Thermal Unfolding Curves Research Articles

Phosphorylation of ovalbumin by dry-heating in the presence of pyrophosphate: Effect of carbohydrate chain on the phosphorylation level and heat stability

Ovalbumin (OVA) derived from egg white (N-OVA) and recombinant OVA (Re-OVA), which was essentially carbohydrate-free, expressed by Escherichia coli, were phosphorylated by dry-heating for 1 day in the presence of pyrophosphate (PP-N- and PP-Re-OVA). The phosphorus contents of N- and Re-OVA were 0.79% and 0.46%, respectively, after phosphorylation. The secondary structural change of both OVAs was small, and the midpoint temperature of both OVAs determined from the thermal unfolding curve was decreased by phosphorylation. In addition, the differential scanning calorimetry thermograms of both OVAs showed a lowering of denaturation temperature by phosphorylation. The stability of both OVA solutions after heating at pH 7.0 was improved, and the degree of stabilization was higher for PP-N-OVA than for PP-Re-OVA. This suggests that the phosphate groups introduced to the carbohydrate chain also play an important role in the heat stability of OVA.

A natural missing link between activated and downhill protein folding scenarios.

We propose protein PTB1 : 4W as a good candidate for engineering into a downhill folder. PTB1 : 4W has a probe-dependent thermal unfolding curve and sub-millisecond T-jump relaxation kinetics on more than one time scale. Its refolding rate in denaturant is a non-linear function of denaturant concentration (curved chevron plot). Yet at high denaturant concentration its unfolding is probe-independent, and the folding kinetics can be fitted to a single exponential decay. The domain appears to fold via a mechanism between downhill folding and activated folding over several small barriers, and when denaturant is added, one of these barriers greatly increases and simplifies the observed folding to apparent two-state kinetics. We predict the simplest free energy function consistent with the thermal denaturation and kinetics experiments by using the singular value Smoluchowski dynamics (SVSD) model. PTB1 : 4W is a natural 'missing link' between downhill and activated folding. We suggest mutations that could move the protein into the downhill folding limit.

Cold Adaptation of Zinc Metalloproteases in the Thermolysin Family from Deep Sea and Arctic Sea Ice Bacteria Revealed by Catalytic and Structural Properties and Molecular Dynamics

Increased conformational flexibility is the prevailing explanation for the high catalytic efficiency of cold-adapted enzymes at low temperatures. However, less is known about the structural determinants of flexibility. We reported two novel cold-adapted zinc metalloproteases in the thermolysin family, vibriolysin MCP-02 from a deep sea bacterium and vibriolysin E495 from an Arctic sea ice bacterium, and compared them with their mesophilic homolog, pseudolysin from a terrestrial bacterium. Their catalytic efficiencies, k(cat)/K(m) (10-40 degrees C), followed the order pseudolysin < MCP-02 < E495 with a ratio of approximately 1:2:4. MCP-02 and E495 have the same optimal temperature (T(opt), 57 degrees C, 5 degrees C lower than pseudolysin) and apparent melting temperature (T(m) = 64 degrees C, approximately 10 degrees C lower than pseudolysin). Structural analysis showed that the slightly lower stabilities resulted from a decrease in the number of salt bridges. Fluorescence quenching experiments and molecular dynamics simulations showed that the flexibilities of the proteins were pseudolysin < MCP-02 < E495, suggesting that optimization of flexibility is a strategy for cold adaptation. Molecular dynamics results showed that the ordinal increase in flexibility from pseudolysin to MCP-02 and E495, especially the increase from MCP-02 to E495, mainly resulted from the decrease of hydrogen-bond stability in the dynamic structure, which was due to the increase in asparagine, serine, and threonine residues. Finally, a model for the cold adaptation of MCP-02 and E495 was proposed. This is the first report of the optimization of hydrogen-bonding dynamics as a strategy for cold adaptation and provides new insights into the structural basis underlying conformational flexibility.

