pH-dependent Conformational Change Research Articles

PH Effects on the Stability and Folding of Monomeric Caspases

Caspases are an ancient class of cysteinyl proteases that play an integral part in cell development and apoptosis as an evolutionarily conserved function. During apoptosis, the intracellular pH decreases from 7.4 to ~ 6.8 and cytosolic acidification affects the activation of caspase‐3 and pH‐dependent conformational changes affect the rate of auto maturation in the dimeric procaspase‐3, which in turn influences the ability of this enzyme to execute the cell. Proteins are required to fold correctly and attain their native conformation which is vital for protein function and the survival of the organism. The protein folding landscape is sculpted through evolution and there exists an evolutionary selection for functional residues by selecting residues that stabilize the interactions to attain the native conformation while repressing the selection of non‐native interactions that take place during folding. Through the course of evolution, how these proteins fold to the native conformation to contribute to the fitness of the organism while maintaining or altering the energy landscape that is guided by trade‐offs between the stability and the activity of the protein remains elusive. To understand how the energy landscapes changed over time, we resurrected an ancestral caspase (existed about 650 milllion years ago) and we examined the folding pathways of caspases from Cnidarians, specifically corals. We studied the equilibrium unfolding of caspases from O.faveolata (species susceptible to disease) and P.astreoides (species resistant to disease) as a function of pH (between pH 3 and pH 10.5) to examine the changes in the context of stability and folding. The data shows that these monomeric proteins have a high secondary structure at the native state and unfold via a three‐state process closer to the physiological pH (pH 6.5~pH8.5), via an intermediate state which is aggregation prone. However, outside of this pH range, the native protein loses its secondary structure and exists in an intermediate state which is stable. Due to this, these proteins undergo a two‐state unfolding mechanism wherein, the protein transitions from an intermediate state to the unfolded state, reversibly. Overall, the free energy change for these primitive coral caspases is ~12.5kcal/mol, which is higher than what is observed for human caspases. This higher stability might be due to the decrease in the activity and the presence of certain stabilizing residues that are not observed in the human caspases.

PH effects on the stability and folding of monomeric caspases

Caspases are an ancient class of cysteinyl proteases that play an integral part in cell development and apoptosis as an evolutionarily conserved function. During apoptosis, the intracellular pH decreases from 7.4 to ∼ 6.8 and cytosolic acidification affects the activation of caspase-3 and pH-dependent conformational changes affect the rate of auto maturation in the dimeric procaspase-3. This protein folding landscape is sculpted through evolution by selection of stabilizing residues, while repressing the non-native ones.

PH-dependent compaction of intrinsically disordered poly-E motif in titin

Intrinsically disordered proteins and regions (IDPs/IDRs) have been found to play a role in a variety of cellular processes. Unlike ordered proteins, many IDPs become more structured under extreme pH conditions as a result of neutralization of the charged amino acids in the IDP. This occurs because a higher net charge per residue (NCPR) results in increased pH-dependent conformational change. However, the correlation between increased NCPR and increased pH sensitivity is variable between IDPs, suggesting that NCPR alone cannot explain the observed variation in pH sensitivity.

Prediction of the Effect of pH on the Aggregation and Conditional Folding of Intrinsically Disordered Proteins with SolupHred and DispHred.

Proteins microenvironments modulate their structures. Binding partners, organic molecules, or dissolved ions can alter the protein's compaction, inducing aggregation or order-disorder conformational transitions. Surprisingly, bioinformatic platforms often disregard the protein context in their modeling. In a recent work, we proposed that modeling how pH affects protein net charge and hydrophobicity might allow us to forecast pH-dependent aggregation and conditional disorder in intrinsically disordered proteins (IDPs). As these approaches showed remarkable success in recapitulating the available bibliographical data, we made these prediction methods available for the scientific community as two user-friendly web servers. SolupHred is the first dedicated software to predict pH-dependent aggregation, and DispHred is the first pH-dependent predictor of protein disorder. Here we dissect the features of these two software applications to train and assist scientists in studying pH-dependent conformational changes in IDPs.

Tuning C-Phycocyanin Photoactivity via pH-Mediated Assembly-Disassembly.

