The foldon substructure of staphylococcal nuclease

To search for submolecular foldon units, the spontaneous reversible unfolding and refolding of staphylococcal nuclease under native conditions was studied by a kinetic native-state hydrogen exchange (HX) method. As for other proteins, it appears that staphylococcal nuclease is designed as an assembly of well-integrated foldon units that may define steps in its folding pathway and may regulate some other functional properties. The HX results identify 34 amide hydrogens that exchange with solvent hydrogens under native conditions by way of large transient unfolding reactions. The HX data for each hydrogen measure the equilibrium stability (ΔG_HX) and the kinetic unfolding and refolding rates (k_op and k_cl) of the unfolding reaction that exposes it to exchange. These parameters separate the 34 identified residues into three distinct HX groupings. Two correspond to clearly defined structural units in the native protein, termed the blue and red foldons. The remaining HX grouping contains residues, not well separated by their HX parameters alone, that represent two other distinct structural units in the native protein, termed the green and yellow foldons. Among these four sets, a last unfolding foldon (blue) unfolds with a rate constant of 6 × 10^− 6 s^− 1 and free energy equal to the protein's global stability (10.0 kcal/mol). It represents part of the β-barrel, including mutually H-bonding residues in the β4 and β5 strands, a part of the β3 strand that H-bonds to β5, and residues at the N-terminus of the α2 helix that is capped by β5. A second foldon (green), which unfolds and refolds more rapidly and at slightly lower free energy, includes residues that define the rest of the native α2 helix and its C-terminal cap. A third foldon (yellow) defines the mutually H-bonded β1-β2-β3 meander, completing the native β-barrel, plus an adjacent part of the α1 helix. A final foldon (red) includes residues on remaining segments that are distant in sequence but nearly adjacent in the native protein. Although the structure of the partially unfolded forms closely mimics the native organization, four residues indicate the presence of some nonnative misfolding interactions. Because the unfolding parameters of many other residues are not determined, it seems likely that the concerted foldon units are more extensive than is shown by the 34 residues actually observed.     

Structural features of interferon-gamma aggregation revealed by hydrogen exchange

Using hydrogen-deuterium exchange (HX) and electrospray ionization mass spectrometry, we have investigated the stability and structural changes of recombinant human interferon-gamma (IFN-gamma) during aggregation induced by guanidine hydrochloride (GdnHCl) and potassium thiocyanate. First, HX labeling was initiated after the amorphous aggregates were formed to probe the tertiary structure of the aggregated state. Second, labeling was performed at low protein concentrations to assess stability under aggregation prone conditions. In 1 M GdnHCl, the stability of IFN-gamma was greatly reduced and much less protection from HX in solution was observed. Exchange under these conditions was slower in helix C than in the rest of the protein. Aggregates formed in 1 M GdnHCl showed a HX pattern consistent with a partially unfolded state with an intact helix C. Although aggregates formed in 0.3 M KSCN exhibited a HX pattern similar to those formed in GdnHCl, the solution phase HX pattern in 0.3 M KSCN was surprisingly comparable to that of the native state. Varying the aggregation time before performing HX revealed that KSCN first precipitated native protein and then facilitated partial unfolding of the precipitated protein. These results show that helix C, which forms the hydrophobic core of the IFN-gamma dimer, is highly protected from HX under native conditions, is more stable in GdnHCl than the rest of the protein and remains intact in both GdnHCl- and KSCN-induced aggregates. This suggests that native-state HX patterns may presage regions of the protein susceptible to unfolding during aggregation.     

Towards a consistent modeling of protein thermodynamic and kinetic cooperativity: how applicable is the transition state picture to folding and unfolding?

