Micronization of lysozyme by supercritical assisted atomization

Supercritical Assisted Atomization (SAA) has been used to produce lysozyme microparticles. Lysozyme has been micronized using water, buffered water at pH 6.2 and water–ethanol mixtures at different volume percentages. Precipitated lysozyme particles were spherical, with a narrow particle size distribution (PSD) ranging from 0.1 to 4 µm. The concentration of lysozyme in the liquid solvent mixture had a nonlinear effect on the particle distribution, with an increase of the X_0.9 from about 1 to 3 µm varying the enzyme concentration from 5 to 20 mg/mL. Precipitation temperature was set as low as possible to avoid enzyme degradation. High‐performance liquid chromatography analysis showed no degradation of lysozyme and the enzyme activity, measured by turbidimetric enzymatic assay, only slightly decreased after SAA processing. Depending on the process conditions lysozyme retained from 95% to 100% of the biological activity compared to the untreated enzyme. Biotechnol. Bioeng. 2009; 104: 1162–1170. © 2009 Wiley Periodicals, Inc.     

Osmotic second virial cross‐coefficient measurements for binary combination of lysozyme,ovalbumin, and α‐amylase in salt solutions

Interactions measurement is a valuable tool to predict equilibrium phase separation of a desired protein in the presence of unwanted macromolecules. In this study, cross‐interactions were measured as the osmotic second virial cross‐coefficients (B₂₃) for the three binary protein systems involving lysozyme, ovalbumin, and α‐amylase in salt solutions (sodium chloride and ammonium sulfate). They were correlated with solubility for the binary protein mixtures. The cross‐interaction behavior at different salt concentrations was interpreted by either electrostatic or hydrophobic interaction forces. At low salt concentrations, the protein surface charge dominates cross‐interaction behavior as a function of pH. With added ovalbumin, the lysozyme solubility decreased linearly at low salt concentration in sodium chloride and increased at high salt concentration in ammonium sulfate. The B₂₃ value was found to be proportional to the slope of the lysozyme solubility against ovalbumin concentration and the correlation was explained by preferential interaction theory. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1203–1211, 2013     

Effects of surfactants on emulsification and secondary structure of lysozyme in aqueous solutions

This paper reports our investigations on the secondary structure of lysozyme in aqueous solution with D₂O and comparing systems emulsified with ionic and non-ionic amphiphiles, respectively. The opposite effect of concentration of surfactants on the lysozyme aqueous system was studied and the activity of lysozyme by the turbidimetric assay with different kinds of surfactant was compared. The effect of different surfactants on emulsification volume formed was observed and discussed. The different interactive patterns between lysozyme and ionic and non-ionic surfactants, such as sodium dodecyl sulfate (SDS), cetyl trimethyl ammonium bromide (CTAB) and Triton-X100 (Isooctoylphenoxypolyethoxy ethanol), respectively, was studied with FTIR. A simple relationship between the secondary structure and denaturation of protein is given for the emulsification of lysozyme in aqueous solutions. It is important for understanding the stabilization and avoiding the emulsions in the extraction process that the interactions between the proteins and surfactants which leads to the formation of such complexes are characterized. The utility of surfactants as a de-emulsifying agent for the commercial process to extract penicillin G from the filtrate of fermentation broth was consistent with these results.     

Ultrasonic absorption in aqueous solution of lysozyme.

The titration curve of ultrasonic absorption at 2.82 MHz in aqueous solutions of lysozyme measured by Zana and Lang [J. Phys. Chem., 74 , 2734 (1970)] is theoretically analyzed. The maxima at pH 3 and pH 11 are describable with proton-transfer reactions of dissociable carboxyl and amino groups by assuming that volume changes due to the reactions are 2.3 and 5.2 cm³/mole, respectively, which are appreciably smaller than those of simple amino acids. The remaining, pH-independent excess absorption over solvent is measured at frequencies ranging from 3 to 150 MHz. The absorption is ascribed to the internal loss of protein. The complex compressibility β′_p ? iβ″_p of lysozyme molecule is evaluated as β′_p = 7.2 × 10^?12 cm²/dyne and β″_P = 4.3 × 10^?14 cm²/dyne from the increments over solvent in absorption as well as in sound velocity.     

