Biomedical subjects

D E Otzen

Publications and source records attributed to D E Otzen.

At least 19 recordsLinked to original sources

PubMed2005

Using micellar mole fractions to assess membrane protein stability in mixed micelles.

The increased focus on the structural and physical properties of membrane proteins has made it critical to develop methods that provide a reliable estimate of membrane protein stability. A simple approach is to monitor the protein's conformational changes in mixed detergent systems, typically consisting of an anionic (denaturing) and non-ionic (non-denaturing) component. Linear correlations between, e.g., the melting temperature and the bulk mole fraction of the anionic component have been observed. However, a potential complication is that the bulk mole fraction is not identical to the mole fraction in the mixed micelle, which is the local environment experienced by the membrane protein. Here, we present an extensive analysis of the thermal stability of the membrane-integrated domain of the outer membrane protein AIDA in the presence of different mixed micelles. In the micelle system SDS-octyl-polyoxyethylene, the melting temperature in the absence of SDS extrapolates to 113 degrees C using bulk mole fractions. However, for mixed micelles involving short-chain detergents or phospholipids, the melting temperature calculated using bulk mole fractions reaches values up to several hundred degrees higher than 113 degrees C and can only be obtained by extrapolation over a narrow mole fraction interval. Furthermore, there is a non-linear relationship between the melting temperature and bulk mole fractions for mixed micelle systems involving cationic detergents (also denaturing). We show that if we instead use the micellar mole fraction as a parameter for denaturing detergent strength, we obtain linear correlations which extrapolate to more or less the same value of the melting temperature. There remains some scatter in the extrapolated values of the melting temperature in different binary systems, which suggest that additional micellar interactions may play a role. Nevertheless, in general terms, the mixed micellar composition is a good parameter to describe the membrane protein's microenvironment. Note, however, that for the mixed micelle system involving SDS and dodecyl maltoside, which has been used by several research groups to determine membrane protein stability, the estimate provided by bulk mole fraction leads to similar values as that of micellar mole fractions.

Adhesins, Escherichia coli↗

PubMed2003

Structural background of cyclodextrin-protein interactions.

Cyclodextrins are cyclic oligosaccharides with the shape of a hollow truncated cone. Their exterior is hydrophilic and their cavity is hydrophobic, which gives cyclodextrins the ability to accommodate hydrophobic molecules/moieties in the cavity. This special molecular arrangement accounts for the variety of beneficial effects cyclodextrins have on proteins, which is widely used in pharmacological applications. We have studied the interaction between beta-cyclodextrin and four non-carbohydrate-binding model proteins: ubiquitin, chymotrypsin inhibitor 2 (CI2), S6 and insulin SerB9Asp by NMR spectroscopy at varying structural detail. We demonstrate that the interaction of beta-cyclodextrin and our model proteins takes place at specific sites on the protein surface, and that solvent accessibility of those sites is a necessary but not compelling condition for the occurrence of an interaction. If this behaviour can be generalized, it might explain the wide range of different effects of cyclodextrins on different proteins: aggregation suppression (if residues responsible for aggregation are highly solvent accessible), protection against degradation (if point of attack of a protease is sterically 'masked' by cyclodextrin), alteration of function (if residues involved in function are 'masked' by cyclodextrin). The exact effect of cyclodextrins on a given protein will always be related to the particular structure of this protein.

Cyclodextrins↗

PubMed2001

Folding of circular permutants with decreased contact order: general trend balanced by protein stability.

To examine the influence of contact order and stability on the refolding rate constant for two-state proteins, we have analysed the folding kinetics of the small beta-alpha-beta protein S6 and two of its circular permutants with relative contact orders of 0.19, 0.15 and 0.12. Data reveal a small but significant increase of the refolding rate constant (log k(f)) with decreasing contact order. At the same time, the decreased contact order is correlated to losses in global stability and alterations of the folding nucleus. When the differences in stability are accounted for by addition of Na2SO4 or by comparison of the folding kinetics at the transition mid-point, the dependence between log k(f) and contact order becomes stronger and follows the general correlation for two-state proteins. The observation emphasizes the combined action of topology and stability in controlling the rate constant of protein folding.