To what extent do general features of folding/unfolding kinetics of small globular proteins follow from their thermodynamic properties? To address this question, we investigate a new simplified protein chain model that embodies a cooperative interplay between local conformational preferences and hydrophobic burial. The present four-helix-bundle 55mer model exhibits protein-like calorimetric two-state cooperativity. It rationalizes native-state hydrogen exchange observations. Our analysis indicates that a coherent, self-consistent physical account of both the thermodynamic and kinetic properties of the model leads naturally to the concept of a native state ensemble that encompasses considerable conformational fluctuations. Such a multiple-conformation native state is seen to involve conformational states similar to those revealed by native-state hydrogen exchange. Many of these conformational states are predicted to lie below native baselines commonly used in interpreting calorimetric data. Folding and unfolding kinetics are studied under a range of intrachain interaction strengths as in experimental chevron plots. Kinetically determined transition midpoints match well with their thermodynamic counterparts. Kinetic relaxations are found to be essentially single-exponential over an extended range of model interaction strengths. This includes the entire unfolding regime and a significant part of a folding regime with a chevron rollover, as has been observed for real proteins that fold with non-two-state kinetics. The transition state picture of protein folding and unfolding is evaluated by comparing thermodynamic free energy profiles with actual kinetic rates. These analyses suggest that some chevron rollovers may arise from an internal frictional effect that increasingly impedes chain motions with more native conditions, rather than being caused by discrete deadtime folding intermediates or shifts of the transition state peak as previously posited.     

The folding energy landscape of the dimerization domain of Escherichia coli Trp repressor: a joint experimental and theoretical investigation

Enhanced structural insights into the folding energy landscape of the N-terminal dimerization domain of Escherichia coli tryptophan repressor, [2-66]2 TR, were obtained from a combined experimental and theoretical analysis of its equilibrium folding reaction. Previous studies have shown that the three intertwined helices in [2-66]2 TR are sufficient to drive the formation of a stable dimer for the full-length protein, [2-107]2 TR. The monomeric and dimeric folding intermediates that appear during the folding reactions of [2-66]2 TR have counterparts in the folding mechanism of the full-length protein. The equilibrium unfolding energy surface on which the folding and dimerization reactions occur for [2-66]2 TR was examined with a combination of native-state hydrogen exchange analysis, pepsin digestion and matrix-assisted laser/desorption mass spectrometry performed at several concentrations of protein and denaturant. Peptides corresponding to all three helices in [2-66]2 TR show multi-layered protection patterns consistent with the relative stabilities of the dimeric and monomeric folding intermediates. The observation of protection exceeding that offered by the dimeric intermediate in segments from all three helices implies that a segment-swapping mechanism may be operative in the monomeric intermediate. Protection greater than that expected from the global stability for a single amide hydrogen in a peptide from the C-helix possibly and another from the A-helix may reflect non-random structure, possibly a precursor for segment swapping, in the urea-denatured state. Native topology-based model simulations that correspond to a funnel energy landscape capture both the monomeric and dimeric intermediates suggested by the HX MS data and provide a rationale for the progressive acquisition of secondary structure in their conformational ensembles.     

Investigation of the four cooperative unfolding units existing in the MICAL-1 CH domain

The solution structure of human MICAL-1 calpolnin homology (CH) domain is composed of six alpha helices and one 3(10) helix. To study the unfolding of this domain, we carry out native-state hydrogen exchange, intrinsic fluorescence and far-UV circular dichroism experiments. The free energy of unfolding, DeltaG(H2O), is calculated to be 7.11+/-0.58 kcal mol(-1) from GuHCl denaturation at pH 6.5. Four cooperative unfolding units are found using native-state hydrogen exchange experiment. Forty-seven slow-exchange residues can be studied by native-state hydrogen exchange experiments. From the concentration dependence of exchange rates, free energy of amide hydrogen with solvent, DeltaG(HX) and m-value (sensitivity of exposure to denaturant) are obtained, which reveal four cooperative unfolding units. The slowest exchanging protons are distributed throughout the whole hydrophobic core of the protein, which might be the folding core. These results will help us understand the structure of MICAL-1 CH domain more deeply.     

OneG: a computational tool for predicting cryptic intermediates in the unfolding kinetics of proteins under native conditions

