Aromatic Amino Acids In Proteins Research Articles

Stabilizing Role of Water Solvation on Anion−π Interactions in Proteins

In this work, anion−π interactions between sulfate groups (SO42–) and protein aromatic amino acids (AAs) (histidine protonated (HisP), histidine neutral (HisN), tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe)) in an aqueous environment have been analyzed using quantum chemical (QC) calculations and molecular dynamics (MD) simulations. Sulfates can occur naturally in solution and can be contained in biomolecules playing relevant roles in their biological function. In particular, the presence of sulfate groups in glycosaminoglycans such as heparin and heparan sulfate has been shown to be relevant for protein and cellular communication and, consequently, for tissue regeneration. Therefore, anion−π interactions between sulfate groups and aromatic residues represent a relevant aspect to investigate. QC results show that such an anion−π mode of interaction between SO42– and aromatic AAs is only possible in the presence of water molecules, in the absence of any other cooperative non-covalent interactions. Protonated histidine stands out in terms of its enhancement in the magnitude of interaction strength on solvation. Other AAs such as non-protonated histidine, tyrosine, and phenylalanine can stabilize anion−π interactions on solvation, albeit with weak interaction energy. Tryptophan does not exhibit any anion−π mode of interaction with SO42–. The order of magnitude of the interaction of aromatic AAs with SO42– on microsolvation is HisP > HisN > Tyr > Trp > Phe. Atoms in molecules (AIM) analysis illustrates the significance of water molecules in stabilizing the divalent SO42– anion over the π surface of the aromatic AAs. MD simulation analysis shows that the order of magnitude of the interaction of SO42– with aromatic AAs in macroscopic solvation is HisP > HisN, Tyr, Trp > Phe, which is very much in line with the QC results. Spatial distribution function analysis illustrates that protonated histidine alone is capable of establishing the anion−π interaction with SO42– in the solution phase. This study sheds light on the understanding of anion−π interactions between SO42– and aromatic AAs such as His and Tyr observed in protein crystal structures and the significance of water molecules in stabilizing such interactions, which is not feasible otherwise.

Modification of insulin amyloid aggregation by Zr phthalocyanines functionalized with dehydroacetic acid derivatives.

Amyloid fibrils are widely studied both as target in conformational disorders and as basis for the development of protein-based functional materials. The three Zr phthalocyanines bearing dehydroacetic acid residue (PcZr(L1)2) and its condensed derivatives (PcZr(L2)2 and PcZr(L3)2) as out-of-plane ligands were synthesized and their influence on insulin fibril formation was studied by amyloid-sensitive fluorescent dye based assay, scanning electron microscopy, fluorescent and absorption spectroscopies. The presence of Zr phthalocyanines was shown to modify the fibril formation. The morphology of fibrils formed in the presence of the Zr phthalocyanines differs from that of free insulin and depends on the structure of out-of-plane ligands. It is shown that free insulin mostly forms fibril clusters with the length of about 0.3–2.1 μm. The presence of Zr phthalocyanines leads to the formation of individual 0.4–2.8 μm-long fibrils with a reduced tendency to lateral aggregation and cluster formation (PcZr(L1)2), shorter 0.2–1.5 μm-long fibrils with the tendency to lateral aggregation without clusters (PcZr(L2)2), and fibril-like 0.2–1.0 μm-long structures (PcZr(L3)2). The strongest influence on fibrils morphology made by PcZr(L3)2 could be explained by the additional stacking of phenyl moiety of the ligand with aromatic amino acids in protein. The evidences of binding of studied Zr phthalocyanines to mature fibrils were shown by absorption spectroscopy (for PcZr(L1)2 and PcZr(L2)2) and fluorescent spectroscopy (for PcZr(L3)2). These complexes could be potentially used as external tools allowing the development of functional materials on protein fibrils basis.

