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H Lilie

Publications and source records attributed to H Lilie.

16 recordsLinked to original sources
PubMed1996

Dynamics of the GroEL-protein complex: effects of nucleotides and folding mutants.

Chaperonins are a ubiquitous class of ring-shaped oligomeric protein complexes that are of crucial importance for protein folding in vivo. Analysis of the underlying functional principles had relied mainly on model proteins the (un)folding of which is dominated by irreversible side-reactions. We used maltose-binding protein (MBP) as a substrate protein for GroEL, since the refolding of this protein is completely reversible and thus allows a detailed analysis of the molecular parameters that determine the interaction of GroEL with non-native protein. We show that MBP folding intermediates are effectively trapped by GroEL in a diffusion-controlled reaction. This complex is stabilized via unspecific hydrophobic interactions. Stabilization energies for wild-type MBP increasing linearly with ionic strength from 50 kJ/mol to 60 kJ/mol. Depending on the intrinsic folding rate and the hydrophobicity of the substrate protein, the interaction of GroEL with MBP folding intermediates leads to a dramatically decreased apparent refolding rate of MBP (wild-type) or a complete suppression of folding (MBP folding mutant Y283D). On the basis of our data, a quantitative kinetic model of the GroEL-mediated folding cycle is proposed, which allows simulation of the partial reactions of the binding and release cycles under all conditions tested. In the presence of ATP and non-hydrolysable analogues, MBP is effectively released from GroEL, since the overall dissociation constant is reduced by three orders of magnitude. Interestingly, binding of nucleotide does not change the off rate by more than a factor of 3. However the on-rate is decreased by at least two orders of magnitude. Therefore, the rebinding reaction is prevented and folding occurs in solution.

Adenine Nucleotides↗
PubMed1996

In vitro folding of inclusion body proteins.

Insoluble, inactive inclusion bodies are frequently formed upon recombinant protein production in transformed microorganisms. These inclusion bodies, which contain the recombinant protein in an highly enriched form, can be isolated by solid/liquid separation. After solubilization, native proteins can be generated from the inactive material by using in vitro folding techniques. New folding procedures have been developed for efficient in vitro reconstitution of complex hydrophobic, multidomain, oligomeric, or highly disulfide-bonded proteins. These protocols take into account process parameters such as protein concentration, catalysis of disulfide bond formation, temperature, pH, and ionic strength, as well as specific solvent ingredients that reduce unproductive side reactions. Modification of the protein sequence has been exploited to improve in vitro folding.

Inclusion Bodies↗
PubMed1995

Interaction of GroEL with a highly structured folding intermediate: iterative binding cycles do not involve unfolding.

The GroE proteins are molecular chaperones involved in protein folding. The general mechanism by which they facilitate folding is still enigmatic. One of the central open questions is the conformation of the GroEL-bound nonnative protein. Several suggestions have been made concerning the folding stage at which a protein can interact with GroEL. Furthermore, the possibility exists that binding of the nonnative protein to GroEL results in its unfolding. We have addressed these issues that are basic for understanding the GroE-mediated folding cycle by using folding intermediates of an Fab antibody fragment as molecular probes to define the binding properties of GroEL. We show that, in addition to binding to an early folding intermediate, GroEL is able to recognize and interact with a late quaternary-structured folding intermediate (Dc) without measurably unfolding it. Thus, the prerequisite for binding is not a certain folding stage of a nonnative protein. In contrast, general surface properties of nonnative proteins seem to be crucial for binding. Furthermore, unfolding of a highly structured intermediate does not necessarily occur upon binding to GroEL. Folding of Dc in the presence of GroEL and ATP involves cycles of binding and release. Because in this system no off-pathway reactions or kinetic traps are involved, a quantitative analysis of the reactivation kinetics observed is possible. Our results indicate that the association reaction of Dc and GroEL in the presence of ATP is rather slow, whereas in the absence of ATP association is several orders of magnitude more efficient. Therefore, it seems that ATP functions by inhibiting reassociation rather than promoting release of the bound substrate.

Chaperonin 60↗
PubMed1995

Association of antibody chains at different stages of folding: prolyl isomerization occurs after formation of quaternary structure.