Understanding the relationships between conformations of proteins and their stabilities is one key to address the protein folding paradigm. The free energy change (ΔG) of unfolding reactions of proteins is measured by traditional denaturation methods and native hydrogen-deuterium (H/D) exchange methods. However, the free energy of unfolding (ΔG(U)) and the free energy of exchange (ΔG(HX)) of proteins are not in good agreement, though the experimental conditions of both methods are well matching to each other. The anomaly is due to any one or combinations of the following reasons: (i) effects of cis-trans proline isomerisation under equilibrium unfolding reactions of proteins (ii) inappropriateness in accounting the baselines of melting curves (iii) presence of cryptic intermediates, which may elude the melting curve analysis and (iv) existence of higher energy metastable states in the H/D exchange reactions of proteins. Herein, we have developed a novel computational tool, OneG, which accounts the discrepancy between ΔG(U) and ΔG(HX) of proteins by systematically accounting all the four factors mentioned above. The program is fully automated and requires four inputs: three-dimensional structures of proteins, ΔG(U), ΔG(U)(*) and residue-specific ΔG(HX) determined under EX2-exchange conditions in the absence of denaturants. The robustness of the program has been validated using experimental data available for proteins such as cytochrome c and apocytochrome b(562) and the data analyses revealed that cryptic intermediates of the proteins detected by the experimental methods and the cryptic intermediates predicted by the OneG for those proteins were in good agreement. Furthermore, using OneG, we have shown possible existence of cryptic intermediates and metastable states in the unfolding pathways of cardiotoxin III and cobrotoxin, respectively, which are homologous proteins. The unique application of the program to map the unfolding pathways of proteins under native conditions have been brought into fore and the program is publicly available at http://sblab.sastra.edu/oneg.html.     

pH dependence of the hydrogen exchange in the SH3 domain of alpha-spectrin

Using nuclear magnetic resonance we have measured the hydrogen exchange (HX) in the Src homology region 3 (SH3) domain of alpha-spectrin as a function of pH*. At very acidic pH* values the exchange of most residues appears to occur via global unfolding, although several residues show abnormally large Gibbs energies of exchange, suggesting the presence of some residual structure in the unfolded state. At higher pH* HX occurs mainly via local or partial unfoldings. We have been able to characterize the coupling between the electrostatic interactions in this domain and the conformational fluctuations occurring under native conditions by analyzing the dependence upon pH* of the Gibbs energy of exchange. The SH3 domain seems to be composed of a central core, which requires large structural disruptions to become exposed to the solvent, surrounded by smaller subdomains, which fluctuate independently.     

Residue-wise conformational stability of DLC8 dimer from native-state hydrogen exchange

Dynein light chain (DLC8) is the smallest subunit of the dynein motor complex, which is known to act as a cargo adaptor in intracellular trafficking. The protein exists as a pure dimer at physiological pH and a completely folded monomer below pH 4. Here, we have determined the energy landscape of the dimeric protein using a combination of optical techniques and native-state hydrogen exchange of amide groups, the former giving the global features and the latter yielding the residue level details. The data indicated the presence of intermediates along the equilibrium unfolding transition. The hydrogen exchange data suggested that the molecule has differential stability in its various segments. We deduce from the free energy data that the antiparallel beta-sheets (beta4 and beta5) that form the hydrophobic core of the protein and the alpha2 helix, all of which are highly protected with regard to hydrogen exchange, contribute significantly to the initial step of the protein folding mechanism. Denaturant-dependent hydrogen exchange indicated further that some amides exchange via local fluctuations, whereas there are others which exchange via global unfolding events. Implications of these to cargo adaptability of the dimer are discussed.     

A statistical mechanical model for hydrogen exchange in globular proteins.

We develop a statistical mechanical theory for the mechanism of hydrogen exchange in globular proteins. Using the HP lattice model, we explore how the solvent accessibilities of chain monomers vary as proteins fluctuate from their stable native conformations. The model explains why hydrogen exchange appears to involve two mechanisms under different conditions of protein stability; (1) a “global unfolding” mechanism by which all protons exchange at a similar rate, approaching that of the denatured protein, and (2) a “stable-state” mechanism by which protons exchange at rates that can differ by many orders of magnitude. There has been some controversy about the stable-state mechanism: does exchange take place inside the protein by solvent penetration, or outside the protein by the local unfolding of a subregion? The present model indicates that the stable-state mechanism of exchange occurs through an ensemble of conformations, some of which may bear very little resemblance to the native structure. Although most fluctuations are small-amplitude motions involving solvent penetration or local unfolding, other fluctuations (the conformational distant relatives) can involve much larger transient excursions to completely different chain folds.     