Carbohydrate – Protein aromatic ring interactions beyond CH/π interactions: A Protein Data Bank survey and quantum chemical calculations

The geometries of the contacts between monosaccharides and aromatic rings of amino acids found in X-ray crystallography structures, in the Protein Data Bank (PDB), were analyzed, while the energies of the interactions were calculated using quantum chemical method. We found 1913 sugar/aromatic ring contacts, 1054 of them (55%) with CH/π interactions and 859 of them (45%) without CH/π interactions. We showed that only the carbohydrate/aromatic contacts with CH/π interactions are preferentially parallel and enable sliding in the plane parallel to aromatic ring. The calculated interaction energies in systems with CH/π interactions are in the range from −1.7 kcal/mol to −6.8 kcal/mol, while in the systems without CH/π interactions are in the range −0.2 to −3.2 kcal/mol. Hence, the binding that does not include CH/π interactions, can also be important for aromatic amino acid and carbohydrate binding processes, since some of these interactions can be as strong as the CH/π interactions. At the same time, these interactions can be weak enough to enable releasing of small carbohydrate fragments after the enzymatic reaction. The analysis of the protein-substrate patterns showed that every second or third carbohydrate unit in long substrates stacks with protein aromatic amino acids.

Gender differences in the fluorescence of human skin in young healthy adults.

Human skin naturally contains many endogenous fluorophores; therefore, fluorescence techniques can be used for monitoring of the human skin even in in vivo mode. The aim of this work was to study skin autofluorescence in vivo regarding the possible effect of gender. Fluorescence emission spectra of young healthy Caucasian adults in 3 anatomical regions (forehead, hand, and inner upper arm) were taken with excitation at 280, 325, or 400nm. Three emission bands were found in the spectra for both men and women: (1) an intensive band peaked at 340/280nm (peak emission/excitation wavelength), corresponding to aromatic amino acids of proteins in epidermis; (2) a broad band with emission between 360 nm and 480 nm (excitation 325nm) with a base peak around 390nm and 2 side peaks at 420 and 450nm, mainly due to collagen cross-links in dermis with a possible weak contribution of elastin and mitochondrial NADPH; (3) a weak but distinct peak at 600/400nm corresponding presumably to skin unmetalled porphyrins. The intensity of skin autofluorescence showed differences between genders and among anatomical regions. The 340 nm intensity was 1.4 times higher in the male group in all 3 anatomical regions studied. The highest intensity of skin autofluorescence for the peaks at 340/280nm and 600/400nm was found on the forehead, whereas the 390/325nm band was most intensive on the inner upper arm in both genders.

Enhanced multiplexing in mass cytometry using osmium and ruthenium tetroxide species.

Mass cytometry facilitates high-dimensional, quantitative, single-cell analysis. The method for sample multiplexing in mass cytometry, called mass-tag cellular barcoding (MCB), relies on the covalent reaction of bifunctional metal chelators with intracellular proteins. Here, we describe the use of osmium and ruthenium tetroxides (OsO4 and RuO4 ) that bind covalently with fatty acids in the cellular membranes and aromatic amino acids in proteins. Both OsO4 and RuO4 rapidly reacted and allowed for MCB with live cells, crosslinked cells, and permeabilized cells. Given the covalent nature of the labeling reaction, isotope leaching was not observed. OsO4 and RuO4 were used in a 20-sample barcoding protocol together with palladium isotopes. As mass channels occupied by osmium and ruthenium are not used for antibody detection the number of masses effectively utilized in a single experiment is expanded. OsO4 and RuO4 can therefore be used as MCB reagents for a wide range of mass cytometry workflows. © 2016 International Society for Advancement of Cytometry.

Aromatic spectral editing techniques for magic-angle-spinning solid-state NMR spectroscopy of uniformly 13C-labeled proteins

The four aromatic amino acids in proteins, namely histidine, phenylalanine, tyrosine, and tryptophan, have strongly overlapping 13C chemical shift ranges between 100 and 160ppm, and have so far been largely neglected in solid-state NMR determination of protein structures. Yet aromatic residues play important roles in biology through π–π and cation–π interactions. To better resolve and assign aromatic residues' 13C signals in magic-angle-spinning (MAS) solid-state NMR spectra, we introduce two spectral editing techniques. The first method uses gated 1H decoupling in a proton-driven spin-diffusion (PDSD) experiment to remove all protonated 13C signals and retain only non-protonated carbon signals in the aromatic region of the 13C spectra. The second technique uses chemical shift filters and 1H–13C dipolar dephasing to selectively detect the Cα, Cβ and CO cross peaks of aromatic residues while suppressing the signals of all aliphatic residues. We demonstrate these two techniques on amino acids, a model peptide, and the microcrystalline protein GB1, and show that they significantly simplify the 2D NMR spectra and both reveal and permit the ready assignment of the aromatic residues' signals.