Environment-triggered protein conformational changes have garnered wide interest in both fundamental research, for deciphering in vivo acclimatory responses, and practical applications, for designing stimuli-responsive probes. Here, we propose a protein-chromophore regulatory mechanism that allows for manipulation of C-phycocyanin (C-PC) from Spirulina platensis by environmental pH and UV irradiation. Using small-angle X-ray scattering, a pH-mediated C-PC assembly-disassembly pathway, from monomers to nonamers, was unraveled. Such flexible protein matrices impart tunability to the embedded tetrapyrroles, whose photochemical behaviors were found to be modulated by protein assembly states. UV irradiation on C-PC triggers pH-dependent singlet oxygen (1O2) generation and conformational changes. Intermolecular photo-crosslinking occurs at pH 5.0 via dityrosine species, which bridges solution-based C-PC oligomers into unprecedented dodecamers and 24-mers. These supramolecular assemblies impart C-PC at pH 5.0, which significantly enhanced 1O2 yield, fluorescence, and photostability relative to those at other pH values, a finding that makes C-PC appealing for tumor-targeted photodynamic therapy.

PH-Dependent Conformational Changes Lead to a Highly Shifted pKa for a Buried Glutamic Acid Mutant of SNase.

Ionizable residues are rarely present in the hydrophobic interior of proteins, but when they are, they play important roles in biological processes such as energy transduction and enzyme catalysis. Internal ionizable residues have anomalous experimental pKa values with respect to their pKa in bulk water. This work investigates the atomistic cause of the highly shifted pKa of the internal Glu23 in the artificially mutated variant V23E of Staphylococcal Nuclease (SNase) using pH replica exchange molecular dynamics (pH-REMD) simulations. The pKa of Glu23 obtained from our calculations is 6.55, which is elevated with respect to the glutamate pKa of 4.40 in bulk water. The calculated value is close to the experimental pKa of 7.10. Our simulations show that the highly shifted pKa of Glu23 is the product of a pH-dependent conformational change, which has been observed experimentally and also seen in our simulations. We carry out an analysis of this pH-dependent conformational change in response to the protonation state change of Glu23. Using a four-state thermodynamic model, we estimate the two conformation-specific pKa values of Glu23 and describe the coupling between the conformational and ionization equilibria.

Structure and regulation of the BsYetJ calcium channel in lipid nanodiscs

BsYetJ is a bacterial homolog of transmembrane BAX inhibitor-1 motif-containing 6 (TMBIM6) membrane protein that plays a key role in the control of calcium homeostasis. However, the BsYetJ (or TMBIM6) structure embedded in a lipid bilayer is uncharacterized, let alone the molecular mechanism of the calcium transport activity. Herein, we report structures of BsYetJ in lipid nanodiscs identified by double electron-electron resonance spectroscopy. Our results reveal that BsYetJ in lipid nanodiscs is structurally different from those crystallized in detergents. We show that BsYetJ conformation is pH-sensitive in apo state (lacking calcium), whereas in a calcium-containing solution it is stuck in an intermediate, inert to pH changes. Only when the transmembrane calcium gradient is established can the calcium-release activity of holo-BsYetJ occur and be mediated by pH-dependent conformational changes, suggesting a dual gating mechanism. Conformational substates involved in the process and a key residue D171 relevant to the gating of calcium are identified. Our study suggests that BsYetJ/TMBIM6 is a pH-dependent, voltage-gated calcium channel.

PH dependent aggregation and conformation changes of rituximab using SAXS and its comparison with the standard regulatory approach of biophysical characterization

Development of biologics and biosimilars involves extensive physical and structural characterization, which underlines the further course of its implementation. These characterization techniques require considerable standardization and are labor intensive. It is therefore, important to have an immediate, independent and affordable characterization strategy that may meet the regulatory guidelines. In this study, we have compared the standard biophysical characterization of an anti-CD 20 antibody with characterization by small angle x ray scattering (SAXS). Aggregation of this mAb was analyzed using standard techniques like size exclusion HPLC, dynamic light scattering and sedimentation velocity - analytical ultracentrifugation, whereas structure analysis was conducted using mass spectrometry, circular dichroism spectroscopy and fluorescence spectroscopy. Our results demonstrated that the inferences about the state of mAb aggregation and its structure deduced using the standard approaches were comparable to the data interpreted using SAXS. The radius of gyration and the P(r) distribution plot obtained using the SAXS scattering data allowed analysis of aggregation and conformation of mAb via a single experiment. Thus, SAXS can be used as an independent technique to complement orthogonal analysis for determining the aggregation profile and structure of mAbs.