A Proteomic-scale, Mass Spec-based Method to Probe Forced Unfolding within Cells

Cysteine is a relatively hydrophobic amino acid and is often buried within protein folds or assemblies. We use Cys labeling of live cells under different conditions, including mechanical stress and temperature, to identify by Mass Spec those proteins and sites that are perturbed. For intracellular proteins, Cys are not tied up in disulfides, and so their reactivity to many fluorescent dyes in solution is readily determined as a function of temperature under both native and high urea conditions. When a protein is folded at low temperature, the reaction rate tends to be faster in urea, and the ratio of rates yields a thermal unfolding curve that looks very similar to results from solution methods such as Circular Dichroism. Unlike these other methods, however, the Cys labeling approach can also be applied to intact cells by using membrane-permeable, cell-viable dyes at low dose. Labeling studies of several cell types, including stem cells, lung cancer cells, and cardiomyocytes, treated under two different conditions, such as stressed vs unstressed or 37C vs fever-like 42C, are followed by electrophoretic separation and detailed LC/MS-MS study. What results is an identification of labeled proteins as well as differentially labeled sites in many proteins, including several structural proteins with major roles in disease and development: Myosin, Filamin, Spectrin, and Vimentin. The Cys shotgun method thus provides unique, proteomic scale measurements of protein structural changes in both solution and intact cells. (Science 317, 663-666, 2007)

Downhill versus Barrier-Limited Folding of BBL 1: Energetic and Structural Perturbation Effects upon Protonation of a Histidine of Unusually Low pKa

A dispersion of melting temperatures at pH5.3 for individual residues of the BBL protein domain has been adduced as evidence for barrier-free downhill folding. Other members of the peripheral subunit domain family fold cooperatively at pH7. To search for possible causes of anomalies in BBL's denaturation behavior, we measured the pH titration of individual residues by heteronuclear NMR. At 298 K, the pKa of His142 was close to that of free histidine at 6.47±0.04, while that of the more buried His166 was highly perturbed at 5.39±0.02. Protonation of His166 is thus energetically unfavorable and destabilizes the protein by ∼1.5 kcal/mol. Changes in Cα secondary shifts at pH5.3 showed a decrease in helicity of the C-terminus of helix 2, where His166 is located, which was accompanied by a measured decrease of 1.1±0.2 kcal/mol in stability from pH7 to 5.3. Protonation of His166 perturbs, therefore, the structure of BBL. Only ∼1% of the structurally perturbed state will be present at the biologically relevant pH7.6. Experiments at pH5.3 report on a near-equal mixture of the two different native states. Further, at this pH, small changes of pH and pKa induced by changes in temperature will have near-maximal effects on pH-dependent conformational equilibria and on propagation of experimental error. Accordingly, conventional barrier-limited folding predicts some dispersion of measured thermal unfolding curves of individual residues at pH5.3.

Experimental Verification of the Crucial Roles of Glu73 in the Catalytic Activity and Structural Stability of Goose Type Lysozyme

The roles of Glu(73), which has been proposed to be a catalytic residue of goose type (G-type) lysozyme based on X-ray structural studies, were investigated by means of its replacement with Gln, Asp, and Ala using ostrich egg-white lysozyme (OEL) as a model. No remarkable differences in secondary structure or substrate binding ability were observed between the wild type and Glu(73)-mutated proteins, as evaluated by circular dichroism (CD) spectroscopy and chitin-coated celite chromatography. Substitution of Glu(73) with Gln or Ala abolished the enzymatic activity toward both the bacterial cell substrate and N-acetylglucosamine pentamer, (GlcNAc)(5), while substitution with Asp did not abolish but drastically reduced the activity of OEL. These results demonstrate that the carboxyl group of Glu(73) is directly involved in the catalytic action of G-type lysozyme. Furthermore, the stabilities of all three mutants, which were determined from the thermal and guanidine hydrochloride (GdnHCl) unfolding curves, respectively, were significantly decreased relative to those of the wild type. The results obtained clearly indicate the crucially important roles of Glu(73) in the structural stability as well as in the catalytic activity of G-type lysozyme.

An Active Enzyme Constructed from a 9-Amino Acid Alphabet

Nature employs a set of 20 amino acids to produce a repertoire of protein structures endowed with sophisticated functions. Here, we combined design and selection to create an enzyme composed entirely from a set of only 9 amino acids that can rescue auxotrophic cells lacking chorismate mutase. The simplified protein captures key structural features of its natural counterpart but appears to be somewhat less stable and more flexible. The potential of a dramatically reduced amino acid alphabet to produce an active catalyst supports the notion that primordial enzymes may have possessed low amino acid diversity and suggests that combinatorial engineering strategies, such as the one used here, may be generally applied to create enzymes with novel structures and functions.