Theoretical insight into sulfur–aromatic interactions with extension to D2 receptor activation mechanism

Contacts between aromatic rings and sulfur-containing amino acids are frequent in proteins. However, little is known about the nature of their interactions particularly if substituents are present on aromatic ring. In this paper, DFT quantum chemical calculations were used to study substituted benzenes in complex with hydrogen sulfide (H2S), methanethiol (CH3SH), and (Methylsulfanyl)methane (CH3SCH3). It was found that SH···π interaction is more stabilizing than the S···π interaction in the case of benzene, but this is changed with increasing electronegativity of the substituent on benzene ring. Although the change of energy of SH···π and S···π interaction follows the conventional model of substituent effect, where S···π interactions are maximized and SH···π interactions are diminished with electron-withdrawing substituent on benzene as a result of changes in the aryl π-system, it was found that it is mainly a consequence of direct electrostatic interaction between substituent and the sulfur-containing molecule. We also investigated the model system of Cys···Trp interaction, adjacent to a cluster of aromatic amino acids, in proteins, using explicit membrane molecular dynamics simulations results of D3 dopamine receptor crystal structure as starting point. It was found that fluorination in aromatic cluster enhances the Cys···Trp interaction. The effect is maximized when transferred through the rest of aromatic system suggesting possible explanation for frequent contacts between sulfur-containing and aromatic amino acids in proteins and their effects on protein folding and stabilization.

Preferential interactions between protein and arginine: Effects of arginine on tertiary conformational and colloidal stability of protein solution

The purpose of this study was to better understand the preferential binding behavior of arginine to protein as well as the impact of arginine on the conformational and colloidal stability of protein solution. Physical stabilities of model proteins, bovine serum albumin (BSA) and ovalbumin (OVA), were investigated by fluorescence-based and dynamic light scattering techniques in the absence and presence of arginine. We investigated the interactions between arginine and tryptophan or tyrosine residues by conducting solubility and fluorescence studies of two amino acid derivatives, N-acetyl-l-tryptophanamide (NATA) and N-acetyl-l-tyrosinamide (NAYA), in arginine solutions. The result showed that arginine preferentially bond to the aromatic amino acids of proteins mainly through hydrogen bonds and Van der Waals’ forces, while the binding constant K of arginine with BSA and OVA at 298K was 41.92 and 5.77L/mol, respectively. The fluorescence quenching, the decreased fluorescence lifetime and the red-shifted ANS peak position revealed that arginine perturbed the local environment of tryptophan and tyrosine residues. We also found the attenuated electrostatic repulsion among BSA and OVA molecules after adding arginine. These findings provided strong evidence that arginine possessed negative effects on tertiary conformational and colloidal stability of BSA and OVA during the preferential binding process.

Photoacoustic spectroscopy of aromatic amino acids in proteins

This paper concerns the use of photoacoustic spectroscopy (PAS) to study the presence of aromatic amino acid in proteins. We examined the aromatic amino acids in six proteins with well-known structures using absorption spectra of near ultraviolet PAS over the wavelength range 240-320 nm. The fundamental understanding of the physical and chemical properties that govern the absorption of light and a subsequent release of heat to generate a transient pressure wave was used to test the concept of monitoring aromatic amino acids with this method. Second derivative spectroscopy in the ultraviolet region of proteins was also used to study the regions surrounding the aromatics and the percentage area in each band was related in order to determine the contribution in function of the respective molar extinction coefficients for each residue. Further investigation was conducted into the interaction between sodium dodecyl sulphate (SDS) and bothropstoxin-I (BthTx-I), with the purpose of identifying the aromatics that participate in the interaction. The clear changes in the second derivative and curve-fitting procedures suggest that initial SDS binding to the tryptophan located in the dimer interface and above 10 SDS an increased intensity between 260 and 320 nm, demonstrating that the more widespread tyrosine and phenylalanine residues contribute to the SDS/BthTx-I interactions. These results demonstrate the potential of near UV-PAS for the investigation of membrane proteins/detergent complexes in which light scattering is significant.