Binding Sites↗

PubMed2001

A simple way to measure protein refolding rates in water.

Refolding of proteins is traditionally carried out either by diluting the denaturant-unfolded protein into buffer (GdmCl-jump) or by mixing the acid-denatured protein with strong buffer (pH-jump). The first method does not allow direct measurement of folding rates in water since the GdmCl cannot be infinitely diluted, and the second method suffers from the limitation that many proteins cannot be pH-denatured. Further, some proteins do not refold reversibly from low pH where they get trapped as aggregation prone intermediates. Here, we present an alternative approach for direct measurement of refolding rates in water, which does not rely on extrapolation. The protein is denatured in SDS, and is then mixed with alpha-cyclodextrin, which rapidly strips SDS molecules from the protein, leaving the naked unfolded protein to refold.

Animals↗

PubMed2000

Designed protein tetramer zipped together with a hydrophobic Alzheimer homology: a structural clue to amyloid assembly.

Limited solubility and precipitation of amyloidogenic sequences such as the Alzheimer peptide (beta-AP) are major obstacles to a molecular understanding of protein fibrillation and deposition processes. Here we have circumvented the solubility problem by stepwise engineering a beta-AP homology into a soluble scaffold, the monomeric protein S6. The S6 construct with the highest beta-AP homology crystallizes as a tetramer that is linked by the beta-AP residues forming intermolecular antiparallel beta-sheets. This construct also shows increased coil aggregation during refolding, and a 14-mer peptide encompassing the engineered sequence forms fibrils. Mutational analysis shows that intermolecular association is linked to the overall hydrophobicity of the sticky sequence and implies the existence of "structural gatekeepers" in the wild-type protein, that is, charged side chains that prevent aggregation by interrupting contiguous stretches of hydrophobic residues in the primary sequence.

Amino Acid Sequence↗

PubMed2000

Model process for separation based on unfolding and refolding of chymotrypsin inhibitor 2 in thermoseparating polymer two-phase systems.

For the design of a new separation process based on unfolding and refolding of protein, the partitioning behaviour of proteins was studied in thermoseparating polymer two-phase systems with varying pH and temperature. Chymotrypsin inhibitor 2 (CI2), which unfolds reversibly in a simple two-state manner, was partitioned in an aqueous two-phase system (ATPS) composed of a random copolymer of ethylene oxide and propylene oxide (Breox) and dextran T-500. Between 25 and 50 degrees C, the partition coefficients of CI2 in Breox-dextran T-500 systems remain constant at neutral pH. However, there is a drastic increase at pH values below 1.7, 2.1, and 2.7 at 25, 40 and 50 degrees C, respectively. The partitioning behavior of CI2 was also investigated in thermoseparating water-Breox systems at 55-60 degrees C, where CI2 was partitioned to the polymer-rich phase at pH values below 2.4. These results on the CI2 partitioning can be explained by the conformational difference between the folded and the unfolded states of the protein, where the unfolded CI2 with a more hydrophobic surface is partitioned to the relatively hydrophobic Breox phase in both systems. A separation process is presented based on the partitioning behavior of unfolded and refolded CI2 by control of pH and temperature in thermoseparating polymer two-phase systems. The target protein can be recovered through (i) selective separation in Breox-dextran systems, (ii) refolding in Breox phase, and (iii) thermoseparation of primary Breox phase.

Chymotrypsin↗

PubMed2000

The spectral and thermodynamic properties of staphylococcal enterotoxin A, E, and variants suggest that structural modifications are important to control their function.