The binding of precipitant ions in the tetragonal crystals of hen egg white lysozyme

Abstract

The bonds between lysozyme molecules and precipitant ions in single crystals grown with chlorides of several metals are analysed on the basis of crystal structure data. Crystals of tetragonal hen egg lysozyme (HEWL) were grown with chlorides of several alkali and transition metals (LiCl, NaCl, KCl, NiCl₂ and CuCl₂) as precipitants and the three-dimensional structures were determined at 1.35?Å resolution by X-ray diffraction method. The positions of metal and chloride ions attached to the protein were located, divided into three groups and analysed. Some of them, in accordance with the recently proposed and experimentally confirmed crystal growth model, provide connections in protein dimers and octamers that are precursor clusters in the crystallization lysozyme solution. The first group, including Cu⁺², Ni⁺² and Na⁺¹ cations, binds specifically to the protein molecule. The second group consists of metal and chloride ions bound inside the dimers and octamers. The third group of ions can participate in connections between the octamers that are suggested as building units during the crystal growth. The arrangement of chloride and metal ions associated with lysozyme molecule at all stages of the crystallization solution formation and crystal growth is discussed.

Communicated by Ramaswamy H. Sarma     

Solubilization limit of lysozyme into DODMAC reverse micelles

Dioctyldimethyl ammonium chloride (DODMAC) was used to form reverse micelles and to extract lysozyme from an aqueous solution into an organic phase. The solubilization behavior of lysozyme into a DODMAC reverse micellar phase was examined in terms of the temperature, the type of cations in the aqueous phase, and the surfactant concentration in the organic phase. Complete removal of lysozyme from the aqueous phase was obtained when the pH was set one unit higher than the pI of the protein. However, it was found that there is a solubilization limit of lysozyme in the organic phase. Not all the lysozyme extracted out of the initial aqueous phase was solubilized into the DODMAC reverse micellar phase, resulting in the formation of white precipitate at the aqueous-organic interface. Temperature has a negligible effect on the solubilization limit of lysozyme. The value of the solubilization limit is a strong function of the type of cations present in the aqueous phase, indicating an important role of lysozyme-cation interactions on the extraction process. An increase in the DODMAC concentration from 100-200 mM resulted in little change in the highest concentration of lysozyme obtained in the organic phase.     

Effects of water activity and aqueous solvent ordering on thermal stability of lysozyme, α-chymotrypsinogen A, and alcohol dehydrogenase

Effects of water activity (a_W) and solvent ordering were separately analyzed on the thermal unfolding of lysozyme and α-chymotrypsinogen A, and also on the thermal deactivation of yeast alcohol dehydrogenase (YADH) in aqueous solutions with various additives. With the coexistence of additives, water activity was the determinant of the extent of the change in the thermal stability of proteins while solvent ordering was the determinant of the direction of the change. The parameter α, determined from the activity coefficient of water, representing the deviation of a_W from that of the ideal solution, was useful as a quantitative index of the solvent ordering showing good correlations with the unfolding temperature and enthalpy of lysozyme and α-chymotrypsinogen A and also with the thermal deactivation rate constant of YADH at a constant a_W. Solvent ordering seemed to affect the thermal stability of proteins mainly through its effect on the intramolecular hydrophobic interaction among amino acid residues in a protein molecule but the contribution of the electrostatic interaction including hydrogen bonding through the change in permittivity of solution was also suggested.     

