Structure-sweetness relationship in egg white lysozyme: role of lysine and arginine residues on the elicitation of lysozyme sweetness

Lysozyme is one of the sweet-tasting proteins. To clarify the structure-sweetness relationship and the basicity-sweetness relationship in lysozyme, we have generated lysozyme mutants with Pichia systems. Alanine substitution of lysine residues demonstrated that two out of six lysine residues, Lys13 and Lys96, are required for lysozyme sweetness, while the remaining four lysine residues do not play a significant role in the perception of sweetness. Arginine substitution of lysine residues revealed that the basicity, but not the shape, of the side chain plays a significant role in sweetness. Single alanine substitutions of arginine residues showed that three arginine residues, Arg14, Arg21, and Arg73, play significant roles in lysozyme sweetness, whereas Arg45, Arg68, Arg125 and chemical modification by 1,2-cyclohexanedione did not affect sweetness. From investigation of the charge-specific mutations, we found that the basicity of a broad surface region formed by five positively charged residues, Lys13, Lys96, Arg14, Arg21, and Arg73, is required for lysozyme sweetness. Differences in the threshold values among sweet-tasting proteins might be caused by the broadness and/or the density of charged residues on the protein surface.     

Critical molecular regions for elicitation of the sweetness of the sweet-tasting protein, thaumatin I

Thaumatin is an intensely sweet-tasting protein. To identify the critical amino acid residue(s) responsible for elicitation of the sweetness of thaumatin, we prepared mutant thaumatin proteins, using Pichia pastoris, in which alanine residues were substituted for lysine or arginine residues, and the sweetness of each mutant protein was evaluated by sensory analysis in humans. Four lysine residues (K49, K67, K106 and K163) and three arginine residues (R76, R79 and R82) played significant roles in thaumatin sweetness. Of these residues, K67 and R82 were particularly important for eliciting the sweetness. We also prepared two further mutant thaumatin I proteins: one in which an arginine residue was substituted for a lysine residue, R82K, and one in which a lysine residue was substituted for an arginine residue, K67R. The threshold value for sweetness was higher for R82K than for thaumatin I, indicating that not only the positive charge but also the structure of the side chain of the arginine residue at position 82 influences the sweetness of thaumatin, whereas only the positive charge of the K67 side chain affects sweetness.     

Understanding the role of internal lysine residues of serum albumins in conformational stability and bilirubin binding

The role of internal lysine residues of different serum albumins, viz. from human, rabbit, goat, sheep and buffalo (HSA, RbSA, GSA, SSA and BuSA), in conformational stability and bilirubin binding was investigated after blocking them using acetylation, succinylation and guanidination reactions. No significant change in the secondary structure was noticed whereas the tertiary structure of these proteins was slightly altered upon acetylation or succinylation as revealed by circular dichroism (CD), fluorescence and gel filtration results. Guanidination did not affect the native protein conformation to a measurable extent. Scatchard analysis, CD and absorption spectroscopic results showed marked reductions (5-21-fold decrease in K(a) and approximately 50% decrease in the CD Cotton effect intensity) in the affinity of albumins for bilirubin upon acetylation or succinylation whereas guanidination produced a small change. Interestingly, monosignate CD spectra of bilirubin complexed with GSA, SSA and BuSA were transformed to bisignate CD spectra upon acetylation or succinylation of internal lysine residues whereas spectra remained bisignate in the case of bilirubin bound to acetylated or succinylated derivatives of HSA and RbSA. When probed by CD spectroscopy, bilirubin bound to acetylated or succinylated derivatives of GSA and SSA rapidly switched over to native albumins and not vice versa. These results suggested that salt linkage(s) contributed by internal lysine residue(s) play an important role in the high-affinity binding of bilirubin to albumin and provide stability to the native three-dimensional conformation of the bound pigment. Chloroform severely decreased the intensity of both positive and negative CD Cotton effects of bilirubin complexed with acetylated or succinylated derivatives of all albumins which otherwise increased significantly in the case of bilirubin complexed with native and guanidinated albumin derivatives, except the bilirubin-RbSA complex which showed a small decrease in intensity. These results suggest that the presence of salt linkage(s) in bilirubin-albumin complexation is(are) crucial to bring about effective and efficient stereochemical changes in the bound pigment by co-binding of chloroform which seems to have at least one conserved binding site on these albumins that is shared with bilirubin.     