The superantigens staphylococcal enterotoxin A and E (SEA and SEE) can activate a large number of T-cells. SEA and SEE have approximately 80% sequence identity but show some differences in their biological function. Here, the two superantigens and analogues were characterized biophysically. SEE was shown to have a substantially higher thermal stability than SEA. Both SEA and SEE were thermally stabilized by 0.1 mM Zn(2+) compared with Zn(2+)-reduced conditions achieved using 1 mM EDTA or specific replacements that affect Zn(2+) coordination. The higher stability of SEE was only partly caused by the T-cell receptor (TCR) binding regions, whereas regions in the vicinity of the major histocompatibility complex class II binding sites affected the stability to a greater extent. SEE exhibited a biphasic denaturation between pH 5.0-6.5, influenced by residues in the TCR binding regions. Interestingly, enzyme-linked immunosorbent assay, isoelectric focusing, and circular dichroism analysis indicated that conformational changes had occurred in the SEA/E chimerical constructs relative to SEA and SEE. Thus, it is proposed that the Zn(2+) binding site is very important for the stability and potency of SEA and SEE, whereas residues in the TCR binding site have a substantial influence on the molecular conformation to control specificity and function.

Amino Acid Sequence↗

PubMed1999

Salt-induced detour through compact regions of the protein folding landscape.

In several cases, inorganic salts have been used to induce partly structured states in protein folding. But what is the nature of these states: Do they represent key stepping stones in the folding process, or are they circumstantial pitfalls in the energy landscape? Here we report that, in the case of the two-state protein S6, the salt-induced collapsed state is off the usual folding routes in the sense that it is prematurely collapsed and slows down folding by several orders of magnitude. Although this species is over-compact, it is not a dead-end trap but may fold by alternative channels to the native state.

Journal Article↗

PubMed1999

Structural changes in the transition state of protein folding: alternative interpretations of curved chevron plots.

The interpretation of folding rates is often rationalized within the context of transition state theory. This means that the reaction rate is linked to an activation barrier, the height of which is determined by the free energy difference between a ground state (the starting point) and an apparent transition state. Changes in the folding kinetics are thus caused by effects on either the ground state, the transition state, or both. However, structural changes of the transition state are rarely discussed in connection with experimental data, and kinetic anomalies are commonly ascribed to ground state effects alone, e.g., depletion or accumulation of structural intermediates upon addition of denaturant. In this study, we present kinetic data which are best described by transition state changes. We also show that ground state effects and transition state effects are in general difficult to distinguish kinetically. The analysis is based on the structurally homologous proteins U1A and S6. Both proteins display two-state behavior, but there is a marked difference in their kinetics. S6 exhibits a classical V-shaped chevron plot (log observed rate constant vs denaturant concentration), whereas U1A's chevron plot is symmetrically curved, like an inverted bell curve. However, S6 is readily mutated to display U1A-like kinetics. The seemingly drastic effects of these mutations are readily ascribed to transition state movements where large kinetic differences result from relatively small alterations of a common free energy profile and broad activation barriers.

Amino Acid Sequence↗

PubMed1999

Analysis of protein-protein interactions by mutagenesis: direct versus indirect effects.

Site-directed mutagenesis, including double-mutant cycles, is used routinely for studying protein-protein interactions. We now present a case analysis of chymotrypsin inhibitor 2 (CI2) and subtilisin BPN' using (i) a residue in CI2 that is known to interact directly with subtilisin (Tyr42) and (ii) two CI2 residues that do not have direct contacts with subtilisin (Arg46 and Arg48). We find that there are similar changes in binding energy on mutation of these two sets of residues. It can thus be difficult to interpret mutagenesis data in the absence of structural information.

Kinetics↗

PubMed1999

A comparative study of the unfolding of the endoglucanase Cel45 from Humicola insolens in denaturant and surfactant.