A comparative study of lysozyme conformation in various reverse micellar systems

The activity and conformation of lysozyme solubilized in apolar solvents via reverse micelles was investigated. The systems used were sodium di-2-ethylhexylsulfosuccinate (AOT)/isooctane/H₂O, cetyltrioctylammoniumbromide (CTAB)/CHCl₃, isooctane/H₂O; tetraethyleneglycoldodecylether (EO₄C₁₂)/isooctane/H₂O, and bulk water. CD spectra of lysozyme in reverse micellar solutions were investigated as a function of w₀ (= [H₂O]/[AOT]) and were compared to the spectra in aqueous solutions. No marked changes were found in the EO₄C₁₂ or in the CTAB systems with respect to water, which indicates that no sizeable conformational changes of the enzyme occurred upon solubilization in the reverse micellar systems. In agreement with previous studies [C. Grandi, R. E. Smith, and P. L. Luisi (1981) J. Biol. Chem. 256 , 837–843] dramatic conformational changes can be inferred in the AOT system on the basis of CD studies. This is taken as an indication that the enzyme denatures in this micellar system. This is particularly striking because the enzyme is fully active in AOT reverse micelles. The apparent paradox is solved by the observation that the native CD spectrum (and by inference, the native conformation) is maintained when lysozyme is bound to NAG or NAG₃, and by inference, when the substrate is bound, e.g., during enzyme turnover. However, in the absence of added NAG, NAG₃, or substrate, the enzyme in the AOT reverse micellar system rapidly denatures. Together with CD studies, fluorescence and nmr data confirm the hypothesis of an irreversible denaturation of lysozyme in the AOT system, the denaturation being slowed down when the substrate is present. The activity of the enzyme has been studied as a function of pH and w₀ using the chromophoric substrate 3,4-dinitrophenyl-tetra-N-acetyl-β-D -chitotetraoside (3,4-DNP-NAG₄). Generally speaking, the kinetic parameters are comparable to those found in bulk water solution. More detailed, in the CTAB system, k_cat tends to be smaller than in aqueous solution (with quite similar K_M), whereas in the EO₄C₁₂ system (at pH 7.0) the turnover number is larger and K_M is smaller than in water. In the AOT system, the kinetic parameters at pH 7.0 are also quite comparable to those found in water.     

Ionic liquids as selectors for controlling the crystallization nucleation of hen egg white lysozyme

Ionic liquids (ILs) have biomaterial applications and are used for protein crystallization. The effect of two imidazolium-based ILs, 1-butyl-3-methylimidazolium chloride ([C₄mim]Cl) and 1,3-dimethylimidazolium iodide ([dmim]I), on the nucleation kinetics of lysozyme was investigated by determining the nucleation induction time, and nucleation parameters were evaluated. The values of interfacial tension calculated for solutions with added 30 g/L ILs [C₄mim]Cl and [dmim]I, and without added ILs were 99.03, 109.7, and 107.3 mJ/m², respectively. Compared with solutions without IL addition, the critical free energy change, size, and molecular number of critical nuclei decreased and the nucleation rate increased after the addition of [C₄mim]Cl. In contrast, the critical free energy change, size, and molecular number of critical nuclei increased and the nucleation rate decreased after the addition of [dmim]I. These new findings provide insights into controlling lysozyme crystallization separation, and present ILs as potentially useful additives for controlling the crystallization of macromolecules.     

Synthesis of hydrophobic nanoparticles for real‐time lysozyme detection using surface plasmon resonance sensor

Diagnostic biomarkers such as proteins and enzymes are generally hard to detect because of the low abundance in biological fluids. To solve this problem, the advantages of surface plasmon resonance (SPR) and nanomaterial technologies have been combined. The SPR sensors are easy to prepare, no requirement of labelling and can be detected in real time. In addition, they have high specificity and sensitivity with low cost. The nanomaterials have also crucial functions such as efficiency improvement, selectivity, and sensitivity of the detection systems. In this report, an SPR‐based sensor is developed to detect lysozyme with hydrophobic poly (N‐methacryloyl‐(L)‐phenylalanine) (PMAPA) nanoparticles. The SPR sensor was first characterized by attenuated total reflection‐Fourier transform infrared, atomic force microscope, and water contact angle measurements and performed with aqueous lysozyme solutions. Various concentrations of lysozyme solution were used to calculate kinetic and affinity coefficients. The equilibrium and adsorption isotherm models of interactions between lysozyme solutions and SPR sensor were determined and the maximum reflection, association, and dissociation constants were calculated by Langmuir model as 4.87, 0.019 nM⁻¹, and 54 nM, respectively. The selectivity studies of SPR sensor were investigated with competitive agents, hemoglobin, and myoglobin. Also, the SPR sensor was used four times in adsorption/desorption/recovery cycles and results showed that, the combination of optical SPR sensor with hydrophobic ionizable PMAPA nanoparticles in one mode enabled the detection of lysozyme molecule with high accuracy, good sensivity, real‐time, label‐free, and a low‐detection limit of 0.66 nM from lysozyme solutions. Lysozyme detection in a real sample was performed by using chicken egg white to evaluate interfering molecules present in the medium.     