The folding pathways of multi-domain proteins are still poorly understood due to the complexity of the reaction involving domain folding, association and, in many cases, prolyl cis/trans isomerization. Here, we have established a kinetic model for the folding of the Fab fragment of the antibody MAK 33 with intact disulfide bonds. Folding of the hetero-dimeric protein from the completely denatured, oxidized state comprises the pairwise association of the two domains of each chain with those of the partner protein. Both the reactivation of the Fab fragment in which the two constituent polypeptide chains were covalently linked via a cystine bond (Fab) and that of a mutant lacking this covalent linkage (Fab/-cys) were monitored by ELISA. Folding of the Fab fragment is a slow process, which can be described by a single exponential term. The kinetic phase reflects a folding step after the association of the two chains. The same reaction was detected in the folding of Fab/-cys but an additional rate-limiting step is involved that is due to a unimolecular step in the folding of the isolated light chain. This implies that, during Fab reactivation, Fd associates with the light chain at the stage of an earlier folding intermediate, thus eliminating the additional slow folding step of the light chain observed with Fab/-cys. Both in Fab and Fab/-cys renaturation, the folding reaction after association is determined by prolyl isomerization. Therefore, at least four different association-competent folding intermediates have to be postulated according to the folding stage of light chain and the configuration of at least one prolyl-peptide bond. Using the different substrate specificities of cyclophilin and FK506 binding protein, we have obtained evidence that Pro159 within the Fd fragment may be responsible for the observed slow folding phase after association, although three other proline residues adopt a cis configuration in the native protein. Furthermore, the data suggest that in the case of the Fab fragment, association is a prerequisite for cis/trans isomerization of prolyl peptide bonds, implying that the quaternary but not the tertiary structure determines the cis-configuration of the prolyl residue in Fd involved in the rate-limiting folding reaction.

Amino Acid Sequence↗
PubMed1995

Transient interaction of Hsp90 with early unfolding intermediates of citrate synthase. Implications for heat shock in vivo.

At normal temperatures, Hsp90 is one of the most abundant proteins in the cytosol of various eucaryotic cells. Upon heat shock, the level of Hsp90 is increased even more, suggesting that it is important for helping cells to survive under these conditions. However, studies so far have been almost exclusively concerned with the function of Hsp90 under non-stress conditions, and therefore only little is known about the role of Hsp90 during heat shock. As a model for heat shock in vitro, we have monitored the inactivation and subsequent aggregation of dimeric citrate synthase (CS) at elevated temperatures. Hsp90 effectively "stabilized" CS under conditions where the enzyme is normally inactivated and finally aggregates very rapidly. A kinetic dissection of the unfolding pathway of CS succeeded in revealing two intermediates which form and subsequently undergo irreversible aggregation reactions. Hsp90 apparently interacts transiently with these highly structured early unfolding intermediates. Binding and subsequent release of the intermediates favorably influences the kinetic partitioning between two competing processes, the further unfolding of CS and the productive refolding to the native state. As a consequence, CS is effectively stabilized in the presence of Hsp90. The significance of this interaction is especially evident in the suppression of aggregation, the major end result of thermal unfolding events in vivo and in vitro. These effects, which are ATP-independent, seem to be a general function of members of the Hsp90 family, since yeast and bovine Hsp90 as well as the Hsp90 homologue from Escherichia coli gave similar results. It seems likely that this function also reflects the role of Hsp90 under heat shock conditions in vivo. We therefore propose that members of the Hsp90 family convey thermotolerance by transiently binding to highly structured early unfolding intermediates, thereby preventing their irreversible aggregation and stabilizing the active species.

Animals↗
PubMed1995

Domain interactions stabilize the alternatively folded state of an antibody Fab fragment.

The structure of the Fab fragment of the monoclonal antibody MAK 33 (kappa/IgG1) at pH 2 was characterized. Spectroscopic and kinetic analysis revealed a molten globule-like state, characterized by elements of secondary structure but less defined tertiary contacts than in the native state. However, some aromatic side chains are in an asymmetrical environment. This structure was not detected using the isolated light chain or a Fab fragment lacking the covalent linkage of the light chain and Fd via the C-terminal disulfide bond. Therefore, interactions between the two chains, stabilized by the interchain disulfide within the Fab fragment, are essential for formation of the alternatively folded state.

Antibodies, Monoclonal↗
PubMed1995

Characterization of a quaternary-structured folding intermediate of an antibody Fab-fragment.

Antibody folding is a complex process comprising folding and association reactions. Although it is usually difficult to characterize kinetic folding intermediates, in the case of the antibody Fab fragment, domain-domain interactions lead to a rate-limiting step of folding, thus accumulating folding intermediates at a late step of folding. Here, we analyzed a late folding intermediate of the Fab fragment of the monoclonal antibody MAK 33 from mouse (kappa/IgG1). As a strategy for accumulation of this intermediate we used partial denaturation of the native Fab by guanidinium chloride. This denaturation intermediate, which can be populated to about 90%, is indistinguishable from a late-folding intermediate with respect to denaturation and renaturation kinetics. The spectroscopic analysis reveals a native-like secondary structure of this intermediate with aromatic side chains only slightly more solvent exposed than in the native state. The respective partner domains are weekly associated. From these data we conclude that the intramolecular association of the two chains during folding, with all domains in a native-like structure, follows a two-step mechanism. In this mechanism, presumably hydrophobic interactions are followed by rearrangements leading to the exact complementarity of the contact sites of the respective domains.