Cellulases are increasingly being used for industrial purposes, particularly in washing powders, yet little is known of the factors governing the stability of proteins in detergent solutions. We present a comparative analysis of the behavior of the cellulase Cel45 from Humicola insolens in the presence of the denaturant guanidinium chloride and the anionic detergent C12-LAS. Although Cel45 unfolds in GdmCl according to a simple two-state model under equilibrium conditions, it accumulates a transient intermediate during refolding. The four disulfide bonds do not contribute detectably to the stability of the native state. Cel45 is unfolded by very low concentrations of C12-LAS (1-4 mM). An analysis of 16 mutants of Cel45 shows a very weak correlation between unfolding rates in denaturant and detergent; mutants that have the same unfolding rate in GdmCl (within a factor of 1.5) vary 1,000-fold in their unfolding rates in C12-LAS. The data support a simple model for unfolding by detergent, in which the introduction of positive charges or removal of negative charges greatly increases detergent sensitivity, while interactions with the hydrophobic detergent tail contribute to a smaller extent. This implies that different detergent-mediated unfolding pathways exist, whose accessibilities depend on individual residues. Double-mutant cycles reveal that mutations in two proximal residues lead to repulsion and a destabilization greater than the sum of the individual mutations as measured by GdmCl denaturation, but they also reduce the affinity for LAS and therefore actually stabilize the protein relative to wild-type. Ligands that interact strongly with the denatured state may therefore alter the unfolding process.

Acid-Base Equilibrium↗

PubMed1998

Folding of circular and permuted chymotrypsin inhibitor 2: retention of the folding nucleus.

The 64-residue chymotrypsin inhibitor 2 (CI2) folds by a two-state nucleation-condensation mechanism, whereby secondary and tertiary structure coalesce concomitantly in the transition state around Ala 16 in the helical N-cap. Permutation of the SH3-domain of alpha-spectrin apparently shifts its folding nucleus to another region of the protein, suggesting that a protein's transition state may be altered by altering the protein's connectivity. We have characterized the structure of the transition state of a circular and a permuted version of CI2 by a protein engineering study encompassing 11 mutations. Circular CI2 was obtained by the introduction of cysteines at residues 3 and 63 and linking them by disulfide bond formation. Subsequent cyanogen-bromide cleavage of the scissile bond, Met 40-Glu 41, yielded permuted CI2. Circular and permuted CI2 also fold according to a two-state mechanism. Permutation does not affect the folding rate constant, but circularization increases it 7-fold. The transition states of circular and permuted CI2 are essentially unchanged from that of wild-type CI2. Importantly, the folding nucleus around Ala16 is retained. These results complement a previous observation that the transition state for association of two CI2 fragments (residues 1-40 and 41-64, generated by CNBr cleavage) is very similar to the folding transition state of intact CI2. The similarity of rate constants for folding of wild-type and permuted CI2, and their value relative to that for the association of fragments, allows us to estimate the gain in entropy of activation on having the separate fragments linked: 18.3 cal M-1 K-1; i.e. an effective molarity of 10(4) M. The contrast between the retention of the folding nucleus on permutation of CI2 and its change for the SH3-domain of alpha-spectrin probably arises because the latter was cleaved in its folding nucleus whereas cleavage at sites other than 40-41 in CI2 is very destabilizing. Whether or not a folding nucleus can be changed probably depends on the specific protein and its permissivity to permutation.

Chymotrypsin↗

PubMed1997

Hydrogen exchange in chymotrypsin inhibitor 2 probed by mutagenesis.

Two-dimensional NMR spectroscopy has been used to monitor hydrogen-deuterium exchange in chymotrypsin inhibitor 2. Application of two independent tests has shown that at pH 5.3 to 6.8 and 33 to 37 degrees C, exchange occurs via an EX2 limit. Comparison of the exchange rates of a number of mutants of CI2 with those of wild-type identifies the pathway of exchange, whether by local breathing, global unfolding or a mixture of the two pathways. For a large number of residues, the exchange rates were unaffected by mutations which destabilized the protein by up to 1.9 kcal mol(-1), indicating that exchange is occurring through local fluctuations of the native state. A small number of residues were found for which the mutations had the same effect on the rate constants for exchange as on the equilibrium constant for unfolding, indicating that these residues exchange by global unfolding. These are residues that have the slowest exchange rates in the wild-type protein. We see no correspondence between these residues and residues involved in the nucleation site for the folding reaction identified by protein engineering studies. Rather, the exchange behaviour of CI2 is determined by the native structure: the most protected amide protons are located in regions of hydrogen bonding, specifically the C terminus of the alpha-helix and the centre of the beta-sheet. A number of the most slowly exchanging residues are in the hydrophobic core of the protein.

