A competitive aggregation model for flash nanoprecipitation.

Flash NanoPrecipitation (FNP) is a novel approach for producing functional nanoparticles stabilized by amphiphilic block copolymers. FNP involves the rapid mixing of a hydrophobic active (organic) and an amphiphilic di-block copolymer with a non-solvent (water) and subsequent co-precipitation of nanoparticles composed of both the organic and copolymer. During this process, the particle size distribution (PSD) is frozen and stabilized by the hydrophilic portion of the amphiphilic di-block copolymer residing on the particle surface. That is, the particle growth is kinetically arrested and thus a narrow PSD can be attained. To model the co-precipitation process, a bivariate population balance equation (PBE) has been formulated to account for the competitive aggregation of the organic and copolymer versus pure organic-organic or copolymer-copolymer aggregation. Aggregation rate kernels have been derived to account for the major aggregation events: free coupling, unimer insertion, and aggregate fusion. The resulting PBE is solved both by direct integration and by using the conditional quadrature method of moments (CQMOM). By solving the competitive aggregation model under well-mixed conditions, it is demonstrated that the PSD is controlled primarily by the copolymer-copolymer aggregation process and that the energy barrier to aggregate fusion plays a key role in determining the PSD. It is also shown that the characteristic aggregation times are smaller than the turbulent mixing time so that the FNP process is always mixing limited.

Population balance modeling of aggregation and breakage in turbulent Taylor-Couette flow.

An experimental and computational study of aggregation and breakage processes for fully destabilized polystyrene latex particles under turbulent-flow conditions in a Taylor-Couette apparatus is presented. To monitor the aggregation and breakage processes, an in situ optical imaging technique was used. Consequently, a computational study using a population balance model was carried out to test the various parameters in the aggregation and breakage models. Very good agreement was found between the time evolution of the cluster size distribution (CSD) calculated with the model and that obtained from experiment. In order to correctly model the left-hand side of the CSD (small clusters), it was necessary to use a highly unsymmetric fragment-distribution function for breakage. As another test of the model, measurements with different solid volume fractions were performed. Within the range of the solid volume fractions considered here, the steady-state CSD was not significantly affected. In order to correctly capture the right-hand side of the CSD (large aggregates) at the higher solid volume fraction, a modified aggregation rate prefactor was used in the population balance model.

Objective decomposition of the stress tensor in granular flows.

A model for the stress tensor in granular flows [Volfson, Tsimring, and Aranson, Phys. Rev. Lett. 90, 254301 (2003)] is correctly generalized to an objective form that is independent of the coordinate system. The objective representation correctly models the isotropic and anisotropic parts of the stress tensor, whereas the original model for stress tensor components is dependent on the coordinate system. This general objective form of the model also relaxes the assumption in the original model that the principal axes of the granular stress tensor be coaxial with that of the "fluid" stress tensor. This generalization expands the applicability of the model to a wider class of granular flows. The objective representation is also useful in analyzing other models based on additive decomposition of the stress tensor in granular flows.

CFD simulation of aggregation and breakage processes in laminar Taylor-Couette flow.

An experimental and computational investigation of the effects of local fluid shear rate on the aggregation and breakage of approximately 10 microm latex spheres suspended in an aqueous solution undergoing laminar Taylor-Couette flow was carried out according to the following program. First, computational fluid dynamics (CFD) simulations were performed and the flow field predictions were validated with data from particle image velocimetry experiments. Subsequently, the quadrature method of moments (QMOM) was implemented into the CFD code to obtain predictions for mean particle size that account for the effects of local shear rate on the aggregation and breakage. These predictions were then compared with experimental data for latex sphere aggregates (using an in situ optical imaging method) and with predictions using spatial average shear rates. The mean particle size evolution predicted by CFD and QMOM using appropriate kinetic expressions that incorporate information concerning the particle morphology (fractal dimension) and the local fluid viscous effects on aggregation collision efficiency match well with the experimental data.

Structural and functional linkages between subunit interfaces in mammalian pyruvate kinase.

