analysis may not always be valid.

Amino Acid Sequence↗