Chemical modification of bovine pancreatic trypsin inhibitor for single site coupling of immunogenic peptides for NMR conformational analysis

Procedures for chemical modification of bovine pancreatic trypsin inhibitor (BPTI) to allow site-specific coupling of immunogenic peptides are reported. Each of the modified proteins has a single free amino group; the other amino groups of lysine or the amino terminus are blocked by acetylation or guanidination. Two of the derivatives were prepared by protecting Lys-15 by complexation with trypsin or chymotrypsin during acetylation with N-hydroxysuccinimide acetate or guanidination with 3,5-dimethylpyrazole-1-carboxamidine nitrate. A third derivative with a free amino group at the amino terminus was prepared by guanidination of the 4 lysine residues with o-methylisourea. The purity and structural integrity of the modified proteins was checked by NMR spectroscopy. Cysteine-containing peptides can be coupled to the single free amino group using several heterobifunctional linking reagents. N-Succinimidyl 3-(2-pyridyldithio)propionate is the most satisfactory coupling reagent for NMR studies because of its high specificity. Two-dimensional NMR spectroscopy shows that the conformation of the modified proteins is almost identical with that of native BPTI. The BPTI derivatives are suitable for use as models for NMR investigations of the conformation of immunogenic peptides conjugated to a carrier protein.     

Hydrogen-deuterium exchange studies on guanidinated pig heart lactate dehydrogenase

Pig heart lactate dehydrogenase becomes more thermostable on increasing the degree of guanidination (conversion of lysine to homoarginine) (Minotani, N., Sekiguchi, T., Bautista, J.G. and Nosoh, Y. (1979) Biochim. Biophys. Acta 581, 334-341). The conformational change of the protein on guanidination was then examined by hydrogen-deuterium (H-2H) exchange reactions. It ws found that (i) the fluctuation degrees of peptides and tyrosine and tryptophan residues in the protein decrease in that order, (ii) two H-2H exchangeable tryptophan residues per subunit are freely accessible to solvent and the fluctuation degrees of the residues does not change on guanidination, (iii) the H-2H exchange detectable tyrosine residues are not freely accessible to solvent and become less fluctuating when 15 lysine residues per subunit are guanidinated, and (iv) the peptides become much less fluctuating on increasing the degree of guanidination. The specific activity of the enzyme decreased on guanidination. The increased thermostability of the protein on guanidination may be related to the decrease in flexibility of the molecular structure by sacrificing the enzyme activity.     

In-gel derivatization of proteins for cysteine-specific cleavages and their analysis by mass spectrometry

As a potential tool for proteomics and protein characterization, in-gel cysteine- and arginine-specific cleavage is demonstrated by means of trypsin or endoproteinase Lys-C for six model proteins (lysozyme, alpha-lactalbumin, beta-lactoglobulin, ribonuclease A, albumin, and transferrin), ranging in size from 14 kDa to 79 kDa. Chemical modifications of cysteine (aminoethylation with bromoethylamine or N-(iodoethyl)-trifluoroacetamide, and subsequent guanidination) and lysine (acetylation) prior to tryptic digestion releases peptides delineated by cysteine or arginine residues. Peptide products are analyzed by MALDI-TOF-MS, ESI-MS, and ESI- and MALDI-MS/MS (with a quadrupole time-of-flight instrument). Complications induced by acrylamide alkylations of cysteines were avoided by substituting lower pH bis-tris polyacrylamide gels for tris-glycine. Sequence coverages from 35 to 86% were obtained and amino acid compositions of generated peptides could be confirmed by comprehensive y- and b-ion series. Detailed information about, in particular, cysteine rich proteins after gel electrophoresis were obtained. The chemistries for modification and cleavage specificities at cysteine residues provide an alternative means to characterize and identify proteins separated by gel electrophoresis.     