Mammalian pyruvate kinase (PK) is a four-domain enzyme that is active as a homo-tetramer. Tissue-specific isozymes of PK exhibit distinct levels of allosteric regulation. PK expressed in muscle tissue (M1-PK) shows hyperbolic steady-state kinetics, whereas PK expressed in kidney tissue (M2-PK) displays sigmoidal kinetics. Rabbit M1 and M2-PK are isozymes whose sequences differ in only 22 out of 530 residues per subunit, and these changes are localized in an inter-subunit interface. Previous studies have shown that a single amino acid mutation to M1-PK at either the Y (S402P) or Z (T340 M) subunit interface can confer a level of allosteric regulation that is intermediate to M1-PK and M2-PK. In an effort to elucidate the roles of the inter-subunit interaction in signal transmission and the functional/structural connectivity between these interfaces, the S402P mutant of M1-PK was crystallized and its structure resolved to 2.8 A. Although the overall S402P M1-PK structure is nearly identical with the wild-type structure within experimental error, significant differences in the conformation of the backbone are found at the site of mutation along the Y interface. In addition, there is a significant change along the Z interface, namely, a loss of an inter-subunit salt-bridge between Asp177 of domain B and Arg341 of domain A of the opposing subunit. Concurrent with the loss of the salt-bridge is an increase in the degree of rotational flexibility of domain B that constitutes the active site. Comparison of previous PK structures shows a correlation between an increase in this domain movement with the loss of the Asp177: Arg341 salt-bridge. These results identify the structural linkages between the Y and Z interfaces in regulating the interconversion of conformational states of rabbit M1-PK.

Functional reconstitution and characterization of recombinant human alpha 1-glycine receptors.

By utilizing a baculoviral expression system described previously (Cascio, M., Schoppa, N. E., Grodzicki, R. L., Sigworth, F. J., and Fox, R. O. (1993) J. Biol. Chem. 268, 22135-22142), functional recombinant homomeric human alpha(1)-glycine receptors (GlyR) were overexpressed in insect cell culture, solubilized, purified, and reconstituted into lipid vesicles via gel filtration. Reconstituted GlyR channels were observed to retain native-like activity in single channel recordings of planar bilayers and in flux assays of small unilamellar vesicles, providing evidence that the recombinant homomeric receptor may be functionally reconstituted. This reconstitution is significant in that it indicates that the overexpressed homomeric receptor is an appropriate substrate for subsequent biophysical characterization aimed at the general elucidation of structure-function. Circular dichroism spectroscopy of reconstituted GlyR indicated a low alpha-helical content and a significant fraction of polyproline structure. The small fraction of observed alpha-helix is insufficient to accommodate the four helical transmembrane domains proposed in models for this receptor. By inference, other members of the homologous ligand-gated channel superfamily, which include the ionotropic gamma-aminobutyric acid, acetylcholine, and serotonin receptors, may also be erroneously modeled, and alternate models should be considered.

Mapping RNA-protein interactions in ribonuclease P from Escherichia coli using disulfide-linked EDTA-Fe.

The protein subunit of Escherichia coli ribonuclease P (which has a cysteine residue at position 113) and its single cysteine-substituted mutant derivatives (S16C/C113S, K54C/C113S and K66C/C113S) have been modified using a sulfhydryl-specific iron complex of EDTA-2- aminoethyl 2-pyridyl disulfide (EPD-Fe). This reaction converts C5 protein, or its single cysteine-substituted mutant derivatives, into chemical nucleases which are capable of cleaving the cognate RNA ligand, M1 RNA, the catalytic RNA subunit of E. coli RNase P, in the presence of ascorbate and hydrogen peroxide. Cleavages in M1 RNA are expected to occur at positions proximal to the site of contact between the modified residue (in C5 protein) and the ribose units in M1 RNA. When EPD-Fe was used to modify residue Cys16 in C5 protein, hydroxyl radical-mediated cleavages occurred predominantly in the P3 helix of M1 RNA present in the reconstituted holoenzyme. C5 Cys54-EDTA-Fe produced cleavages on the 5' strand of the P4 pseudoknot of M1 RNA, while the cleavages promoted by C5 Cys66-EDTA-Fe were in the loop connecting helices P18 and P2 (J18/2) and the loop (J2/4) preceding the 3' strand of the P4 pseudoknot. However, hydroxyl radical-mediated cleavages in M1 RNA were not evident with Cys113-EDTA-Fe, perhaps indicative of Cys113 being distal from the RNA-protein interface in the RNase P holoenzyme. Our directed hydroxyl radical-mediated footprinting experiments indicate that conserved residues in the RNA and protein subunit of the RNase-P holoenzyme are adjacent to each other and provide structural information essential for understanding the assembly of RNase P.

