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E Dürr

Publications and source records attributed to E Dürr.

10 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↗
PubMed2000

Thermodynamic analysis of cavity creating mutations in an engineered leucine zipper and energetics of glycerol-induced coiled coil stabilization.

Protein stability in vitro can be influenced either by introduction of mutations or by changes in the chemical composition of the solvent. Recently, we have characterized the thermodynamic stability and the rate of folding of the engineered dimeric leucine zipper A(2), which has a strengthened hydrophobic core [Dürr, E., Jelesarov, I., and Bosshard, H. R. (1999) Biochemistry 38, 870-880]. Here we report on the energetic consequences of a cavity introduced by Leu/Ala substitution at the tightly packed dimeric interface and how addition of 30% glycerol affects the folding thermodynamics of A(2) and the cavity mutants. Folding could be described by a two-state transition from two unfolded monomers to a coiled coil dimer. Removal of six methylene groups by Leu/Ala substitutions destabilized the dimeric coiled coil by 25 kJ mol(-1) at pH 3.5 and 25 degrees C in aqueous buffer. Destabilization was purely entropic at around room temperature and became increasingly enthalpic at elevated temperatures. Mutations were accompanied by a decrease of the unfolding heat capacity by 0.5 kJ K(-1) mol(-1). Addition of 30% glycerol increased the free energy of folding of A(2) and the cavity mutants by 5-10 kJ mol(-1) and lowered the unfolding heat capacity by 25% for A(2) and by 50% for the Leu/Ala mutants. The origin of the stabilizing effect of glycerol varied with temperature. Stabilization of the parent leucine zipper A(2) was enthalpic with an unfavorable entropic component between 0 and 100 degrees C. In the case of cavity mutants, glycerol induced enthalpic stabilization below 50 degrees C and entropic stabilization above 50 degrees C. The effect of glycerol could not be accounted for solely by the enthalpy and entropy of transfer or protein surface from water to glycerol/water mixture. We propose that in the presence of glycerol the folded coiled coil dimer is better packed and displays less intramolecular fluctuations, leading to enhanced enthalpic interactions and to an increase of the entropy of folding. This work demonstrates that mutational and solvent effects on protein stability can be thermodynamically complex and that it may not be sufficient to only analyze changes of enthalpy and entropy at the unfolding temperature (T(m)) to understand the mechanisms of protein stabilization.

Acids↗
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

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

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↗
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↗
PubMed1995

Characterization of leucine zipper complexes by electrospray ionization mass spectrometry.

The development of "soft" ionization methods has enabled the mass spectrometric analysis of higher-order structural features of proteins. We have applied electrospray ionization mass spectrometry (ESI-MS) to the analysis of the number and composition of polypeptide chains in homomeric and heteromeric leucine zippers. In comparison with other methods that have been used to analyze leucine zippers, such as analytical ultracentrifugation, gel chromatography, or electrophoretic band shift assays, ESI-MS is very fast and highly sensitive and provides a straightforward way to distinguish between homomeric and heteromeric coiled-coil structures. ESI-MS analyses were carried out on the parallel dimeric leucine zipper domain GCN4-p1 of the yeast transcription factor GCN4 and on three synthetic peptides with the sequences Ac-EYEALEKKLAAX1EAKX2QALEKKLEALEHG-amide: peptide LZ (X1, X2 = Leu), peptide LZ(12A) (X1 = Ala, X2 = Leu), and peptide LZ(16N) (X1 = Leu, X2 = Asn). Equilibrium ultracentrifugation analysis showed that LZ forms a trimeric coiled coil and this could be confirmed unequivocally by ESI-MS as could the dimeric nature of GCN4-p1. The formation of heteromeric two- and three-stranded leucine zippers composed of chains from LZ and LZ(12A), or from GCN4-p1 and LZ, was demonstrated by ESI-MS and confirmed by fluorescence quenching experiments on fluorescein-labeled peptides. The results illustrate the adaptability and flexibility of the leucine zipper motif, properties that could be useful to the design of specific protein assemblies by way of coiled-coil domains.

Amino Acid Sequence↗
PubMed1995

Mass spectrometric approaches to molecular characterization of protein-nucleic acid interactions.

The recent development of 'soft' ionization-desorption methods has lead to a breakthrough for the mass spectrometric analysis of biomacromolecules such as proteins and nucleic acids. In particular, the feasibility of electrospray-ionization mass spectrometry (ESI-MS) for the direct characterization of non-covalent supramolecular complexes is opening new analytical perspectives. Examples hitherto analyzed by ESI-MS include enzyme-substrate and -inhibitor complexes, homo- and heterodimers/trimers of leucine zipper polypeptides, and several other DNA- and RNA-binding proteins. Furthermore, the characterization of double-stranded and higher-order oligo- and polynucleotide complexes by negative-ion ESI has been demonstrated. Ions specific of non-covalent protein and oligonucleotide complexes can be selectively dissociated by changing the solution conditions and by increasing the desolvation potential. These results form the basis for the molecular characterization of protein-nucleotide interactions, thus complementing protein-chemical approaches, and other methods of structure determination.

Amino Acid Sequence↗
PubMed1983

Presence and absence of virus particles in hybridomas secreting monoclonal antibodies against gliomas.

Hybridoma clones were produced by fusion of splenocytes from glioma-immunized hosts and the X63-Ag8.653 mouse myeloma line and Y3-Ag.1.2.3. rat myeloma line. Oncornavirus particles were found in all clones descending from the mouse myeloma line. No virus particles could be found in either the spleens of immunized Balb/c mice and Fischer rats or in the rat myeloma line and the hybridomas derived from it.

Animals↗