Characterization of Localization, Ligand Binding, and pH-Dependent Conformational Changes of Two Chemosensory Proteins Expressed in the Antennae of the Japanese Carpenter Ant, Camponotus Japonicus.

Camponotus japonicus uses basiconic antennal sensilla (s. basiconica) to sense a colony-specific blend of species-specific cuticular hydrocarbons (CHCs). The inner portion of the s. basiconica is filled with sensillar lymph and chemosensory proteins (CSPs) presumed to transport CHCs to olfactory neuron receptors. Although 12 CSPs have been found in C. japonicus antennae, we focused on CjapCSP1 and CjapCSP13. The molecular basis of CSP1 function was explored by observation of its structure in solution at pH 4.0 and 7.0 through circular dichroism (CD) and X-ray solution scattering. Although the secondary structure did not vary with pH change, the radius of gyration (Rg) was larger by 5.3% (0.74 Å increase) at pH 4.0 than at pH 7.0. The dissociation constant (Kd) for CjapCSP1 measured with a fluorescent probe, 1-N-phenylnaphthylamine, was larger at pH 4.0 than at pH 7.0, suggesting that acidic pH triggers ligand dissociation. In contrast to CjapCSP1, the Rg of CjapCSP13 was slightly smaller at pH 4.0 than at pH 7.0. Western blotting and immunohistochemistry with protein-specific antisera revealed that both CjapCSP1 and CjapCSP13 are detected in the antennae, but differ in their specific internal localization. Binding to four compounds, including the ant CHC (z)-9-tricosene, was examined. Although both CjapCSP1 and CjapCSP13 bound to (z)-9-tricosene, CjapCSP13 bound with higher affinity than CjapCSP1 and showed different binding properties. CjapCSP1 and CjapCSP13 are synthesized by the same cells of the antenna, but function differently in CHC distribution due to differences in their localization and binding characteristics.

Nitric Oxide Synthase Oxygenase in Thin Layer-By-Layer Polymeric Films: Does the pH at Which the Film Is Built Affect the Direct Electron Transfer or the Related NOS-Mediated Catalysis?

We prepared thin films containing nitric oxide synthase (NOS) oxygenase enzymes and polyethyleneimine (PEI) using the layer-by-layer film building strategy. The charge-based layer-by-layer electrostatic adsorption allows for assembly of multi-component protein/PEI films. During the film building process, we used NOS oxygenase enzyme from solutions at two distinct pHs (pH=7.0 and pH=8.6) to study the effect of pH on enzyme loading in the thin polymeric film. We studied direct electron transfer to NOS oxygenase in the two kinds of films at physiologic pH. We show that NOS oxygenase enzymes maintain their overall integral structure in PEI films in both pH conditions, and exhibit relatively fast but similar electron transfer rates. Using thin film coulometry, we show that the enzyme loading from pH 8.6 is almost 4-times more than the amount loaded in films prepared using NOS oxygenase solutions at pH=7.0.To closely examine the effect of pH during the layer-by-layer film building on the trapped NOS oxygenase enzymes, we studied the catalytic reduction of exogenous nitric oxide analyte mediated by NOS oxygenases in PEI films. We used this property to compare and contrast the catalytic activity of the films of NOS enzymes prepared at pH 7.0 and those prepared at pH 8.6. Our results show that there is a distinct increase in catalytic activity of enzymes adsorbed from pH 8.6 compared to those prepared at pH=7.0. We also show that the measured differential loading amnounts in the two kinds of films cannot explain the observed increase in mediated catalytic activity. In this regard, we show that NOS oxygenase films constructed at pH 8.6 exhibit significantly higher catalytic efficiency (kcat /KM) compared to films constructed at pH 7.0. To better understand the change in catalytic activity, we used detailed Fourier Transform Infrared (FTIR) spectroscopic analysis of the thin films to closely examine the structural integrity of the trapped NOS oxygenases in the two sets of films. Deconvolution methods using FTIR data suggest that although NOS oxygenase enzymes maintain their overall structure, we observe distinct pH-dependent conformational changes, which includes a decrease in α-helix contributions in the NOS protein adsorbed at pH 8.6. The observed changes in the secondary structure apparently add to the disordering of protective loops that affect a hydrophobic pocket trap that is critical for NO binding during catalysis.This finding is in line with the observed change in NO affinity for the trapped NOS enzymes as evidenced by the distinct change in catalytic efficiency of NOS oxygenases in the two sets of films. This work shows that while the electron transfer to NOS enzymes in PEI films is not affected by pH in which the layer-by-layer film fabrication occurs, the subtle pH-induced backbone changes provides a structural basis for the higher specific catalytic electrochemical reduction of NO by NOS oxygenases in PEI films constructed at pH 8.6.