Directed cleavage of RNA with protein-tethered EDTA-Fe.

There are several methods for locating the RNA site where a protein binds. One of the less common methods is directed cleavage of the RNA by an EDTA-Fe reagent tethered to the protein. The reaction of the EDTA-Fe(III) with ascorbate or hydrogen peroxide produces reactive oxygen species, such as hydroxyl radicals, localized within a 10-A radius of the iron center. The reactive oxygen species will attack the ribose or deoxyribose of nucleic acids as well as proximal polypeptide backbones. One EDTA-Fe reagent, (EDTA-2-aminoethyl)-2-pyridyl disulfide complexed to iron (EPD-Fe), has been tethered to several proteins through a disulfide linkage to engineered cysteine thiols and used to cleave DNA, proteins, and RNA. A second tethered EDTA-Fe reagent, 1-(p-bromoacetamidobenzyl)-EDTA-Fe, or BABE, has also been used to cleave RNA. Here we describe the issues involved in using these reagents with any RNA binding protein.

Stability studies of amino acid substitutions at tyrosine 27 of the staphylococcal nuclease beta-barrel.

In order to help determine the extent to which side chain interactions within the staphylococcal nuclease beta-barrel affect its global stability, a full set of point mutants was generated for residue 27. Intrinsic tryptophan fluorescence was monitored during solvent denaturation with guanidine hydrochloride (GuHCl) and was used to calculate DeltaGH2O unfolding and m values for each mutant. In the wild type protein, residue 27 is a tyrosine which is at the first position of a type I' beta-turn, and which participates in both hydrophobic interactions and side chain to side chain hydrogen bonding. The hydrophobicity of the mutant residue was found to be the dominant factor in determining global protein stability within this series of nuclease mutants.

Comparison of straight chain and cyclic unnatural amino acids embedded in the core of staphylococcal nuclease.

We have determined by X-ray crystallography the structures of several variants of staphylococcal nuclease with long flexible straight chain and equivalent length cyclic unnatural amino acid side chains embedded in the protein core. The terminal atoms in the straight side chains are not well defined by the observed electron density even though they remain buried within the protein interior. We have previously observed this behavior and have suggested that it may arise from the addition of side-chain vibrational and oscillational motions with each bond as a side chain grows away from the relatively rigid protein main chain and/or the population of multiple rotamers (Wynn R, Harkins P, Richards FM. Fox RO. 1996. Mobile unnatural amino acid side chains in the core of staphylococcal nuclease. Protein Sci 5:1026-1031). Reduction of the number of degrees of freedom by cyclization of a side chain would be expected to constrain these motions. These side chains are in fact well defined in the structures described here. Over-packing of the protein core results in a 1.0 A shift of helix 1 away from the site of mutation. Additionally, we have determined the structure of a side chain containing a single hydrogen to fluorine atom replacement on a methyl group. A fluorine atom is intermediate in size between methyl group and a hydrogen atom. The fluorine atom is observed in a single position indicating it does not rotate like methyl hydrogen atoms. This change also causes subtle differences in the packing interactions.

Native-like structure of a protein-folding intermediate bound to the chaperonin GroEL.

The chaperonin GroEL binds nonnative proteins in its central channel through hydrophobic interactions and initiates productive folding in this space underneath bound co-chaperone, GroES, in the presence of ATP. The questions of where along the folding pathway a protein is recognized by GroEL, and how much structure is present in a bound substrate have remained subjects of discussion, with some experiments suggesting that bound forms are fully unfolded and others suggesting that bound species are partially structured. Here we have studied a substrate protein, human dihydrofolate reductase (DHFR), observing in stopped-flow fluorescence experiments that it can rapidly bind to GroEL at various stages of folding. We have also analyzed the structure of the GroEL-bound protein using hydrogen-deuterium exchange and NMR spectroscopy. The pattern and magnitude of amide proton protection indicate that the central parallel beta-sheet found in native DHFR is present in a moderately stable state in GroEL-bound DHFR. Considering that the strands are derived from distant parts of the primary structure, this suggests that a native-like global topology is also present. We conclude that significant native-like structure is present in protein-folding intermediates bound to GroEL.

Mobile unnatural amino acid side chains in the core of staphylococcal nuclease.