Structure-sweetness relationship in thaumatin: importance of lysine residues

To clarify the structural basis for the sweetness of thaumatin I, lysine-modified derivatives and carboxyl-group-modified derivatives were prepared by chemical modification followed by chromatographic purification. The sweetness of derivatives was evaluated by sensory analysis. Phosphopyridoxylation of lysine residues Lys78, Lys97, Lys106, Lys137 and Lys187 markedly reduced sweetness. The intensity of sweetness was returned to that of native thaumatin by dephosphorylation of these phosphopyridoxylated lysine residues except Lys106. Pyridoxamine modification of the carboxyl group of Asp21, Glu42, Asp60, Asp129 or Ala207 (C-terminal) did not markedly change sweetness. Analysis by far-UV circular dichroism spectroscopy indicated that the secondary structure of all derivatives remained unchanged, suggesting that the loss of sweetness was not a result of major disruption in protein structure. The five lysine residues, modification of which affected sweetness, are separate and spread over a broad surface region on one side of the thaumatin I molecule. These lysine residues exist in thaumatin, but not in non-sweet thaumatin-like proteins, suggesting that these lysine residues are required for sweetness. These lysine residues may play an important role in sweetness through a multipoint interaction with a putative thaumatin receptor.     

The Binding Groups in Ovomucin-lysozyme Interaction

The ovomucin-lysozyme aggregation was remarkably affected by pH or ionic strength. The extent of interaction of F-ovomucin with lysozyme was much larger than that of S-ovomucin. The ovomucin-lysozyme interaction decreased correspondingly, at a rate depending on the time at which ovomucin was modified by neuraminidase. On the other hand, the ovomucin-lysozyme interaction disappeared completely by acetylation, succinilation, or carbamylation of lysyl ε-amino groups in lysozyme, but it was not greatly affected by guanidination of lysyl ε-amino groups in lysozyme. From these results, it was confirmed that the electrostatic interaction between the negative charges of the terminal sialic acid in ovomucin and the positive charges of lysyl ε-amino groups in lysozyme is essential for the ovomucin-lysozyme interaction.     

EFFECT OF ACETYLATION AND METHYLATION ON THE SWEETNESS INTENSITY OF THAUMATIN I

The lysine residues in thaumatin I were chemically modifiedby acetylation with acetic anhydride and by reductive methylation,under various conditions. The acetylated and methylated thaumatinswere isolated by ion-exchange chromatography. The number ofremaining free amino groups was determined by trinitrophenylation. At least four acetylated thaumatins with either one, two, threeor four acetylated amino groups were obtained as well as onemethylated thaumatin with six dimethyl lysine residues and onemonomethyl lysine residue. The sweetness intensity of the acetylated thaumatins decreasedwith the increasing number of acetylated amino groups; the sweettaste had disappeared completely when four amino groups wereacetylated. The methylated thaumatin with seven modified lysineresidues had a sweetness intensity practically equal to thatof the original thaumatin. The total net change, i.e. the isoelectric point of thaumatin,might play a role in the physiological behaviour of thaumatincausing a sweet taste sensation.     

Electrostatic interactions play an essential role in the binding of oleic acid with α‐lactalbumin in the HAMLET‐like complex: A study using charge‐specific chemical modifications

H uman α ‐lactalbumin m ade le thal to t umor cells (HAMLET) and its analogs are partially unfolded protein‐oleic acid (OA) complexes that exhibit selective tumoricidal activity normally absent in the native protein itself. To understand the nature of the interaction between protein and OA moieties, charge‐specific chemical modifications of lysine side chains involving citraconylation, acetylation, and guanidination were employed and the biophysical and biological properties were probed. Upon converting the original positively‐charged lysine residues to negatively‐charged citraconyl or neutral acetyl groups, the binding of OA to protein was eliminated, as were any cytotoxic activities towards osteosarcoma cells. Retention of the positive charges by converting lysine residues to homoarginine groups (guanidination); however, yielded unchanged binding of OA to protein and identical tumoricidal activity to that displayed by the wild‐type α‐lactalbumin‐oleic acid complex. With the addition of OA, the wild‐type and guanidinated α‐lactalbumin proteins underwent substantial conformational changes, such as partial unfolding, loss of tertiary structure, but retention of secondary structure. In contrast, no significant conformational changes were observed in the citraconylated and acetylated α‐lactalbumins, most likely because of the absence of OA binding. These results suggest that electrostatic interactions between the positively‐charged basic groups on α‐lactalbumin and the negatively‐charged carboxylate groups on OA molecules play an essential role in the binding of OA to α‐lactalbumin and that these interactions appear to be as important as hydrophobic interactions. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Characterization of lysozyme-estrone glucuronide conjugates. The effect of the coupling reagent on the substitution level and sites of acylation.