Transient Deprotonation of the Chromophore Affects Protein Dynamics Proximal and Distal to the Linear Tetrapyrrole Chromophore in Phytochrome Cph1.

Phytochromes are biological red/far-red light sensors found in many organisms. Prototypical phytochromes, including Cph1 from the cyanobacterium Synechocystis 6803, act as photochemical switches that interconvert between stable red (Pr)- and metastable far-red (Pfr)-absorbing states induced by photoisomerization of the bilin chromophore. The connection between photoconversion and the cellular output signal involves light-mediated global structural changes in the interaction between the photosensory module (PAS-GAF-PHY) and the C-terminal transmitter (output) module, usually a histidine kinase, as in the case of Cph1. The chromophore deprotonates transiently during the Pr → Pfr photoconversion in association with extensive global structural changes required for signal transmission. Here, we performed equilibrium studies in the Pr state, involving pH titration of the linear tetrapyrrole chromophore in different Cph1 constructs, and measurement of pH-dependent structural changes at various positions in the protein using picosecond time-resolved fluorescence anisotropy. The fluorescent reporter group was attached at positions 371 (PHY domain), 305 (GAF domain), and 120 (PAS domain), as well as at sites in the PAS-GAF bidomain. We show direct correlation of chromophore deprotonation with pH-dependent conformational changes in the various domains. Our results suggest that chromophore deprotonation is closely associated with a higher protein mobility (conformational space) both in proximal and in distal protein sites, implying a causal relationship that might be important for the global large protein arrangements and thus intramolecular signal transduction.

Structural and conformational behavior of MurE ligase from Salmonella enterica serovar Typhi at different temperature and pH conditions

MurE ligase is known to play a significant role in peptidoglycan biosynthesis. It catalyzes the addition of meso-diaminopimelic acid to nucleotide precursor. The protein can adopt different conformations for its proper functioning. Different environmental conditions can alter the stability and function of enzyme due to their ability to disrupt interactions between different domains. We have explored the pH and temperature dependent conformational changes in MurE ligase from Salmonella Typhi and estimated the protein stability. The study enabled us to decipher the effect of different milieu condition in the enzyme activity. At acidic pH 3.0, StMurE ligase forms molten globule (MG) state and at alkaline pH it is in unfolded state. The different states of StMurE ligase were characterized using various spectroscopic techniques. These techniques including near-UV CD, far-UV CD, ANS fluorescence, differential scanning calorimetry and fluorescence spectroscopy helped to determine the secondary structural changes and detect local conformational modifications. The structural analysis using StMurE ligase homology model revealed the variations in ionization states of catalytic amino acid residues involved in substrate binding. This study provides an insight into the dynamics states of StMurE ligase at different environmental conditions during bacterial pathogenesis.

Structural Analysis of Rabies Virus Glycoprotein Reveals pH-Dependent Conformational Changes and Interactions with a Neutralizing Antibody

Rabies virus (RABV), the etiological agent for the lethal disease of rabies, is a deadly zoonotic pathogen. The RABV glycoprotein (RABV-G) is a key factor mediating virus entry and the major target of neutralizing antibodies. Here, we report the crystal structures of RABV-G solved in the free form at ∼pH-8.0 and in the complex form with a neutralizing antibody 523-11 at ∼pH-6.5, respectively. RABV-G has three domains, and the basic-to-acidic pH change results in large domain re-orientations and concomitant domain-linker re-constructions, switching it from a bent hairpin conformation into an extended conformation. During such low-pH-induced structural transitions, residues located in the domain-linker are found to play important roles in glycoprotein-mediated membrane fusion. Finally, the antibody interacts with RABV-G mainly through its heavy chain and binds to a bipartite conformational epitope in the viral protein for neutralization. These structures provide valuable information for vaccine and drug design.