The structures of several variants of staphylococcal nuclease with long flexible unnatural amino acid side chains in the hydrophobic core have been determined by X-ray crystallography. The unnatural amino acids are disulfide moieties between the lone cysteine residue in V23C nuclease and methane, ethane, 1-n-propane, 1-n-butane, 1-n-pentane, and 2-hydroxyethyl thiols. We have examined changes in the core packing of these mutants. Side chains as large as the 1-n-propyl cysteine disulfide can be incorporated without perturbation of the structure. This is due, in part, to cavities present in the wild-type protein. The longest side chains are not well defined, even though they remain buried within the protein interior. These results suggest that the enthalpy-entropy balance that governs the rigidity of protein interiors favors tight packing only weakly. Additionally, the tight packing observed normally in protein interiors may reflect, in part, the limited numbers of rotamers available to the natural amino acids.

Mapping the structure of a non-native state of staphylococcal nuclease.

Non-native states of proteins populated at extremes of pH, or by mutation or truncation of the protein sequence, are thought to be equilibrium models for kinetic intermediates on the folding pathway. While the global physical properties of these molecules have been well characterized, analysis of their structure by NMR spectroscopy has proven difficult. Here we report the use of a new chemical cleavage technique, based on reactive oxygen species, to map the backbone fold of a truncated form of staphylococcal nuclease in a non-native state. The fragment adopts a native-like fold, however the technique also reveals regions of non-native structure.

Interactions in nonnative and truncated forms of staphylococcal nuclease as indicated by mutational free energy changes.

Several mixed disulfide variants of staphylococcal nuclease have been produced by disulfide bond formation between nuclease V23C and methane, ethane, 1-propane, 1-n-butane, and 1-n-pentane thiols. Although CD spectroscopy shows that the native state is largely unperturbed, the stability toward urea-induced unfolding is highly dependent on the nature of the group at this position, with the methyl disulfide protein being the most stable. The variant produced by modification with iodoacetic acid, however, gives a CD spectrum indicative of an unfolded polypeptide. Thiol-disulfide exchange equilibrium constants between nuclease V23C and 2-hydroxyethyl disulfide have been measured as a function of urea concentration. Because thiol-disulfide exchange and unfolding are thermodynamically linked, the effects of a mutation (disulfide exchange) can be partitioned between various conformational states. In the case of unmodified V23C and the 2-hydroxyethyl protein mixed disulfide, significant effects in the nonnative states of nuclease are observed. Truncated forms of staphylococcal nuclease are thought to be partially folded and may be good models for early folding intermediates. We have characterized a truncated form of nuclease comprised of residues 1-135 with a V23C mutation after chemical modification of the cysteine residue. High-resolution size-exclusion chromatography indicates that modification brings about significant changes in the Stokes radius of the protein, and CD spectroscopy indicates considerable differences in the amount of secondary structure present. Measurement of the disulfide exchange equilibrium constant between this truncated protein and 2-hydroxyethyl disulfide indicate significant interactions between position 23 and the rest of the protein when the urea concentration is lower than 1.5 M.(ABSTRACT TRUNCATED AT 250 WORDS)

Capillary electrophoresis of S. nuclease mutants.

The electrophoretic migration behavior of 12 S. nuclease variants from Staphylococcus aureus with small but well defined structural differences from site directed mutation was investigated in free solution capillary electrophoresis at pH 2.8 to 9.5. The nucleases are basic proteins; the pI and the M(r) of the wild type are 10.3 and 16.811 kd, respectively. With specially selected oligoamino buffers and with an inert, hydrophilic wall coating in 75 microns I.D. quartz capillary tubes, most of the proteins could be separated by CZE without interference by wall adsorption even at pH 9.5 where the selectivity was the highest. At pH 2.8, 4.1 and 7.0, S. nucleases are known to be in the random coil, "swollen" and the tight native state. Assuming that in a given state, i.e., at a certain pH, the molecular radii of the nucleases are the same, their hydrodynamic radii were calculated from their pertinent electrophoretic mobilities. The respective radii of 50.1, 26.8, and 25.0 Angstrum thus obtained agreed very well with the corresponding radii of gyration obtained from X-ray scattering. In fact, from the electrophoretic mobilities at pH 9.5, the existence of a hitherto unknown swollen basic state of the nuclease having a hydrodynamic radius of 30.5 Angstrum was postulated. In addition, a method was described to evaluate the valence of the protein at different pH from their pertinent electrophoretic mobilities. A general advantage of this method is that only the differences between the valences of the mutants and the wild type are needed; and for none of the proteins is required the knowledge of the actual valence. The results of the methods allowed the construction of a pH profile of the protein's valence. For the wild type, this profile was compared to the H+ titration curve and the agreement was excellent. Both methods employed some novel structure-electrophoretic mobility relationships and the predicted protein properties compared remarkably well to the values obtained by exoelectrophoretic methods such as pH titration and X-ray scattering. Surprisingly, certain S. nucleases having the same valence could also be readily separated by CZE in some cases under the same conditions used for the others. Close examination of appropriate X-ray crystallography and/or NMR data indicated subtle differences in the molecular structure of these proteins that could be responsible for slight alteration in their hydrodynamic radii.(ABSTRACT TRUNCATED AT 400 WORDS)