Natural selection for kinetic stability is a likely origin of correlations between mutational effects on protein energetics and frequencies of amino acid occurrences in sequence alignments

It appears plausible that natural selection constrains, to some extent at least, the stability in many natural proteins. If, during protein evolution, stability fluctuates within a comparatively narrow range, then mutations are expected to be fixed with frequencies that reflect mutational effects on stability. Indeed, we recently reported a robust correlation between the effect of 27 conservative mutations on the thermodynamic stability (unfolding free energy) of Escherichia coli thioredoxin and the frequencies of residues occurrences in sequence alignments. We show here that this correlation likely implies a lower limit to thermodynamic stability of only a few kJ/mol below the unfolding free energy of the wild-type (WT) protein. We suggest, therefore, that the correlation does not reflect natural selection of thermodynamic stability by itself, but of some other factor which is linked to thermodynamic stability for the mutations under study. We propose that this other factor is the kinetic stability of thioredoxin in vivo, since( i) kinetic stability relates to irreversible denaturation, (ii) the rate of irreversible denaturation in a crowded cellular environment (or in a harsh extracellular environment) is probably determined by the rate of unfolding, and (iii) the half-life for unfolding changes in an exponential manner with activation free energy and, consequently, comparatively small free energy effects can have deleterious consequences for kinetic stability. This proposal is supported by the results of a kinetic study of the WT form and the 27 single-mutant variants of E. coli thioredoxin based on the global analyses of chevron plots and equilibrium unfolding profiles determined from double-jump unfolding assays. This kinetic study suggests, furthermore, one of the factors that may contribute to the high activation free energy for unfolding in thioredoxin (required for kinetic stability), namely the energetic optimization of native-state residue environments in regions, which become disrupted in the transition state for unfolding.     

Mapping Transient Partial Unfolding by Protein Engineering and Native-State Proteolysis

Transient partial unfolding of proteins under native conditions may have significant consequences in the biochemical and biophysical properties of proteins. Native-state proteolysis offers a facile way to investigate the thermodynamic and kinetic accessibilities of partially unfolded forms (cleavable forms) under native conditions. However, determination of the structure of the cleavable form, which is populated only transiently, remains challenging. Although in some cases partially cleaved products from proteolysis provide information on the structure of this elusive form, proteolysis of many proteins does not accumulate detectable intermediates. Here, we describe a systematic approach to determining structures of cleavable forms by protein engineering and native-state proteolysis. By devising φ_c analysis, which is analogous to conventional φ analysis, we have determined the structure of the cleavable form of Escherichia coli maltose-binding protein (MBP), which does not accumulate any partially cleaved products. We mutated 10 buried residues in MBP to alanine and determined φ_c values from the effects of the mutations on global stability and proteolytic susceptibility. The result of this analysis suggests that two C-terminal helices in MBP are unfolded in their cleavable form. The effect of ligand binding on proteolytic susceptibility and C-terminal deletion mutations also confirms the proposed structure. Our approach and methodology are generally applicable not only in elucidating the mechanism of proteolysis but also in investigating other important processes involving partial unfolding under native conditions such as protein misfolding and aggregation.     

Characterization of the unfolding of ribonuclease a by a pulsed hydrogen exchange study: evidence for competing pathways for unfolding

The unfolding of ribonuclease A was studied in 5.2 M guanidine hydrochloride at pH 8 and 10 degrees C using multiple optical probes, native-state hydrogen exchange (HX), and pulse labeling by hydrogen exchange. First, native-state HX studies were used to demonstrate that the protein exists in two slowly interconverting forms under equilibrium native conditions: a predominant exchange-incompetent N form and an alternative ensemble of conformations, N(I), in which some amide hydrogens are fully exposed to exchange. Pulsed HX studies indicated that, during unfolding, the rates of exposure to exchange with solvent protons were similar for all backbone NH probe protons. It is shown that two parallel routes of unfolding are available to the predominant N conformation as soon as it encounters strong unfolding conditions. A fraction of molecules appears to rapidly form N(I) on one route. On the other route an exchange-incompetent intermediate state ensemble, I(U)(2), is formed. The kinetics of unfolding measured by far-UV circular dichroism (CD) were faster than those measured by near-UV CD and intrinsic tyrosine fluorescence of the protein. The logarithms of the rate constants of the unfolding reaction measured by all three optical probes also showed a nonlinear dependence on GdnHCl concentration. All of the data suggest that N(I) and I(U)(2) are nativelike in their secondary and tertiary structures. While N(I) unfolds directly to the fully exchange-competent unfolded state (U), I(U)(2) forms another intermediate I(U)(3) which then unfolds to U. I(U)(3) is devoid of all native alpha-helical secondary structure and has only 30% of the tertiary interactions still intact. Since the rates of global unfolding measured by near-UV CD and fluorescence agree well with the rates of exposure determined for all of the backbone NH probe protons, it appears that the rate-limiting step for the unfolding of RNase A is the dissolution of the entire native tertiary structure and penetration of water into the hydrophobic core.     

