Multiple methionine substitutions are tolerated in T4 lysozyme and have coupled effects on folding and stability

In order to further explore the tolerance of proteins to amino acid substitutions within the interior, a series of core residues was replaced by methionine within the C-terminal domain of T4 lysozyme. By replacing leucine, isoleucine, valine and phenylalanine residues a total of 10 methionines could be introduced, which corresponds to a third of the residues that are buried in this domain. As more methionines are incorporated the protein gradually loses stability. This is attributed in part to a reduction in hydrophobic stabilization, in part to the increased entropic cost of localizing the long, flexible methionine sidechains, and in part to steric clashes. The changes in structure of the mutants relative to the wildtype protein are modest but tend to increase in an additive fashion as more methionines are included. In the most extreme case, namely the 10-methionine mutant, much of the C-terminal domain remains quite similar to wildtype (root-mean-square backbone shifts of 0.56 Å), while the F and G helices undergo rotations of approximately 20° and center-of-mass shifts of approximately 1.4 Å. For up to six methionine substitutions the changes in stability are additive. Beyond this point, however, the multiple mutants are somewhat more stable than suggested from the sum of their constituents, especially for those including the replacement Val111→Met. This is interpreted in terms of the larger structural changes associated with this substitution. The substituted sidechains in the mutant structures have somewhat higher crystallographic thermal factors than their counterparts in WT*. Nevertheless, the interiors of the mutant proteins retain a well-defined structure with little suggestion of molten-globule characteristics. Lysozymes in which selenomethionine has been incorporated rather than methionine tend to have increased stability. At the same time they also fold faster. This provides further evidence that, at the rate-limiting step in folding, the structure of the C-terminal domain of T4 lysozyme is similar to that of the fully folded protein.     

Selective labeling of selenomethionine residues in proteins with a fluorescent derivative of benzyl bromide

Benzyl bromide is used as a reagent for the selective modification of methionine residues in proteins. We here explored the suitability of the bromobenzyl moiety as a reactive group for the targeted fluorescent labeling of methionine and selenomethionine residues in proteins. A novel labeling reagent (N,N',N'-trimethyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- N'-(p-bromomethylbenzyl)-ethylenediamine, NBD-BBr) was synthesized and tested for reactivity with two model proteins containing single methionine or selenomethionine residues. The amounts of reagent and reactions times required for modification of methionine resulted in side reactions with other amino acid residues, a finding which was also confirmed for benzyl bromide itself. However, with selenomethionine, lower concentrations and shorter reaction times were sufficient for NBD-BBr modification. Under these conditions, labeling was confined to selenomethionine residues with one but not the other model protein. Where applicable, the protein labeling strategy characterized here is rapid and efficient. It should be useful in combination with cysteine-specific labeling if dual site-specific modification is desired.     

Selenomethionyl analog of recombinant human choriogonadotropin

Selenomethionyl and high mannose type analog of recombinant human choriogonadotropin (hCG) to solve the crystallization and phase problems has been obtained by gene transfer methodology. SF9 insect cells were infected with the recombinant viruses containing hCG alpha and hCG beta cDNAs in selenomethionine containing methionine-free Grace's medium. The selenomethionyl hCG (SehCG) was purified from the culture medium by one step immunoaffinity chromatography using an immobilized monoclonal antibody against hCG beta. The presence of selenomethionine was demonstrated by amino acid analysis of SehCG. The amino acid composition indicated that more than 84% of methionine residues were substituted by selenomethionine. Its sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis yielded a single 38-kDa protein band under nonreducing conditions. The carbohydrate analysis of SehCG was consistent with the presence of four N-linked high mannose type carbohydrates and four O-linked simple disaccharide chains. The in vitro immunological and biological studies of SehCG indicated that selenomethionine substitution had no effect on the immunopotency, receptor binding, and steroidogenic activities of the hormone.     

Methionine Changes in Bacteriorhodopsin Detected by FTIR and Cell-Free Selenomethionine Substitution

Bacteriorhodopsin (BR) is an integral membrane protein, which functions as a light-driven proton pump in Halobacterium salinarum. We report evidence that one or more methionine residues undergo a structural change during the BR→M portion of the BR photocycle. Selenomethionine was incorporated into BR using a cell-free protein translation system containing an amino acid mixture with selenomethionine substituted for methionine. BR→M FTIR difference spectra recorded for unlabeled and selenomethionine-labeled cell-free expressed BR closely resemble the spectra of in vivo expressed BR. However, reproducible changes occur in two regions near 1284 and 900 cm⁻¹ due to selenomethionine incorporation. Isotope labeled tyrosine was also co-incorporated with selenomethionine in order to confirm these assignments. Based on recent x-ray crystallographic studies, likely methionines which give rise to the FTIR difference bands are Met-118 and Met-145, which are located inside the retinal binding pocket and in a position to constrain the motion of retinal during photoisomerization. The assignment of methionine bands in the FTIR difference spectrum of BR provides a means to study methionine-chromophore interaction under physiological conditions. More generally, combining cell-free incorporations of selenomethionine into proteins with FTIR difference spectroscopy provides a useful method for investigating the role of methionines in protein structure and function.     