Proline cis-trans isomerization in staphylococcal nuclease: multi-substrate free energy perturbation calculations.

Staphylococcal nuclease A exists in two folded forms that differ in the isomerization state of the Lys 116-Pro 117 peptide bond. The dominant form (90% occupancy) adopts a cis peptide bond, which is observed in the crystal structure. NMR studies show that the relatively small difference in free energy between the cis and trans forms (delta Gcis-->trans approximately 1.2 kcal/mol) results from large and nearly compensating differences in enthalpy and entropy (delta Hcis-->trans approximately delta TScis-->trans approximately 10 kcal/mol). There is evidence from X-ray crystal structures that the structural differences between the cis and the trans forms of nuclease are confined to the conformation of residues 112-117, a solvated protein loop. Here, we obtain a thermodynamic and structural description of the conformational equilibrium of this protein loop through an exhaustive conformational search that identified several substates followed by free energy simulations between the substrates. By partitioning the search into conformational substates, we overcame the multiple minima problem in this particular case and obtained precise and reproducible free energy values. The protein and water environment was implicitly modeled by appropriately chosen nonbonded terms between the explicitly treated loop and the rest of the protein. These simulations correctly predicted a small free energy difference between the cis and trans forms composed of larger, compensating differences in enthalpy and entropy. The structural predictions of these simulations were qualitatively consistent with known X-ray structures of nuclease variants and yield a model of the unknown minor trans conformation.

Crystallization and preliminary X-ray investigation of the recombinant Trypanosoma brucei rhodesiense calmodulin.

Bipyramidal crystals of the recombinant calmodulin from Trypanosoma brucei rhodesiense were obtained by vapor diffusion against 55% (v/v) 2-methyl-2,4-pentanediol in 0.05 M cacodylate buffer, pH 5.6. When few nucleation events occurred, crystals grew to 0.25 x 0.25 x 1.20 mm. The space group of the crystal is I4(1)22, with unit cell dimensions a = b = 56.88 A, c = 230.11 A, alpha = beta = gamma = 90 degrees, z = 16. The molecular mass and volume of the unit cell suggest that there is one molecule in the asymmetric unit. The I/sigma (I) ratio for data at 3.0 A resolution was 3.67, indicating that the final structure can be refined at higher resolution. Molecular replacement methods and the PC-refinement technique have not yet yielded the structure under a variety of search conditions. We are currently investigating the multiple isomorphous replacement approach to determine this crystal structure.

Charge and size effects in the capillary zone electrophoresis of nuclease A and its variants.

The migration behavior of nuclease A from Staphylococcus aureus and 11 of its variants in capillary zone electrophoresis (CZE) was investigated in the light of their three-dimensional structure known from X-ray crystallography and nuclear magnetic resonance (NMR) measurements. Nuclease A (molecular mass 16.8 kDa, pKa 10.3) and the variants differ only in a single amino acid residue and have a very similar crystal structure. With the use of coated quartz capillaries and suitable buffers, the protein migration was investigated at pH from 2.8 to 9.5 without interference by wall adsorption. Although the selectivity of the electrophoretic system for the proteins was mainly determined by their charge differences, certain variants having the same net charge could also be readily separated under nondenaturing conditions. For instance, the mobility of variant K116A was sufficiently higher than that of K116G so that they could be separated by CZE. The structures of both variants are the same except for the solvent-exposed loop containing residue 116. For this reason, the difference in electrophoretic mobilities can be attributed to the fact that in K116G the backbone of the 112 to 117 amino acids protrudes slightly from the protein, with a concomitant increase in the hydrodynamic radius with respect to that of K116A. Consequently, K116G shows a smaller mobility than K116A due to its larger hydrodynamic radius despite its smaller molecular mass. The interpretation of the experimentally measured mobilities of such closely related proteins therefore requires not only consideration of their electrostatic charge but also the fine details of their molecular structures.