Binding Sites↗

PubMed1997

The rate of isomerisation of peptidyl-proline bonds as a probe for interactions in the physiological denatured state of chymotrypsin inhibitor 2.

There are four peptidyl-proline bonds in the 64-residue protein chymotrypsin inhibitor 2 (CI2), all of which are in the trans conformation in the native structure. The isomerisation of one or more of these peptidyl-proline bonds to the cis conformation in the denatured state gives rise to heterogeneity, leading to both fast and slow-folding species. The refolding of the fast-folding species, which has all trans peptidyl-proline bonds, is much faster than that of the slow-folding species, which have one or more cis peptidyl-proline bonds. In CI2, the slow-folding species can be classified into two groups by their rates of refolding, temperature-dependence, pH-dependence and [GdmCl]-dependence of the rate constants and the effect of peptidyl-prolyl isomerase on the rate constants. The replacement of Pro6 by Ala removes one of the slow refolding phases, suggesting that the cis peptidyl-Pro6 conformation is solely responsible for one of the slow-folding species. Pro6 is located in a region of the protein where non-random interactions have been found in a series of N-terminal fragments of CI2 (residues 1 to 13, 1 to 25, 1 to 28 and 1 to 40). In addition, NMR studies on a mutant fragment, (1-40)T3A, have confirmed that this non-native interaction is associated with the bulky side-chain of Trp5. The atypical rate of cis to trans isomerisation of the peptidyl-Pro bond is indicative of the presence of a similar hydrophobic cluster in the physiological denatured state of intact CI2.

Amino Acid Isomerases↗

PubMed1996

Structure of the transition state for folding of a protein derived from experiment and simulation.

Independent experimental and theoretical studies of the unfolding of barley chymotrypsin inhibitor 2 (CI2) are compared in an attempt to derive plausible three-dimensional structural models of the transition states. A very simple structure index is calculated along the sequence for the molecular dynamics-generated transition state models to facilitate comparison with the phi F values. The two are in good agreement overall (correlation coefficient = 0.87), which suggests that the theoretical models should provide a structural framework for interpretation of the phi F values. Both experiment and simulation indicate that the transition state is a distorted form of the native state in which the alpha-helix is weakened but partially intact and the beta-sheet is quite disrupted. As inferred from the phi f values and observed directly in the simulations, the unfolding of CI2 is cooperative and there is a "folding core" comprising a patch on the alpha-helix and a portion of the beta-sheet, nucleated by interactions between Ala16, Ile49 and other neighbouring residues. The protein becomes less structured radiating away from this core. Overall the data indicate that CI2 folds by a nucleation-collapse mechanism. In the absence of experimental information, we have little confidence that the molecular dynamics simulations are correct, especially when only one or a few simulations are performed. On the other hand, even though the experimentally derived phi values may reflect the extent of overall structure formation, they do not provide an actual atomic-resolution three dimensional structure of the transition state. By combining the two approaches, however, we have a framework for interpreting phi F values and can hopefully arrive at a more trustworthy model of the transition state. The process is in some ways similar to the combination of molecular dynamics and NMR data to solve the tertiary structure of proteins.

Amino Acid Sequence↗

PubMed1995

Conformational pathway of the polypeptide chain of chymotrypsin inhibitor-2 growing from its N terminus in vitro. Parallels with the protein folding pathway.