Acrylamides↗
PubMed1994

Influence of protein disulfide isomerase (PDI) on antibody folding in vitro.

The role of eucaryotic protein disulfide isomerase (PDI) in the folding and reoxidation of proteins in vitro was investigated using an antibody Fab fragment as a model substrate, since PDI is known to participate in the disulfide bond formation of immunoglobulins in vivo. PDI has no effect on the folding of the Fab fragment with intact disulfide bonds, suggesting that, at least in this system, PDI is not able to influence the folding process in a chaperone-like manner. Instead, the role of PDI is limited to disulfide bond formation as demonstrated for the folding of the denatured and reduced Fab fragment. Here, PDI influences the yield of reactivation enormously with a maximum effect at about stoichiometric amounts of PDI and Fab. Furthermore, PDI changes the redox dependence of the reaction. In the presence of PDI, formation of the correct disulfide bonds is possible at higher oxidizing conditions compared to the spontaneous reaction. The requirements both for stoichiometric amounts of PDI and for the presence of PDI during the first seconds of refolding suggest that there is a kinetic competition between rapid structure formation of the antibody domains and interaction of PDI with cysteine residues in the folding protein.

Animals↗
PubMed1993

Prolyl isomerases catalyze antibody folding in vitro.

Some slow-folding phases in the in vitro refolding of proteins originate from the isomerization of prolyl-peptide bonds, which can be accelerated by a class of enzymes called prolyl isomerases (PPIs). We used the in vitro folding of an antibody Fab fragment as a model system to study the effect of PPI on a folding reaction that is only partially reversible. We show here that members of both subclasses of PPIs, cyclophilin and FK 506 binding protein (FKBP), accelerate the refolding process and increase the yield of correctly folded molecules. An acceleration of folding was not observed in the presence of the specific inhibitor cyclosporin A, but still the yield of correctly folded molecules was increased. Bovine serum albumin (BSA) increased the yield comparable to cyclophilin but, in contrast, did not influence the rate of reactivation. These effects were observed only when cyclophilin or BSA were present during the first few seconds of refolding. However, the rate-limiting reactivation reaction is still accelerated when PPI is added several minutes after starting refolding. In contrast, the prokaryotic chaperone GroEL influences the refolding yield when added several minutes after initiating refolding. The results show that PPIs influence the folding of Fab in two different ways. (1) They act as true catalysts of protein folding by accelerating the rate-limiting isomerization of Xaa-Pro peptide bonds. Proline isomerization is obviously a late folding step and has no influence on the formation of aggregates within the first seconds of the refolding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Isomerases↗
PubMed1992

[The refusal to consent to blood transfusion. Legal and medical aspects using Jehovah's Witnesses as an example].

The limited understanding of a patient, such as a Jehovah's Witness, who has consented to an operation but refuses a blood transfusion for personal or religious reasons, places the physician in a moral dilemma. According to Article 2 II of the German Constitution, the fact that the patient has withheld or expressly refused his consent, i.e., in writing, mandates that the legal right of physical integrity be upheld with final legal effect, even in the case of an emergency. If this right to self-determination is abused, the person giving treatment is guilty of bodily harm in the sense of section 223 of the German Penal Code. Intraoperative haemodilution, the cell-save procedure, and colloidal and crystalloid volume replacement represent alternative methods of blood transfusion for Jehovah's Witnesses.

Blood Transfusion↗
PubMed1991

Alternatively folded states of an immunoglobulin.

Well-defined, non-native protein structures of low stability have been increasingly observed as intermediates in protein folding or as equilibrium structures populated under specific solvent conditions. These intermediate structures, frequently referred to as molten globule states, are characterized by the presence of secondary structure, a lack of significant tertiary contacts, increased hydrophobicity and partial specific volume as compared to native structures, and low cooperativity in thermal unfolding. The present study demonstrates that under acidic conditions (pH less than 3) the antibody MAK33 can assume a folded stable conformation. This A-state is characterized by a high degree of secondary structure, increased hydrophobicity, a native-like maximum wavelength of fluorescence emission, and a tendency toward slow aggregation. A prominent feature of this low-pH conformation is the stability against denaturant and thermal unfolding that is manifested in highly cooperative reversible phase transitions indicative of the existence of well-defined tertiary contacts. These thermodynamic results are corroborated by the kinetics of folding from the completely unfolded chain to the alternatively folded state at pH 2. The given data suggest that MAK33 at pH 2 adopts a cooperative structure that differs from the native immunoglobulin fold at pH 7. This alternatively folded state exhibits certain characteristics of the molten globule but differs distinctly from it by its extraordinary structural stability that is characteristic for native protein structures.

Amino Acids↗