Estrone glucuronide conjugates of hen egg white lysozyme were prepared by the mixed anhydride and active ester coupling procedures. Both methods gave good yields of conjugates, but the active ester procedure gave a more diverse range of products, making it less suitable for preparing conjugates for homogeneous enzyme immunoassay. Conjugation of lysozyme with estrone glucuronide by the mixed anhydride procedure gave one major derivative exclusively acylated at lysine residue 33 whereas conjugation by the active ester method gave six derivatives which were acylated at one or more of lysine residues 33, 97, and 116. None of the lysine residues 1, 13, and 96, or the N-terminal alpha-amino group, were acylated in any of the conjugates isolated. The correlation of the conjugate structures with the protein environments of the amino groups in the crystal structure of lysozyme suggested that the sites of acylation were determined not only by the chemical nature of the acylating reagent but also by the surface accessibility and nucleophilicity of the individual lysine residues.     

Unfolding of nucleosome cores induced by chemical acetylation of histones

Relative accessibility of nucleosomal histones to acetic anhydride during acetylation has been studied as a function of concentration, pH and ionic strength of the solution using high-resolution gel-electrophoresis. It was shown that about 80% of lysine residues in nucleosomal histones and 100% of the same residues in histone complexes without DNA in 2 M NaCl are accessible to the modification, which is proved by the localization of the majority of lysine residues in nucleosomes near the surface of the histone octamer, by their participation in ionic interactions with DNA and, probably, in histone-histone contacts. Gel-electrophoretic experiments with nucleosomes and studies of the histone resistance to mild trypsinolysis indicated that neither nucleosomes themselves nor histone octamers are affected even though 50% of lysine residues in histones have been acetylated. The process of acetylation is accompanied by the growing tendency of histones to participate in mild trypsinolysis and by a gradual decline in electrophoretic mobility and in the value of the sedimentation constant. The circular dichroism spectra and the microscopic appearance of nucleosomes are also markedly changed. These results suggest that a gradual unfolding of nucleosomes occurs when 5 or more lysine residues in the nucleosomal histones have been acetylated.     

Titration study of acetylated lysozyme.

To study the interaction between carboxyl groups and amino groups in native lysozyme [EC 3.2.1.17], and to identify the positions and the pK values of the abnormal carboxyl groups, N-acetylated lysozyme was prepared. The acetylation did not affect the molecular shape of the enzyme, but changed six amino groups to a non-ionizable form, leaving one amino group free; this was determined to be Lys 33. In addition, pH titration of the acetylated lysozyme in 0.2 or 0.02 M KCl aqueous solution indicated fewer titratable groups with pK(int) of 7.8 or 10.4 compared with the native protein, though the number of titratable carboxyl groups was not affected by the acetylation. From the pH titration results and structural considerations, the unititratable carboxyl groups were suggested to be Asp 48, Asp 66, and Asp 87. On the other hand, spectrophotometric titration in 0.2 M KCl showed that all three tyrosine residues are titratable in the acetylated protein, although an abnormal tyrosine residue exists in the native state. Tyr 20 was suggested to be untitratable in the pH range of 8-12.6.     

Difference absorption spectra, circular dichroism, and disulfide cleavage of hen and turkey lysozymes in the alkaline pH region.