Kinetics of unfolding and folding from amide hydrogen exchange in native ubiquitin

Amide hydrogen (NH) exchange is one of the few experimental techniques with the potential for determining the thermodynamics and kinetics of conformational motions at nearly every residue in native proteins. Quantitative interpretation of NH exchange in terms of molecular motions relies on a simple two-state kinetic model: at any given slowly exchanging NH, a closed or exchange-incompetent conformation is in equilibrium with an open or exchange-competent conformation. Previous studies have demonstrated the accuracy of this model in measuring conformational equilibria by comparing exchange data with the thermodynamics of protein unfolding. We report here a test of the accuracy of the model in determining the kinetics of conformational changes in native proteins. The kinetics of folding and unfolding for ubiquitin have been measured by conventional methods and compared with those derived from a comprehensive analysis of the pH dependence of exchange in native ubiquitin. Rate constants for folding and unfolding from these two very different types of experiments show good agreement. The simple model for NH exchange thus appears to be a robust framework for obtaining quantitative information about molecular motions in native proteins.     

Thermodynamic and kinetic exploration of the energy landscape of Borrelia burgdorferi OspA by native-state hydrogen exchange

We report a native-state hydrogen-exchange (HX) method to simultaneously obtain both thermodynamic and kinetic information on the formation of multiple excited states in a folding energy landscape. Our method exploits the inherent dispersion and pH dependence of the intrinsic HX rates to cover both the EX2 (thermodynamic) and EX1 (kinetic) regimes. At each concentration of denaturant, HX measurements are performed over a range of pH values. Using this strategy, we dissected Borrelia burgdorferi OspA, a predominantly beta-sheet protein containing a unique single-layer beta-sheet, into five cooperative units and postulated excited states predominantly responsible for HX. More importantly, we determined the interconversion rates between these excited states and the native state. The use of both thermodynamic and kinetic information from native-state HX enabled us to construct a folding landscape of this 28kDa protein, including local minima and maxima, and to discriminate on-pathway and off-pathway intermediates. This method, which we term EX2/EX1 HX, should be a powerful tool for characterizing the complex folding mechanisms exhibited by the majority of proteins.     

Energetics of the native energy landscape of a two-domain calcium sensor protein: distinct folding features of the two domains

The protein folding energy landscape allows a thorough understanding of the protein folding problem which in turn helps in understanding various aspects of biological functions. Characterizing the cooperative unfolding units and the intermediates along the folding funnel of a protein is a challenging task. In this paper, we investigated the native energy landscape of EhCaBP, a calcium sensor, belonging to the same EF-hand superfamily as calmodulin. EhCaBP is a two-domain EF-hand protein consisting of two EF-hands in each domain and binding to four Ca2+ cations. Native-state hydrogen exchange (HX) was used to assess the folding features of the landscape and also to throw light on the structure-folding function paradigm of calcium sensor proteins. HX measurements under the EX2 regime provided the thermodynamic information about the protein folding events under native conditions. HX studies revealed that the unfolding of EhCaBP is not a two-state process. Instead, it proceeds through cooperative units. The C-terminal domain exhibits less denaturant dependence than the N-terminal domain, suggesting that the former is dominated by local fluctuations. It is interesting to note that the N- and C-terminal domains of EhCaBP have distinct folding features. In fact, these observed differences can regulate the domain-dependent target recognition of two-domain Ca2+ sensor proteins.     