Calcium Regulates S100A12 Zinc Sequestration by Limiting Structural Variations.

Antimicrobial proteins such as S100A12 and S100A8/A9 are highly expressed and secreted by neutrophils during infection and participate in human immune response by sequestering transition metals. At neutral pH, S100A12 sequesters Zn2+ with nanomolar affinity, which is further enhanced upon calcium binding. We investigated the pH dependence of human S100A12 zinc sequestration by using Co2+ as a surrogate. Apo-S100A12 exhibits strong Co2+ binding between pH 7.0 and 10.0 that progressively diminishes as the pH is decreased to 5.3. Ca2+ -S100A12 can retain nanomolar Co2+ binding up to pH 5.7. NMR spectroscopic measurements revealed that calcium binding does not alter the side-chain protonation of the Co2+ /Zn2+ binding histidine residues. Instead, the calcium-mediated modulation is achieved by restraining pH-dependent conformational changes to EF loop 1, which contains Co2+ /Zn2+ binding Asp25. This calcium-induced enhancement of Co2+ /Zn2+ binding might assist in the promotion of antimicrobial activities in humans by S100 proteins during neutrophil activation under subneutral pH conditions.

The dimeric ectodomain of the alkali-sensing insulin receptor–related receptor (ectoIRR) has a droplike shape

Insulin receptor-related receptor (IRR) is a receptor tyrosine kinase of the insulin receptor family and functions as an extracellular alkali sensor that controls metabolic alkalosis in the regulation of the acid-base balance. In the present work, we sought to analyze structural features of IRR by comparing them with those of the insulin receptor, which is its closest homolog but does not respond to pH changes. Using small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM), we investigated the overall conformation of the recombinant soluble IRR ectodomain (ectoIRR) at neutral and alkaline pH. In contrast to the well-known inverted U-shaped (or λ-shaped) conformation of the insulin receptor, the structural models reconstructed at different pH values revealed that the ectoIRR organization has a "droplike" shape with a shorter distance between the fibronectin domains of the disulfide-linked dimer subunits within ectoIRR. We detected no large-scale pH-dependent conformational changes of ectoIRR in both SAXS and AFM experiments, an observation that agreed well with previous biochemical and functional analyses of IRR. Our findings indicate that ectoIRR's sensing of alkaline conditions involves additional molecular mechanisms, for example engagement of receptor juxtamembrane regions or the surrounding lipid environment.

Ultrafast pH-jump two-dimensional infrared spectroscopy.

We present a pH-jump two-dimensional infrared (2D IR) spectrometer to probe pH-dependent conformational changes from nanoseconds to milliseconds. The design incorporates a nanosecond 355nm source into a pulse-shaper-based 2D IR spectrometer to trigger dissociation of a caged proton prior to probing subsequent conformational changes with femtosecond 2D IR spectroscopy. We observe a blue shift in the amide I mode (C═O stretch) of diglycine induced by protonation of the terminal amine. This method combines the bond-specific structural sensitivity of ultrafast 2D IR with triggered conformational dynamics, providing structural access to multiscale biomolecular transformations such as protein folding.

PH-Dependent Thermal Stability of Vibrio cholerae L-asparaginase.

pH is one of the decisive macromolecular properties of proteins that significantly affects enzyme structure, stability and reaction rate. Change in pH may protonate or deprotonate the side group of aminoacid residues in the protein, thereby resulting in changes in chemical and structural features. Hence studies on the kinetics of enzyme deactivation by pH are important for assessing the bio-functionality of industrial enzymes. L-asparaginase is one such important enzyme that has potent applications in cancer therapy and food industry. The objective of the study is to understand and analyze the influence of pH on deactivation and stability of Vibrio cholerae L-asparaginase. Kinetic studies were conducted to analyze the effect of pH on stability and deactivation of Vibrio cholerae L-asparaginase. Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) studies have been carried out to understand the pH-dependent conformational changes in the secondary structure of V. cholerae L-asparaginase. The enzyme was found to be least stable at extreme acidic conditions (pH< 4.5) and exhibited a gradual increase in melting temperature from 40 to 81 °C within pH range of 4.0 to 7.0. Thermodynamic properties of protein were estimated and at pH 7.0 the protein exhibited ΔG37of 26.31 kcal mole-1, ΔH of 204.27 kcal mole-1 and ΔS of 574.06 cal mole-1 K-1. The stability and thermodynamic analysis revealed that V. cholerae L-asparaginase was highly stable over a wide range of pH, with the highest stability in the pH range of 5.0-7.0.