Bacteriophage P22 Tail Accessory Factor GP26 Is a Long Triple-stranded Coiled-coil

P22 is a well characterized tailed bacteriophage that infects Salmonella enterica serovar Typhimurium. It is characterized by a "short" tail, which is formed by five proteins: the dodecameric portal protein (gp1), three tail accessory factors (gp4, gp10, gp26), and six trimeric copies of the tail-spike protein (gp9). We have isolated the gene encoding tail accessory factor gp26, which is responsible for stabilization of viral DNA within the mature phage, and using a variety of biochemical and biophysical techniques we show that gp26 is very likely a triple stranded coiled-coil protein. Electron microscopic examination of purified gp26 indicates that the protein adopts a rod-like structure approximately 210 angstroms in length. This trimeric rod displays an exceedingly high intrinsic thermostability (T(m) approximately 85 degrees C), which suggests a potentially important structural role within the phage tail apparatus. We propose that gp26 forms the thin needle-like fiber emanating from the base of the P22 neck that has been observed by electron microscopy of negatively stained P22 virions. By analogy with viral trimeric coiled-coil class I membrane fusion proteins, gp26 may represent the membrane-penetrating device used by the phage to pierce the host outer membrane.

Structural Characteristics of Hen Egg Ovalbumin Expressed in YeastPichia pastoris

The recombinant ovalbumin (OVA) produced in yeast Pichia pastoris was purified from the culture medium by anion exchange chromatography, and its structural characteristics were compared with those of hen egg OVA, mainly from the point of view of posttranslational modification. The expressed OVA consisted of two molecular species immmunoreactive with antibody for hen egg OVA. The two molecular species, 45 and 47 kDa in molecular size, were thought to correspond to mono-glycosylated form and di-glycosylated form respectively. The non-glycosylated form was not produced in the system. The other posttranslational modifications (N-terminal acetylation and phosphorylation) observed in hen egg OVA were not detected in either of the molecular species. The two recombinant proteins displayed almost exactly the same circular dichroism and intrinsic tryptophan fluorescence spectra as hen egg OVA. The melting temperature, Tm, which was determined from the thermal unfolding curve, was almost identical in the two recombinant proteins, despite the difference in glycosylation levels, while it decreased by about 2.5 degrees C as compared with that of hen egg OVA (77.3 degrees C). These data indicate that the additional glycosylation to Asn-311 in the recombinant protein does not affect protein conformation or thermostability.

Equilibrium Structure and Folding of a Helix-Forming Peptide: Circular Dichroism Measurements and Replica-Exchange Molecular Dynamics Simulations

We have performed experimental measurements and computer simulations of the equilibrium structure and folding of a 21-residue α-helical heteropeptide. Far ultraviolet circular dichroism spectroscopy is used to identify the presence of helical structure and to measure the thermal unfolding curve. The observed melting temperature is 296 K, with a folding enthalpy of −11.6 kcal/mol and entropy of −39.6 cal/(mol K). Our simulations involve 45 ns of replica-exchange molecular dynamics of the peptide, using eight replicas at temperatures between 280 and 450 K, and the program CHARMM with a continuum solvent model. In a 30-ns simulation started from a helical structure, conformational equilibrium at all temperatures was reached after 15 ns. This simulation was used to calculate the peptide melting curve, predicting a folding transition with a melting temperature in the 330–350 K range, enthalpy change of −10 kcal/mol, and entropy change of −30 cal/(mol K). The simulation results were also used to analyze the peptide structural fluctuations and the free-energy surface of helix unfolding. In a separate 15-ns replica-exchange molecular dynamics simulation started from the extended structure, the helical conformation was first attained after ∼2.8 ns, and equilibrium was reached after 10 ns of simulation. These results showed a sequential folding process with a systematic increase in the number of hydrogen bonds until the helical state is reached, and confirmed that the α-helical state is the global free-energy minimum for the peptide at low temperatures.

Bacterial IscU is a well folded and functional single domain protein.