Role of aromatic amino acids in protein–nucleic acid recognition

Statistical analysis of structures from the PBD has been used to examine the role that the aromatic amino acids play in protein-nucleic acid recognition. In protein-DNA complexes, the residues Phe and His are found to bind selectively to the DNA chain--Phe to A and T, and His to T and G. The preferred binding modes are identified, and the interactions involving Phe are shown to be important in the transcription process. In protein-RNA complexes, Phe is found to occur far less often and is instead replaced by Trp, which binds selectively to C and G, offering a possible mechanism for differentiation between the two nucleic acids. SASA analysis of the two sets of complexes suggests that all of the aromatic amino acids are more heavily involved in binding than would be expected on the balance of probability. Phe and Tyr occur approximately equal in both sets of data, whereas the proportions of His and Trp vary considerably, supporting the idea that these residues may be involved in differentiating between the two nucleic acids.

Isolation and characterization of estuarine dissolved organic matter: Comparison of ultrafiltration and C 18 solid-phase extraction techniques

Characterization of dissolved organic matter (DOM) from aquatic environments has always been constrained by the ability to obtain a representative fraction of the DOM pool for analysis. Ultrafiltration or extraction, commonly using XAD or C 18 sorbents, is therefore generally used to concentrate and desalt DOM samples for further analyses. In this study, we compared ultrafiltration and C 18 solid-phase extraction disks (SPE) as DOM isolation methods for estuarine samples. We also evaluated the use of the C 18 SPE disks to isolate low-molecular-weight DOM (LMW-DOM) in the filtrate from ultrafiltration. The isolates from both methods and the LMW-DOM C 18-extracts were characterized using Fourier transform infrared spectroscopy (FTIR) and direct temperature-resolved mass spectrometry (DT-MS). Based on mass balance and blank measurements, we found that the C 18 SPE disks can be used to isolate bulk DOM and LMW-DOM from estuarine samples. FTIR and DT-MS analysis show that C 18-extracted DOM and ultrafiltered high-molecular-weight DOM (HMW-DOM) differ markedly in chemical composition. The HMW-DOM is enriched in (degraded) polysaccharides along with aminosugars when compared with the C 18-extracted DOM. The C 18-extracted DOM appears enriched in aromatic compounds, probably from lignin and/or aromatic amino acids in proteins. C 18 SPE of LMW-DOM samples from ultrafiltration increases the recovery of DOM from the total sample up to about 70%, compared to around 50% using ultrafiltration alone. Thus, a majority of the DOM can be isolated from estuarine samples by a combination of these techniques.

Trace analysis of oxidized, nitrated, and chlorinated aromatic amino acids by capillary electrophoresis with electroosmotic flow modification allowing large-volume sample stacking

A capillary electrophoresis method has been developed for the simultaneous analysis of the oxidized, nitrated, and chlorinated aromatic amino acids, as well as their parent compounds. These modifications of the aromatic amino acids in proteins or free form are induced by the attack of reactive, mainly free radical species generated during cell stress, and these stable products may serve as biomarkers of cell damage. The analytes tyrosine, phenylalanine, dihydroxyphenylalanine, tryptophan, 3-nitrotyrosine, 3-chlorotyrosine, ortho-tyrosine, meta-tyrosine, 3-hydroxyphenylacetic acid (internal standard 1), and alpha-methyltyrosine (internal standard 2) were separated in their anionic forms in alkaline borate buffer. The polyamine spermine was used as electroosmotic flow (EOF) modifier. Adsorbing to the capillary wall, spermine can either suppress or even reverse the EOF depending on its concentration and the pH. The effects of the pH of the separation buffer, the spermine concentration, the temperature, and the applied field strength on the separation were examined. The modified aromatic amino acids are present in biological fluids in a much lower concentration than their parent compounds, thus high detection sensitivity of the analytical method is required. To achieve good detection sensitivity, field-amplified sample stacking of large injection volumes was applied. Omitting polyamine from the sample buffer allowed local reversal of the EOF, thus removal of the low conductivity sample buffer at the capillary inlet. In this way, 100% of the capillary to the detection window could be filled with the sample, and the detection limits achieved for the modified aromatic amino acids were in the range of 2.5-10 nM.