The difference absorption spectra of hen and turkey lysozymes in the alkaline pH region had three maxima at around 245, 292, and 300 nm and had no isosbestic points. The ratio of the extinction difference at 245 nm to that at 295 nm changed with pH. These spectral features are quite different from those observed when only tyrosyl residues are ionized, and it was impossible to determine precisely the pK values of the tyrosyl residues in lysozyme by spectrophotometric titration. A time-dependent spectral change was observed above about pH 12. This is not due to exposure of a buried tyrosyl residue on alkali denaturation. The disulfide bonds and the peptide bonds in the lysozyme molecule were cleaved by alkali above about pH 11. The intrinsic pK value of Tyr 23 of hen lysozyme was determined to be 10.24 (apparent pK 9.8) at 0.1 ionic strength and 25 degrees C from the CD titration data. Comparison of the CD titration of turkey lysozyme with that of hen lysozyme suggested that Tyr 3 and Tyr 23 in turkey lysozyme have apparent pK values of 11.9 and 9.8, respectively.     

Basis of thermostability in pig heart lactate dehydrogenase treated with O-methylisourea

Acetamidination of pig heart lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) with ethyl acetimidate resulted in an increase of thermostability, and covalent bridge formation between pairs of lysine residues is observed. Guanidination with O-methylisourea of the enzyme also increases the thermostability, but such a bridge seems not to be formed. Increased thermostability of guanidinated enzyme is considered to be due to the shift of the pK values of the lysine residues from 10.5 to 12.5 after guanidination. Modification experiments with carbodiimide reveals that the enzyme contains 4.6 pairs of neighboring lysine and carboxyl residues per subunit, and amide bonding between 3.2 pairs results in an increase of thermostability. Guanidination of 4.6 Lys/subunit of the enzyme yields an enzyme derivative with considerably increased thermostability. Salt bridge formation between the 4.6 pairs of neighboring carboxyl and guanidinated lysine residues per subunit might make a major contribution to the increased thermostability of the guanidinated enzyme.     

Affecting proton mobility in activated peptide and whole protein ions via lysine guanidination

We have evaluated the effect of lysine guanidination in peptides and proteins on the dissociation of protonated ions in the gas phase. The dissociation of guanidinated model peptide ions compared to their unmodified forms showed behavior consistent with concepts of proton mobility as a major factor in determining favored fragmentation channels. Reduction of proton mobility associated with lysine guanidination was reflected by a relative increase in cleavages occurring C-terminal to aspartic acid residues as well as increases in small molecule losses. To evaluate the effect of guanidination on the dissociation behavior of whole protein ions, bovine ubiquitin was selected as a model. Essentially, all of the amide bond cleavages associated with the +10 charge state of fully guanidinated ubiquitin were observed to occur C-terminal to aspartic acid residues, unlike the dissociation behavior of the +10 ion of the unmodified protein, where competing cleavage N-terminal to proline and nonspecific amide bond cleavages were also observed. The +8 and lower charge states of the guanidinated protein showed prominent losses of small neutral molecules. This overall fragmentation behavior is consistent with current hypotheses regarding whole protein dissociation that consider proton mobility and intramolecular charge solvation as important factors in determining favored dissociation channels, and are also consistent with the fragmentation behaviors observed for the guanidinated model peptide ions. Further evaluation of the utility of condensed phase guanidination of whole proteins is necessary but the results described here confirm that guanidination can be an effective strategy for enhancing C-terminal aspartic acid cleavages. Gas phase dissociation exclusively at aspartic acid residues, especially for whole protein ions, could be useful in identifying and characterizing proteins via tandem mass spectrometry of whole protein ions.     

Multifunctionality of lipoamide dehydrogenase: lysine residue and cationic environment

Chemical modifications are carried out to investigate cationic residues of lipoamide dehydrogenase. Amidinations with imidoesters which introduce amidino groups with various substituents, alter the dehydrogenase activity without significantly affecting other functional activities. Correlation analyses of kinetic parameters (lipoamide reduction catalyzed by amidinated enzymes) for substituent effects offer a useful technique for studying structure-function relationship of the lysine residues. The specificity of phosphopyridoxylation and subsequent photoinactivation of the phosphopyridoxylated enzyme enable us to identify the lysine residue at the proximity of the active site histidine. Sensitized photoinactivation of glyoxalated enzyme together with relevant results suggest that the lysine residue provides cationic environment to the hydrophobic active site, and thereby, affects the reactivity of the active site histidine in the dehydrogenase reaction.