Lessons from the lysozyme of phage T4

An overview is presented of some of the major insights that have come from studies of the structure, stability, and folding of T4 phage lysozyme. A major purpose of this review is to provide the reader with a complete tabulation of all of the variants that have been characterized, including melting temperatures, crystallographic data, Protein Data Bank access codes, and references to the original literature. The greatest increase in melting temperature (T_m) for any point mutant is 5.1°C for the mutant Ser 117 → Val. This is achieved in part not only by hydrophobic stabilization but also by eliminating an unusually short hydrogen bond of 2.48 Å that apparently has an unfavorable van der Waals contact. Increases in T_m of more than 3–4°C for point mutants are rare, whereas several different types of destabilizing substitutions decrease T_m by 20°C or thereabouts. The energetic cost of cavity creation and its relation to the hydrophobic effect, derived from early studies of “large‐to‐small” mutants in the core of T4 lysozyme, has recently been strongly supported by related studies of the intrinsic membrane protein bacteriorhodopsin. The L99A cavity in the C‐terminal domain of the protein, which readily binds benzene and many other ligands, has been the subject of extensive study. Crystallographic evidence, together with recent NMR analysis, suggest that these ligands are admitted by a conformational change involving Helix F and its neighbors. A total of 43 nonisomorphous crystal forms of different monomeric lysozyme mutants were obtained plus three more for synthetically‐engineered dimers. Among the 43 space groups, P2₁2₁2₁ and P2₁ were observed most frequently, consistent with the prediction of Wukovitz and Yeates.     

Influence of mixed electrolytes on the adsorption of lysozyme,PEG, and PEGylated lysozyme on a hydrophobic interaction chromatography resin

In a recent work (Werner A and Hasse H in J Chromatogr A 2013;1315:135) the influence of mixed electrolytes on the adsorption of the macromolecules lysozyme, PEG and di‐PEGylated lysozyme on a hydrophobic resin has been studied, but only at one overall ionic strength (3000 mM). The present work, therefore, extends these studies to other ionic strengths (2400 and 2700 mM), and explores the application of a model to predict the entire data set. The adsorbent is Toyopearl PPG‐600M. The solvent is a 25 mM aqueous sodium phosphate buffer at pH 7.0. The studied salts are sodium chloride, sodium sulfate, ammonium chloride and ammonium sulfate. Pure salts as well as binary and ternary mixtures of these salts with varying ratios of the amounts of the salts are studied at 25 °C. The loading of the adsorbent increases with increasing salt concentration for all macromolecules. Synergetic effects of the mixed electrolytes are observed. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1104–1115, 2017     

Static and dynamic quenching of protein fluorescence. II. lysozyme.

The fluorescence parameters—lifetime, relative quantum yield, wavelength of maximum fluorescence intensity, half-width, and polarization—of 0.01% lysozyme were measured at 15°C in aqueous solution, in glycerol–water mixtures (0–90% v/v glycerol), in aqueous urea (0–8M) solutions, and in aqueous guanidine hydrochloride (0–6.4M) solutions. The changes in the static and dynamic quenching of lysozyme fluorescence, monitored by the quantum yield and lifetime measurements, were correlated with the other fluorescence parameters and compared with our earlier results with bovine serum albumin. The results were interpreted in terms of induced conformational changes. The various perturbants altered the fluorescence parameters of lysozyme and bovine serum albumin very differently. The differences were shown to be entirely consistent with our earlier conclusion that bovine serum albumin fluorophores are nonsurface residues and with the conclusion of others that lysozyme fluorophores are surface residues. Unlike their effects on bovine serum albumin, urea and guanidine hydrochloride affect lysozyme structure quite differently, both in nature and degree. We have suggested that the affect of urea on lysozyme fluorescence is an indirect result of reduction in the size of the cleft brought about by the structure-breaking action of urea on water in the cleft. 4M Urea is sufficient for this reaction. Large decreases in the polarization of the fluorescence of lysozyme in the 0.8–1.6M and 3.2–4.8M guanidine hydrochloride ranges demonstrated two guanidine hydrochloride-induced conformation changes. A red shift of the fluorescence maximum to 354 nm indicated that the second transition completely exposes all fluorescing tryptophan residues of lysozyme to mobile solvent water. However, even 6.4M guanidine hydrochloride did not completely unravel the lysozyme molecule at 15°C, as evidenced by its failure to cause any of the tyrosine residues to become fluorescent.     