Stabilization of a strained protein loop conformation through protein engineering.

Staphylococcal nuclease is found in two folded conformations that differ in the isomerization of the Lys 116-Pro 117 peptide bond, resulting in two different conformations of the residue 112-117 loop. The cis form is favored over the trans with an occupancy of 90%. Previous mutagenesis studies have shown that when Lys 116 is replaced by glycine, a trans conformation is stabilized relative to the cis conformation by the release of steric strain in the trans form. However, when Lys 116 is replaced with alanine, the resulting variant protein is identical to the wild-type protein in its structure and in the dominance of the cis configuration. The results of these studies suggested that any nuclease variant with a non-glycine residue at position 116 should also favor the cis form because of steric requirements of the beta-carbon at this position. In this report, we present a structural analysis of four nuclease variants with substitutions at position 116. Two variants, K116E and K116M, follow the "beta-carbon" hypothesis by favoring the cis form. Furthermore, the crystal structure of K116E is nearly identical to that of the wild-type protein. Two additional variants, K116D and K116N, provide exceptions to this simple "beta-carbon" rule in that the trans conformation is stabilized relative to the cis configuration by these substitutions. Crystallographic data indicate that this stabilization is effected through the addition of tertiary interactions between the side chain of position 116 with the surrounding protein and water structure. The detailed trans conformation of the K116D variant appears to be similar to the trans conformation observed in the K116G variant, suggesting that these two mutations stabilize the same conformation but through different mechanisms.

Mapping staphylococcal nuclease conformation using an EDTA-Fe derivative attached to genetically engineered cysteine residues.

Six single cysteine variants of staphylococcal nuclease were reacted with the iron complex of (EDTA-2-aminoethyl) 2-pyridyl disulfide (EPD-Fe) [Ermácora, M. R., Delfino, J. M., Cuenoud, B., Schepartz, A., & Fox, R. O. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6383-6387] and used to assess the ability of this cleavage reagent to faithfully report on the structure of nonnative protein states. The act of mutation and modification did not significantly alter the protein's global structure, as measured by CD and enzymatic activity, and only modestly affected its stability. The reaction was conformation dependent and generated specific cleavage products that mapped tertiary interactions present in the folded state. Several parameters relevant to the cleavage reaction and its use as a conformational probe were analyzed. Proximity and solvent accessibility are the most important parameters in determining the cleavage pattern and can be used to predict cleavage sites in the native protein. The cleavage reaction can be performed in the presence of high denaturant concentration, in the presence of SDS, and under a wide range of pH values; thus it can readily be applied to the study of equilibrium folding intermediates. Mass spectrometric analysis combined with N-terminal sequencing identified cleavage products consistent with a single cleavage event per protein molecule and revealed one cleavage mechanism which was not previously considered for protein oxidative degradation, although it was reported for hydroxyl radical induced cleavage of small peptides. Identification of the cleavage sites obtained from each variant allowed a nearest-neighbor mapping of the secondary structural elements of nuclease. Quantitation of specific cleavage products was used to monitor the disruption of the interaction between helices H2 and H3 in equilibrium unfolding experiments. The resulting unfolding curve revealed a local conformational heterogeneity at low denaturant concentration which was not observed when the same transition was monitored by the change in fluorescence of a single nuclease tryptophan. Interestingly, the midpoint of the transition and the second half of the unfolding curve were the same, as monitored by the two probes. This indicates that the lifetime of the reactive oxygen species generated by the cleavage reagent is short compared to the unfolding equilibrium rate constants and that the cleavage technique identifies a native-like folding intermediate not detected by fluorescence. The experiments presented herein demonstrate that EPD-Fe-mediated protein cleavage is an appropriate technique for the study of nonnative protein structure.(ABSTRACT TRUNCATED AT 400 WORDS)