We have obtained a series of fragments growing from the N terminus of the protein chymotrypsin inhibitor-2 (C12) in order to study the development of structure on elongation of the polypeptide in solution. We present an extensive biophysical characterization of ten fragments using different conformational probes. Small fragments up to residue 40 of the 64-residue protein are disordered. Fragment (1-40) has non-native local hydrophobic clusters, but nevertheless does not bind 8-anilinonaphthalene-1-sulphonate (ANS). Hydrophobic regions in longer fragments become gradually more capable of binding ANS as the chain grows to completion, with a tendency to form native structures. Major changes in secondary structure and accessibility to hydrophobic sites occur in parallel, between (1-40) and (1-53), together with changes in hydrodynamic volume and flexibility. NMR studies of (1-53), the first fragment displaying tertiary interactions, show that a subcore is fully formed and the alpha-helix (residues 12 to 24) is of fluctuating structure. Fragments (1-53) and (1-60) share many properties with molten globule-like structures, with varying degrees or order. Fluorescence properties of the native fold are gradually recovered from fragments (1-60) to full-length C12, together with a decrease in hydrophobic exposure. A small degree of co-operativity of formation of structure appears when residue 60 is added, gradually increasing as residue 62 is added, but a full two-state co-operative transition appears only on addition of Arg62 and Val63. We believe this is the result of correct side-chain packing of the hydrophobic core, capping the major elements of secondary structure in C12 at this late stage, which is probed by the complete recovery of the fluorescence of the unique Trp5. The structures that develop as the polypeptide chain increases in length parallel the structural features present in the nucleus for the folding of intact protein, which develops in the transition state. The folding nucleus consists of much of the helix and the interactions made by Ala16 in the helix with residues in the core, especially with Leu49 and Ile57, with the rest of the structure being formed only very weakly in the transition state.

Amino Acid Sequence↗

PubMed1995

The structure of the transition state for folding of chymotrypsin inhibitor 2 analysed by protein engineering methods: evidence for a nucleation-condensation mechanism for protein folding.

The 64-residue protein chymotrypsin inhibitor 2 (CI2) is a single module of structure. It folds and unfolds as a single co-operative unit by simple two-state kinetics via a single rate determining transition state. This transition state has been characterized at the level of individual residues by analysis of the rates and equilibria of folding of some 100 mutants strategically distributed at 45 sites throughout the protein. Only one residue, a helical residue (Ala16) buried in the hydrophobic core, has its full native interaction energy in the transition state. The only region of structure which is well developed in the transition state is the alpha-helix (residues 12 to 24). But, the interactions within it are weakened, especially at the C-terminal region. The rest of the protein has varying degrees of weakly formed structure. Thus, secondary and tertiary interactions appear to form concurrently. These data, reinforced by studies on the structures of peptide fragments, fit a "nucleation-condensation" model in which the overall structure condenses around an element of structure, the nucleus, that itself consolidates during the condensation. The high energy transition state is composed of the whole of the molecule making a variety of weak interactions, the nucleus being those residues that make the strongest interactions. The nucleus here is part of the alpha-helix and some distant residues in the sequence with which it makes contacts. The remainder of the protein has to be sufficiently ordered that it provides the necessary interactions to stabilize the nucleus. The nucleus is only weakly formed in the denatured state but develops in the transition state. The onrush of stability as the nucleus consolidates its local and long range interactions is so rapid that it is not yet fully formed in the transition state. The formation of the nucleus is thus coupled with the condensation. These results are consistent with a recent simulation of the folding of a computer model protein on a lattice which is found to proceed by a nucleation-growth mechanism. We suggest that the mechanism of folding of CI2 may be a common theme in protein folding whereby fundamental folding units of larger proteins, which are modelled by the folding of CI2, form by nucleation-condensation events and coalesce, perhaps in a hierarchical manner.

Chymotrypsin↗

PubMed1995

Nature and consequences of GroEL-protein interactions.

The importance of chaperonin-protein interactions has been investigated by analyzing the refolding of the barley chymotrypsin inhibitor 2 in the presence of GroEL. The chaperonin retards the rate of refolding of wild type and 32 representative point mutants. The retardation of the rate drops to a finite level at saturating concentrations of GroEL, being lowered by a factor of 3-100, depending on the mutation. It is seen qualitatively that truncation of large hydrophobic side chains to smaller side chains weakens binding. Analysis of the magnitude of the rates of retardation shows further that hydrophobic and positively charged side chains tend to interact favorably with GroEL whereas negatively charged side chains tend to repel. There is an inverse correlation between the strength of hydrophobic interactions and the rate constant for refolding of the GroEL-complexed protein: the better the binding, the slower the folding. This shows directly that hydrophobic (and other favorable) interactions between the chaperonin and substrate are weakened during the refolding process and implies that unfolding can be catalyzed by the gain of such interactions.

Chaperonin 60↗