Studies of lysozyme binding to histamine as a ligand for hydrophobic charge induction chromatography

Histamine was immobilized on Sepharose CL‐6B (Sepharose) for use as a ligand of hydrophobic charge induction chromatography (HCIC) of proteins. Lysozyme adsorption onto Histamine‐Sepharose (HA‐S) was studied by adsorption equilibrium and calorimetry to uncover the thermodynamic mechanism of the protein binding. In both the experiments, the influence of salt (ammonium sulfate and sodium sulfate) was examined. Adsorption isotherms showed that HA‐S exhibited a high salt tolerance in lysozyme adsorption. This property was well explained by the combined contributions of hydrophobic interaction and aromatic stacking. The isotherms were well fitted to the Langmuir equation, and the equilibrium parameters for lysozyme adsorption were obtained. In addition, thermodynamic parameters (ΔH_ads, ΔS_ads, and ΔG_ads) for the adsorption were obtained by isothermal titration calorimetry by titrating lysozyme solutions into the adsorbent suspension. Furthermore, free histamine was titrated into lysozyme solution in the same salt‐buffers. Compared with the binding of lysozyme to free histamine, lysozyme adsorption onto HA‐S was characterized by a less favorable ΔG_ads and an unfavorable ΔS_ads because histamine was covalently attached to Sepharose via a three‐carbon‐chain spacer. Consequently, the immobilized histamine could only associate with the residues on the protein surface rather than those in the hydrophobic pocket, causing a less favorable orientation between histamine and lysozyme. Further comparison of thermodynamic parameters indicated that the unfavorable ΔS_ads was offset by a favorable ΔH_ads, thus exhibiting typical enthalpy‐entropy compensation. Moreover, thermodynamic analyses indicated the importance of the dehydration of lysozyme molecule and HA‐S during the adsorption and a substantial conformational change of the protein during adsorption. The results have provided clear insights into the adsorption mechanisms of lysozyme onto the new HCIC material. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Protein-protein interaction on lysozyme crystallization revealed by rotational diffusion analysis

Intermolecular interactions between protein molecules diffusing in various environments underlie many biological processes as well as control protein crystallization, which is a crucial step in x-ray protein structure determinations. Protein interactions were investigated through protein rotational diffusion analysis. First, it was confirmed that tetragonal lysozyme crystals containing fluorescein-tagged lysozyme were successfully formed with the same morphology as that of native protein. Using this nondisruptive fluorescent tracer system, we characterized the effects of sodium chloride and ammonium sulfate concentrations on lysozyme-lysozyme interactions by steady-state and time-resolved fluorescence anisotropy measurements and the introduction of a novel interaction parameter, k_rot. The results suggested that the specific attractive interaction, which was reflected in the retardation of the protein rotational diffusion, was induced depending on the salt type and its concentration. The change in the attractive interactions also correlated with the crystallization/precipitation behavior of lysozyme. Moreover, we discuss the validity of our rotational diffusion analysis through comparison with the osmotic second virial coefficient, B₂₂, previously reported for lysozyme and those estimated from k_rot.     

Thermodynamics of the denaturation of lysozyme in alcohol--water mixtures.

The thermal denaturation of lysozyme was studied at pH 2 in aqueous mixtures of methanol, ethanol, and 1-propanol by high sensitivity differential scanning calorimetry (DSC). The most obvious effect of alcohols was the lowering of Td, the temperature of denaturation, increasingly with higher alcohol concentration and longer alkyl chain. Both the calorimetric and van't Hoff enthalpies of denaturation initially increased and then decreased with increasing alcohol concentration, the ratio of the two enthalpies being nearly unity, 1.007 +/- 0.011, indicating the validity of the two-state approximation for the unfolding of lysozyme in these solvent systems. The reversibility of the denaturation was demonstrated by the reversibility of the DSC curves and the complete recovery of enzymic activity on cooling. The changes in heat capacity on unfolding decreased with increasing alcohol concentration for each alcohol. Experimentally determined values of denaturation temperature and of entropy and heat capacity changes were used to derive the additional thermodynamic parameters delta G degrees and delta S degrees for denaturation as a function of temperature for each alcohol--water mixture. Comparison of the thermodynamic parameters with those reported [Pfeil, W., & Privalov, P.L. (1976) Biophys. Chem. 4, 23--50] in aqueous solution at various values of pH and guanidine hydrochloride concentration showed that these latter changes have no effect on the heat capacity changes, whereas the addition of alcohols causes a sharp decrease.     