Protective Effects of N-Acetylcysteine and Vitamin E Derivative U83836E on Proteins Modifications Induced by Methanol Intoxication

Methanol is oxidized into the formaldehyde and formate and these processes are accompanied by free radicals' generation. Formaldehyde and free radicals induce chemical modifications of proteins, leading to changes in their structure and function. The aim of this paper has been to evaluate the effect of N-acetylcysteine and vitamin E derivative U83836E on free radicals' generation and protein modifications induced during acute methanol intoxication. U83836E is an analog of α-tocopherol and similarly protects cells against oxidative damage. Moreover, this compound has hydrophilic properties and can be dissolved in an aqueous phase of blood and interstitial fluid, and next, membranes readily take it up. This compound belonging to the benzopyran family contains the reactive trolox ring and possesses antioxidant properties. The ESR determination indicates the increase in free radicals' signal 6 and 12 h after intoxication. Methanol ingestion causes a significant decrease in GSH level (by about 35%) and a significant increase in the lipid peroxidation product malondialdehyde (by about 25%). During methanol metabolism the aromatic amino acids of proteins are modified—the amount of carbonyl groups is increased (by about 42%) and fluorescence intensity of tryptophan is statistically decreased (by about 30%). The increase (by about 200%) in bityrosine fluorescence is also observed. Moreover, a significant decrease in free sulphydryl (by about 40%) and amino groups (by about 30%) in liver proteins is observed during intoxication. This is accompanied by the loss of lysosomal protease-cathepsin B activity (by about 25%). N-acetylcysteine (in dose 150 mg/kg body weight) and U83836E (in dose 10 mg/kg body weight) prevent free radicals' generation to a similar degree. U83836E protects membrane phospholipids against peroxidation a little stronger than N-acetylcysteine (concentration of MDA is decreased by 9 to 20% in the U83836 group and by 7 to 14% in the N-acetylcysteine group compared to methanol group). However after treating methanol-intoxicated rats with N-acetylcysteine, the changes in protein modification parameters are significantly smaller than in the group receiving methanol alone and they are a little smaller than after U83836E application.These findings suggest that N-acetylcysteine and to a smaller degree U83836E protect protein from modification in methanol intoxication, which can prevent liver pathologies.

The nitration of proteins in platelets

Nitric oxide has many important physiological functions, but it may also form an important oxidant, peroxynitrite, as a consequence of its reaction with superoxide anions. Peroxynitrite is capable of nitrating the aromatic amino acids in proteins, particularly tyrosine. Nitrated proteins are found in tissues of a variety of diseases where inflammation occurs. However, our recent work suggests that more selective nitration of specific proteins may occur during normal physiological processes, such as platelet activation by collagen. It is not yet clear what role this may play in the normal cell biology, but there is potential to be a role in signal transduction mechanisms, possibly by influencing tyrosine phosphorylation or dephosphorylation.

New 224 nm Hollow Cathode Laser-UV Raman Spectrometer

We have developed an optimized high-throughput UV Raman spectrometer that utilizes a simple, inexpensive new 224.3 nm hollow cathode laser. This quasi-continuous wave (CW) 224.3 nm laser can be used to detect sub-ppm concentrations of aromatic and polycyclic aromatic hydrocarbons in aqueous solutions. This excitation is also useful for studying aromatic amino acids in proteins. We demonstrate the utility of this spectrometer to study the environments of tyr and trp in horse heart myoglobin.

π-Stacking Interactions: ALIVE AND WELL IN PROTEINS

A representative set of high resolution x-ray crystal structures of nonhomologous proteins have been examined to determine the preferred positions and orientations of noncovalent interactions between the aromatic side chains of the amino acids phenylalanine, tyrosine, histidine, and tryptophan. To study the primary interactions between aromatic amino acids, care has been taken to examine only isolated pairs (dimers) of amino acids because trimers and higher order clusters of aromatic amino acids behave differently than their dimer counterparts. We find that pairs (dimers) of aromatic side chain amino acids preferentially align their respective aromatic rings in an off-centered parallel orientation. Further, we find that this parallel-displaced structure is 0.5-0.75 kcal/mol more stable than a T-shaped structure for phenylalanine interactions and 1 kcal/mol more stable than a T-shaped structure for the full set of aromatic side chain amino acids. This experimentally determined structure and energy difference is consistent with ab initio and molecular mechanics calculations of benzene dimer, however, the results are not in agreement with previously published analyses of aromatic amino acids in proteins. The preferred orientation is referred to as parallel displaced pi-stacking.