Iron-sulfur clusters are widely represented in most organisms, but the mechanism of their formation is not fully understood. Of the two main proteins involved in cluster formation, NifS/IscS and NifU/IscU, only the former has been well studied from a structural point of view. Here we report an extensive structural characterization of Escherichia coli IscU. We show by a variety of physico-chemical techniques that E. coli IscU construct can be expressed to high purity as a monomeric protein, characterized by an alphabeta fold with high alpha-helix content. The high melting temperature and the reversibility of the thermal unfolding curve (as measured by CD spectroscopy) hint at a well ordered stable fold. The excellent dispersion of cross peaks in the (1)H-(15)N correlation spectrum is consistent with these observations. Monomeric E. coli IscU is able to provide a scaffold for Iron-sulfur cluster assembly, but has no direct interaction with either Fe(II) or Fe(III) ions, suggesting the need of further partners to achieve a stable interaction.

Detection and Characterization of Partially Unfolded Oligomers of the SH3 Domain of α-Spectrin

For the purpose of equilibrium and kinetic folding-unfolding studies, the SH3 domain of α-spectrin (spc-SH3) has long been considered a classic two-state folding protein. In this work we have indeed observed that the thermal unfolding curves of spc-SH3 measured at pH 3.0 by differential scanning calorimetry, circular dichroism, and NMR follow apparently the two-state model when each unfolding profile is considered individually. Nevertheless, we have found that protein concentration has a marked effect upon the thermal unfolding profiles. This effect cannot be properly explained in terms of the two-state unfolding model and can only be interpreted in terms of the accumulation of intermediate associated states in equilibrium with the monomeric native and unfolded states. By chemical cross-linking and pulsed-field gradient NMR diffusion experiments we have been able to confirm the existence of associated states formed during spc-SH3 unfolding. A three-state model, in which a dimeric intermediate state is assumed to be significantly populated, provides the simplest interpretation of the whole set of thermal unfolding data and affords a satisfactory explanation for the concentration effects observed. Whereas at low concentrations the population of the associated intermediate state is negligible and the unfolding process consequently takes place in a two-state fashion, at concentrations above ∼0.5 mM the population of the intermediate state becomes significant at temperatures between 45°C and 80°C and reaches up to 50% at the largest concentration investigated. The thermodynamic properties of the intermediate state implied by this analysis fall in between those of the unfolded state and the native ones, indicating a considerably disordered conformation, which appears to be stabilized by oligomerization.

Long-range interactions stabilize the fold of a non-natural oligomer.

Toward designing nonbiological polymers that fold into predictable tertiary structures, we report a "beta-oligomer" composed of beta-amino acids that adopts a cooperatively folded structure. We have computationally designed a C(2)-symmetrical pair of interacting 14-helical beta-oligomers stabilized via long-range interhelical interactions and stapled together by a disulfide bond. The reduced (BHBred) and oxidized (BHBox) forms of the synthetic beta-oligomer represent the individual isolated helices and the two-helix bundle, respectively. We also prepared a third monomeric synthetic beta-oligomer (BHBmon) to avoid inadvertent disulfide formation during characterization. Circular dichroism spectroscopy revealed that BHBox showed a 2-fold increase in secondary structure, relative to the monohelical controls, BHBred and BHBmon. Further, BHBox showed a sigmoidal thermal unfolding curve with a per-residue van't Hoff enthalpy of approximately 0.7 kcal/(mol.residue), analogous to folded proteins. In contrast, BHBmon shows a broad thermal transition, typical of multistate unfolding for monomeric helices. Also, analytical ultracentrifugation showed that BHBmon and BHBox were monomeric at concentrations < or =800 and 280 microM, respectively. Therefore, the enhanced helicity of BHBox could be attributed to intramolecular helix-helix interactions.

Sulfate anion stabilization of native ribonuclease A both by anion binding and by the Hofmeister effect.

Data are reported for T(m), the temperature midpoint of the thermal unfolding curve, of ribonuclease A, versus pH (range 2-9) and salt concentration (range 0-1 M) for two salts, Na(2)SO(4) and NaCl. The results show stabilization by sulfate via anion-specific binding in the concentration range 0-0.1 M and via the Hofmeister effect in the concentration range 0.1-1.0 M. The increase in T(m) caused by anion binding at 0.1 M sulfate is 20 degrees at pH 2 but only 1 degree at pH 9, where the net proton charge on the protein is near 0. The 10 degrees increase in T(m) between 0.1 and 1.0 M Na(2)SO(4), caused by the Hofmeister effect, is independent of pH. A striking property of the NaCl results is the absence of any significant stabilization by 0.1 M NaCl, which indicates that any Debye screening is small. pH-dependent stabilization is produced by 1 M NaCl: the increase in T(m) between 0 and 1.0 M is 14 degrees at pH 2 but only 1 degree at pH 9. The 14 degree increase at pH 2 may result from anion binding or from both binding and Debye screening. Taken together, the results for Na(2)SO(4) and NaCl show that native ribonuclease A is stabilized at low pH in the same manner as molten globule forms of cytochrome c and apomyoglobin, which are stabilized at low pH by low concentrations of sulfate but only by high concentrations of chloride.