Pilot-scale separation of lysozyme from hen egg white by integrating aqueous two-phase partitioning and membrane separation processes

A novel, cost-effective method of lysozyme separation from hen egg white was studied. This method integrates aqueous two-phase partitioning in the system EO₅₀PO₅₀/phosphates with membrane separation processes. The experiments were carried out in a pilot-scale on crude hen egg white.Initially, by forming an aqueous two-phase system, lysozyme was selectively extracted to the upper, polymer-rich phase while the other egg white proteins partitioned to the lower, phosphate-rich phase. Then, in order to recover lysozyme, thermoseparation of polymer-rich phase was applied. A novel approach for the simultaneous thermoseparation of the polymer-rich phase as well as for the recovery of the lysozyme was proposed, using a cross-flow microfiltration. Additionally, recovery of proteins by ultrafiltration from lower, phosphate-rich phase was also investigated.Lysozyme could be obtained after the thermal phase separation by means of microfiltration at a total recovery over the extraction steps of 47.5 and the purification factor of 10.5. The specific activity of lysozyme preparations was 34 188 U/mg of protein. Using cross-flow membrane techniques, it was found that the recovery of the polymer by microfiltration from the top phase was 83.9.     

Kinetics and equilibria of lysozyme precipitation and crystallization in concentrated ammonium sulfate solutions

The kinetics and thermodynamics of lysozyme precipitation in ammonium sulfate solutions at pH 4 and 8 and room temperature were studied. X-ray powder diffraction (XRD) was used to characterize the structure of lysozyme precipitates. It was found that, if sufficient time was allowed, microcrystals developed following an induction period after initial lysozyme precipitation, even up to ionic strengths of 8 m and at acidic pH, where lysozyme is refractory to crystallization in ammonium sulfate. The full set of precipitation and crystallization data allowed construction of a phase diagram of lysozyme, showing the ammonium sulfate dependence. It suggests that precipitation may reflect a frustrated metastable liquid-liquid phase separation, which would allow this process to be understood within the framework of the generic phase diagram for proteins. The results also demonstrate that XRD, more frequently used for characterizing inorganic and organic polycrystalline materials, is useful both in characterizing the presence of crystals in the dense phase and in verifying the crystal form of proteins.     

Cloud-point temperature and liquid-liquid phase separation of supersaturated lysozyme solution

The detailed understanding of the structure of biological macromolecules reveals their functions, and is thus important in the design of new medicines and for engineering molecules with improved properties for industrial applications. Although techniques used for protein crystallization have been progressing greatly, protein crystallization may still be considered an art rather than a science, and successful crystallization remains largely empirical and operator-dependent. In this work, a microcalorimetric technique has been utilized to investigate liquid-liquid phase separation through measuring cloud-point temperature T(cloud) for supersaturated lysozyme solution. The effects of ionic strength and glycerol on the cloud-point temperature are studied in detail. Over the entire range of salt concentrations studied, the cloud-point temperature increases monotonically with the concentration of sodium chloride. When glycerol is added as additive, the solubility of lysozyme is increased, whereas the cloud-point temperature is decreased.     

A comparison of the denatured states of alpha-lactalbumin and lysozyme

We have carried out a study of the denaturation of bovine α-lactalbumin by LiClO₄, LiCl, GuCl and urea, using difference spectroscopy, viscometry, polarimetry, and optical rotatory dispersion. These denaturants give rise to three different denatured states (GuCl and urea give the same state), which cannot be related to each other as members of a simple linear progression from the native state to the completely disordered state. The data require that LiClO₄ unfolds one part of the molecule, LiCl another, and GuCl or urea the whole molecule. There are striking parallels between the denaturation behaviour of lactalbumin and earlier observations on hen egg-white lysozyme, a protein with which it is about 40% homologous, and we believe the mass of the data supports the hypothesis, advanced by Browne et al. (1969), that the two proteins have similar backbone conformations.