Assigning the NMR spectra of aromatic amino acids in proteins: analysis of two Ets pointed domains

The measurement of interproton nuclear Overhauser enhancements (NOEs) and dihedral angle restraints of aromatic amino acids is a critical step towards determining the structure of a protein. The complete assignment of the resonances from aromatic rings and the subsequent resolution and identification of their associated NOEs, however, can be a difficult task. Shown here is a strategy for assigning the 1H, 13C, and 15N signals from the aromatic side chains of histidine, tryptophan, tyrosine, and phenylalanine using a suite of homo- and hetero-nuclear scalar and NOE correlation experiments, as well as selective deuterium isotope labelling. In addition, a comparison of NOE information obtained from homonuclear NOE spectroscopy (NOESY) and 13C-edited NOESY - heteronuclear single quantum correlation experiments indicates that high-resolution homonuclear two-dimensional NOESY spectra of selectively deuterated proteins are invaluable for obtaining distance restraints to the aromatic residues.Key words: NMR assignment, aromatic residue, transcription factor, NOE, dihedral angle.

Assigning the NMR spectra of aromatic amino acids in proteins: analysis of two Ets pointed domains.

The measurement of interproton nuclear Overhauser enhancements (NOEs) and dihedral angle restraints of aromatic amino acids is a critical step towards determining the structure of a protein. The complete assignment of the resonances from aromatic rings and the subsequent resolution and identification of their associated NOEs, however, can be a difficult task. Shown here is a strategy for assigning the 1H, 13C, and 15N signals from the aromatic side chains of histidine, tryptophan, tyrosine, and phenylalanine using a suite of homo- and hetero-nuclear scalar and NOE correlation experiments, as well as selective deuterium isotope labelling. In addition, a comparison of NOE information obtained from homonuclear NOE spectroscopy (NOESY) and 13C-edited NOESY-heteronuclear single quantum correlation experiments indicates that high-resolution homonuclear two-dimensional NOESY spectra of selectively deuterated proteins are invaluable for obtaining distance restraints to the aromatic residues.

Theoretical study of binding of tetramethylammonium ion with aromatics

Ab initio computations including correlation have been performed in a comparative study of complexes of tetramethylammonium (TMA) with benzene, pyrrole, pyridine, and imidazole, using polarized Gaussian basis sets of different accuracies. With the best basis (optimized on molecular polarizabilities), the BSSE-corrected binding energies in the most stable complexes of these four ligands are 9.1, 10.7, 13.3, and 16.3 kcal/mol, respectively, with benzene and pyrrole binding in a plane perpendicular to the TMA axis, and pyridine and imidazole inserting their nitrogen lone pair essentially along the TMA axis. The characteristics of secondary sites of binding of benzene are also determined and the overall results are discussed in connection with the possible role of aromatic amino acids in proteins. © 1997 John Wiley & Sons, Inc. J Comput Chem 18: 2012–2022, 1997

Transient Changes of the Conformation of Hemagglutinin of Influenza Virus at Low pH Detected by Time-resolved Circular Dichroism Spectroscopy

Membrane fusion of influenza virus is mediated by a conformational change of the viral membrane protein hemagglutinin (HA) triggered by low pH. By near UV CD spectroscopy, which is sensitive to the arrangement and mobility of aromatic amino acids in proteins, we have monitored continuously with a time resolution of 5 s the kinetics of structural alterations of the ectodomain of HA isolated from different influenza virus strains (H1 (A/PR 8/34), H2 (A/Japan), and H3 (X31)). To establish a functional correlation to structural alterations of the HA ectodomain reflected by the CD, we have measured the kinetics of the virus-erythrocyte fusion and of the inactivation of fusion by low pH preincubation of viruses. At acidic pH we found a multiphasic behavior of the CD signal recorded at 283 nm. Upon lowering the pH we detected first an increase of the CD amplitude, which is associated with the formation of a fusion-competent state of HA. The initial increase was followed by a continuous decline of CD amplitude, which can be ascribed to a transformation into a fusion-inactivated conformation that is in its early phase reversible as found for A/Japan. The half-time of the different phases of the CD signal depended on the virus strain, the temperature, and the acidic pH. The results support recent hypotheses that the fusion-competent conformation is an intermediate of the fusion-inactivated structure of HA.