Structural basis of the pH-dependent conformational change of the N-terminal region of human mannose receptor/CD206

Mannose receptor (MR, CD206) is an immune receptor highly expressed on macrophages and plays important roles in glycoprotein clearance, immune response and matrix turnover. Previous studies have shown that MR recognizes multiple ligands and recycles between cell surface and endosomes, and the conformation and ligand binding of MR are regulated by environmental pH. However, due to the lack of high-resolution details, the mechanisms of the pH-dependent properties of MR have not been fully understood. Here we investigate the pH-dependent conformational change of MR by solving a series of crystal structures of MR N-terminal fragments (CysR~CTLD2/3) at pH ranging from 4.0 to 8.5. The results show that the CTLD3 domain plays a critical role in regulating the conformational change of the N-terminal region of MR by forming interactions with the CTLD2 domain specifically at acidic pH. Moreover, the structural data also show the conformational changes of the 4-SO4-GalNAc binding pocket at the CysR domain, which might be relevant to the binding and release of the ligand. Overall, these results provide a model for the pH-dependent conformational change of the N-terminal region of MR that may help to understand its functional mechanism at molecular level.

The pH-dependent conformational change of eukaryotic translation initiation factor 5: Insights into partner-binding manner

In the process of eukaryotic translation, the formation of preinitiation complex 43S, which consists of a 40S subunit, the eIF2–GTP–Met-tRNAiMet ternary complex, eIF3, eIF1, eIF1A, and eIF5, is essential for translational quality control. Of those factors, eIF5 promotes the hydrolysis of eIF2-bound GTP to release eIF2-GDP in the complex for the recycling of eIF2. eIF5 appears to bind to the β subunit of eIF2 (eIF2β) via an interaction between aromatic/acidic residue-rich regions (AA-boxes) in the C-terminal domain of eIF5 (eIF5CTD) and three lysine clusters (K-boxes) in the N-terminal domain of eIF2β (eIF2βNTD). However, the details of this interaction are unclear, due to the lack of a structure for the eIF5-eIF2β complex, and the unavailability of an intact structure of eIF5, in which the AA-boxes are always disordered, with high flexibility. In this study, we solved two crystal structures of eIF5CTD from Candida albicans, which for the first time showed the AA-box2 of eIF5 presenting as an ordered helical structure. The structures exhibited different arrangements of AA-box2 under different pH values, which may reflect the dynamic nature of the interactions of eIF5CTD, and eIF2βNTD in the preinitiation complex.

Quantitative Photo-crosslinking Mass Spectrometry Revealing Protein Structure Response to Environmental Changes.

Protein structures respond to changes in their chemical and physical environment. However, studying such conformational changes is notoriously difficult, as many structural biology techniques are also affected by these parameters. Here, the use of photo-crosslinking, coupled with quantitative crosslinking mass spectrometry (QCLMS), offers an opportunity, since the reactivity of photo-crosslinkers is unaffected by changes in environmental parameters. In this study, we introduce a workflow combining photo-crosslinking using sulfosuccinimidyl 4,4′-azipentanoate (sulfo-SDA) with our recently developed data-independent acquisition (DIA)-QCLMS. This novel photo-DIA-QCLMS approach is then used to quantify pH-dependent conformational changes in human serum albumin (HSA) and cytochrome C by monitoring crosslink abundances as a function of pH. Both proteins show pH-dependent conformational changes resulting in acidic and alkaline transitions. 93% and 95% of unique residue pairs (URP) were quantifiable across triplicates for HSA and cytochrome C, respectively. Abundance changes of URPs and hence conformational changes of both proteins were visualized using hierarchical clustering. For HSA we distinguished the N–F and the N–B form from the native conformation. In addition, we observed for cytochrome C acidic and basic conformations. In conclusion, our photo-DIA-QCLMS approach distinguished pH-dependent conformers of both proteins.