Determination of RNase A/2′-Cytidine Monophosphate Binding Affinity and Enthalpy by a Global Fit of Thermal Unfolding Curves

A spectropolarimeter was used to measure the thermal response curves of RNase A in the presence and absence of the ligand cytidine 2′-monophosphate. A coupled equilibrium model was used to describe the dissociation of the protein–ligand complex (NL ⇄ N + L) and the thermal unfolding of the free protein (N ⇄ U). The unfolding curves of the protein in the presence of several different concentrations of ligand were fit to this coupled equilibrium model using global linkage analysis. The best-fitted values for the thermal unfolding of the apo-protein were 60.9 ± 0.2°C (Tm) and 105.5 ± 1.4 kcal/mol (ΔH), while the fitted values for the dissociation of the protein–ligand complex were 1.6 ± 0.4 μM (KD) and 18.7 ± 1.0 kcal mol−1 (ΔHL). These values were in excellent agreement with values obtained by other methods. The sensitivity of the data fitting was compared using linear or quadratic temperature dependence for the response curves of the free ligand (L), native apo-protein (N), native ligand–bound protein (NL), and unfolded apo-protein (U). There was no significant improvement in the precision of the fitted data when the temperature-dependent response for each species (N, L, NL, and U) was expressed as quadratic functions of temperature.

The Two-Domain NK1 Fragment of Plasminogen: Folding, Ligand Binding, and Thermal Stability Profile

The two-domain fragment N+K1 (rNK1) [Glu(1)-Glu(163)] of human plasminogen was expressed in E. coli as a hexahistidine-tagged fusion protein and chromatographically purified. The (1)H NMR spectrum supports proper folding of the K1 component within the refolded rNK1 construct (rNK1/K1). The functional properties of rNK1/K1 were investigated via intrinsic fluorescence titration with kringle-specific omega-aminocarboxylic acid ligands. The affinities closely match those previously measured for the isolated K1, which indicates that the N-domain does not significantly affect the interaction of ligands with the lysine binding site of K1. Far-UV CD spectra recorded for the N-domain suggest conformational plasticity and flexibility for the module. Two classes of spectra, referred to as types A and B, were identified with the type A spectrum reflecting a higher secondary structure content than that estimated for the type B spectrum. Subtracting the CD spectrum of rK1 from that of rNK1 yields a spectrum (Delta) which reflects the conformation of the N-domain within the rNK1 construct (rNK1/N). Delta resembles the type A spectrum, suggesting that rNK1/N adopts a relatively more ordered conformation, stabilized by the adjacent rNK1/K1 domain. In contrast, thermal unfolding curves determined via CD indicate that the rNK1/N slightly lowers the melting temperature (T(m)) of rNK1/K1. Independence of the two domains within rNK1 was tested by monitoring the thermal unfolding of rNK1/K1 when in the presence of the kringle-specific ligand AMCHA, which left the rNK1/N T(m) essentially unaffected, while increasing that of the rNK1/K1 by approximately 10 degrees C.

Selection of potent chymotrypsin and elastase inhibitors from M13 phage library of basic pancreatic trypsin inhibitor (BPTI)