Beyond the EX1 limit: probing the structure of high-energy states in protein unfolding

Hydrogen exchange kinetics in native solvent conditions have been used to explore the conformational fluctuations of an immunoglobulin domain (CD2.domain1). The global folding/unfolding kinetics of the protein are unaltered between pH 4.5 and pH 9.5, allowing us to use the pH-dependence of amide hydrogen/deuterium exchange to characterise conformational states with energies up to 7.2kcal/mol higher than the folded ground state. The study was intended to search for discreet unfolding intermediates in this region of the energy spectrum, their presence being revealed by the concerted exchange behaviour of subsets of amide groups that become accessible at a given free energy, i.e. the spectrum would contain discreet groupings. Protection factors for 58 amide groups were measured across the pH range and the hydrogen-exchange energy profile is described.More interestingly, exchange behaviour could be grouped into three categories; the first two unremarkable, the third unexpected. (1) In 33 cases, amide exchange was dominated by rapid fluctuation, i.e. the free energy difference between the ground state and the rapidly accessed open state is sufficiently low that the contribution from crossing the unfolding barrier is negligible. (2) In 18 cases exchange is dominated by the global folding transition barrier across the whole pH range measured. The relationship between hydroxyl ion concentration and observed exchange rate is hyperbolic, with the limiting rate being that for global unfolding; the so-called EX1 limit. For these, the free energy difference between the folded ground state and any rapidly-accessed open state is too great for the proton to be exchanged through such fluctuations, even at the highest pH employed in this study. (3) For the third group, comprising five cases, we observe a behaviour that has not been described. In this group, as in category 2, the rate of exchange reaches a plateau; the EX1 limit. However, as the intrinsic exchange rate (k(int)) is increased, this limit is breached and the rate begins to rise again. This unintuitive behaviour does not result from pH instability, rather it is a consequence of amide groups experiencing two processes; rapid fluctuation of structure and crossing the global barrier for unfolding. The boundary at which the EX1 limit is overcome is determined by the equilibrium distribution of the fluctuating open and closed states (K(O/C)) and the rate constant for unfolding (k(u)). This critical boundary is reached when k(int)K(O/C)=k(u). Given that, in a simple transition state formalism: k(u)=K(#)k' (where K(#) describes the equilibrium distribution between the transition and ground state and k' describes the rate of a barrierless rearrangement), it follows that if the pH is raised to a level where k(int)=k', then the entire free energy spectrum from ground state to transition state could be sampled.     

NMR investigations on residue level unfolding thermodynamics in DLC8 dimer by temperature dependent native state hydrogen exchange

Understanding protein stability at residue level detail in the native state ensemble of a protein is crucial to understanding its biological function. At the same time, deriving thermodynamic parameters using conventional spectroscopic and calorimetric techniques remains a major challenge for some proteins due to protein aggregation and irreversibility of denaturation at higher temperature values. In this regard, we describe here the NMR investigations on the conformational stabilities and related thermodynamic parameters such as local unfolding enthalpies, heat capacities and transition midpoints in DLC8 dimer, by using temperature dependent native state hydrogen exchange; this protein aggregates at high (>65°C) temperatures. The stability (free energy) of the native state was found to vary substantially with temperature at every residue. Significant differences were found in the thermodynamic parameters at individual residue sites indicating that the local environments in the protein structure would respond differently to external perturbations; this reflects on plasticity differences in different regions of the protein. Further, comparison of this data with similar data obtained from GdnHCl dependent native state hydrogen exchange indicated many similarities at residue level, suggesting that local unfolding transitions may be similar in both the cases. This has implications for the folding/unfolding mechanisms of the protein. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Stopped-flow NMR measurement of hydrogen exchange rates in reduced horse cytochrome c under strongly destabilizing conditions

A procedure to measure exchange rates of fast exchanging protein amide hydrogens by time-resolved NMR spectroscopy following in situ initiation of the reaction by diluting a native protein solution into an exchanging deuterated buffer is described. The method has been used to measure exchange rates of a small set of amide hydrogens of reduced cytochrome c, maintained in a strictly anaerobic atmosphere, in the presence of an otherwise inaccessible range of guanidinium deuterochloride concentrations. The results for the measured protons indicate that hydrogen exchange in the unfolding transition region of cytochrome c reach the EX2 limit, but emphasize the difficulty in interpretation of the exchange mechanism in protein hydrogen exchange studies. Comparison of free energies of structure opening for the measured hydrogens with the global unfolding free energy monitored by far-UV CD measurements has indicated the presence of at least one partially unfolded equilibrium species of reduced cytochrome c. The results provide the first report of measurement of free energy of opening of structure to exchange in the 0–2-kcal/mol range. Proteins 32:241–247, 1998. © 1998 Wiley-Liss, Inc.