The combinatorial approach offered by phage display has proved to be powerful in obtaining novel variants of canonical inhibitors of serine proteinases that show new binding patterns. We applied this strategy to search for variants of basic pancreatic trypsin inhibitor (BPTI) that would be strong inhibitors of two serine proteinases: bovine α-chymotrypsin and porcine pancreatic elastase. BPTI only moderately inhibits the first and does not inhibit the second enzyme. A representative library of 3.2×10 4 BPTI variants, randomized at P 1, P 1′, P 2′ and P 3′ positions of the proteinase binding loop, was displayed on the surface of phage M13. After four to five rounds of selection on the target proteinase consensus sequences of the inhibitor binding loop were obtained. In both cases, the variants selected differed from BPTI at two to four positions, with a strong preference for selection of hydrophobic residues. Nevertheless, five of these variants expressed in a free form appeared to be correctly folded, stable proteins, and did not aggregate during thermal denaturation. The midpoints of the thermal unfolding curves of these variants were lowered by 5–20°C as compared to BPTI. The expressed variants proved to be new potent inhibitors of the target enzymes with association constants up to 6.9×10 9 M −1 and 3.7×10 10 M −1 for elastase and chymotrypsin, respectively. Thus, the inhibitory properties of BPTI were improved by as much as 7×10 6-fold towards elastase and 420-fold towards chymotrypsin.

Structural and physical properties of gelatin extracted from different marine species: a comparative study

Gelatin from skins of several marine species were compared on the basis of their rheological characteristics (viscoelasticity and gel strength) and chemical/structural properties (amino acid composition, molecular weight distribution and triple helix formation). Gelatins from flat-fish species (sole and megrim) presented the best gelling ability and the gels were more thermostable than those from cold-adapted fish (cod and hake). This different behaviour may be explained considering the amino acid composition, the α 1/ α 2 collagen-chain ratio, and the molecular weight distribution. Thus, cod gelatin presented a lower alanine and imino acid content, and a decreased proline hydroxylation degree; cod and hake gelatins presented a low α 1/ α 2 ratio (∼1); hake gelatin showed a highly significant decrease in β-components and other aggregates. The squid gelatin presented the most significant changes regarding amino acid composition and molecular weight distribution, most of these differences arising from the low solubility of the squid connective tissue. However, the squid gelatin showed viscoelastic properties intermediate between those from flat-fish and cold-adapted fish species. Circular dichroism analysis reveals that gelling involves a refolding of denatured collagen chains into the typical triple helix conformation and, conversely, unfolding upon reheating. Thermal folding and unfolding curves were similar to those of viscoelastic properties but showing a shift towards lower or higher temperatures upon cooling and heating, respectively. The folding process seem to be directly related in the stabilisation of the gels without disregarding its role in triggering the gelation process. Finally, gel strength evaluation revealed the importance of slow cold maturation.

Theoretic information approach to protein stabilization by solvent engineering

AbstractA novel approach based on molecular thermodynamics and the information theory is proposed to quantify the influence of water‐miscible additives on protein stability. According to the two‐state mechanism of inactivation, solvent effects are described in terms of perturbation of the equilibrium between the folded and unfolded protein forms. The model provides the dependence of the protein's melting temperature on the additive concentration. Effects of the latter are accounted for by an empirical parameter related to the free energy of transfer of the protein from the pure to the mixed solvent. The model was tested using experimental data relative to the influence of hydroxylic and aminoacidic additives on the thermal unfolding of hen egg lysozyme and erythrocyte carbonic anhydrase. Fitting parameters were correlated in terms of a theoretic information index characterizing the additive's molecule and incorporating an atomic‐composition term and a topological contribution. Model calculations agreed very well with experimental data, suggesting that the molecular information content of the additive can be used effectively to correlate solvent‐induced perturbations of stability. The procedure was also used to predict melting temperatures in systems containing binary mixtures of additives and to reconstruct thermal unfolding curves in the different media.

Evolution of binding affinity in a WW domain probed by phage display.

The WW domain is an approximately 38 residue peptide-binding motif that binds a variety of sequences, including the consensus sequence xPPxY. We have displayed hYAP65 WW on the surface of M13 phage and randomized one-third of its three-stranded antiparallel beta-sheet. Improved binding to the hydrophobic peptide, GTPPPPYTVG (WW1), was selected in the presence of three different concentrations of proteinase K to simultaneously drive selection for improved stability as well as high-affinity binding. While some of the selected binders show cooperative unfolding transitions, others show noncooperative thermal unfolding curves. Two novel WW consensus sequences have been identified, which bind to the xPPxY motif with higher affinity than the wild-type hYAP65 WW domain. These WW domain sequences are not precedented in any natural WW domain sequence. Thus, there appear to be a large number of motifs capable of recognizing the target peptide sequence, only a subset of which appear to be used in natural proteins.