Selenomethionine and selenocysteine double labeling strategy for crystallographic phasing

A protocol for the quantitative incorporation of both selenomethionine and selenocysteine into recombinant proteins overexpressed in Escherichia coli is described. This methodology is based on the use of a suitable cysteine auxotrophic strain and a minimal medium supplemented with selenium-labeled methionine and cysteine. The proteins chosen for these studies are the cathelin-like motif of protegrin-3 and a nucleoside-diphosphate kinase. Analysis of the purified proteins by electrospray mass spectrometry and X-ray crystallography revealed that both cysteine and methionine residues were isomorphously replaced by selenocysteine and selenomethionine. Moreover, selenocysteines allowed the formation of unstrained and stable diselenide bridges in place of the canonical disulfide bonds. In addition, we showed that NDP kinase contains a selenocysteine adduct on Cys122. This novel selenium double-labeling method is proposed as a general approach to increase the efficiency of the MAD technique used for phase determination in protein crystallography.     

Tolerance of point substitution of methionine for isoleucine in hen egg white lysozyme

X-ray structure determination of proteins by using the multiple-wavelength anomalous dispersion method targeting selenomethionine is now widely employed. Isoleucine was examined for the second choice of the substitution of methionine next to leucine. We performed a systematic mutational study of the substitutions of methionine for isoleucine. All mutated lysozymes were less stable than the wild-type by about 1 kcal/mol and it is suggested that this instability was caused by the change in residual hydrophobicity from isoleucine to methionine. The X-ray structures of all mutant lysozymes were very similar to that of the wild-type. In addition, both the accessible surface areas and the conformation of the side chain of methionine in all mutant lysozymes were similar to those of the side chain at the respective isoleucine in the wild-type. Therefore, it is suggested that the mutation from isoleucine to methionine in a protein can be considered as a "safe" substitution.     

Comparing the effect on protein stability of methionine oxidation versus mutagenesis: steps toward engineering oxidative resistance in proteins.

The biological activity of some proteins is known to be sensitive to oxidative damage caused by a variety of oxidants. The model protein staphylococcal nuclease was used to explore the effect on protein structural stability of oxidizing methionine to the sulfoxide form. These effects were compared with the effects of substituting methionines with isoleucine and leucine, a potential strategy for stabilizing proteins against oxidative damage. Wild-type nuclease and various mutants were oxidized with hydrogen peroxide. Stabilities of both oxidized and unoxidized proteins were determined by guanidine hydrochloride denaturation. Oxidation destabilized the wild-type protein by over 4 kcal/mol. This large loss of stability supports the idea that in some cases loss of biological activity is linked to disruption of the protein native state. Comparison of mutant protein's stability losses upon oxidation showed that methionines 65 and 98 had a much greater destabilizing effect when oxidized than methionines 26 or 32. While substitution of methionine 98 carried as great an energetic penalty as oxidation, substitution at position 65 was less disruptive than oxidation. Thus a simple substitution mutagenesis strategy to protect a protein against oxidative destabilization is practical for some methionine residues.     

Context-dependent protein stabilization by methionine-to-leucine substitution shown in T4 lysozyme.

The substitution of methionines with leucines within the interior of a protein is expected to increase stability both because of a more favorable solvent transfer term as well as the reduced entropic cost of holding a leucine side chain in a defined position. Together, these two terms are expected to contribute about 1.4 kcal/mol to protein stability for each Met --> Leu substitution when fully buried. At the same time, this expected beneficial effect may be offset by steric factors due to differences in the shape of leucine and methionine. To investigate the interplay between these factors, all methionines in T4 lysozyme except at the amino-terminus were individually replaced with leucine. Of these mutants, M106L and M120L have stabilities 0.5 kcal/mol higher than wild-type T4 lysozyme, while M6L is significantly destabilized (-2.8 kcal/mol). M102L, described previously, is also destabilized (-0.9 kcal/mol). Based on this limited sample it appears that methionine-to-leucine substitutions can increase protein stability but only in a situation where the methionine side chain is fully or partially buried, yet allows the introduction of the leucine without concomitant steric interference. The variants, together with methionine-to-lysine substitutions at the same sites, follow the general pattern that substitutions at rigid, internal sites tend to be most destabilizing, whereas replacements at more solvent-exposed sites are better tolerated.     

P99-T A Simple Method to Quantitate Selenomethionine Incorporation into Proteins

Selenomethionine substitution is the preferred method for preparing heavy-atom derivatives of proteins for crystal structure determination using the multi-wavelength anomalous diffraction phasing method. This approach allows researchers to take advantage of the anomalous signal from a number of diverse atoms. We recently published a protocol describing a number of variables that play a role in determining incorporation efficiency of selenomethionine into mammalian expression systems.¹ Here we describe, in detail, a simple method for assessing selenomethionine substitution by replacement of methionine in homogeneous protein preparations. Using matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF) technology following trypsin proteolysis of the recombinant protein, we are able to evaluate variables that play roles in affecting selenomethionine incorporation. Examples will illustrate (a) the ease of identification of modified peptides containing the selenomethione and (b) relative quantitation of such peptides when compared with the control, unmodified peptides.     

Incorporation and distribution of selenium into thiolase from Clostridium kluyveri

Clostridium kluyveri incorporates selenium as selenomethionine into its acetoacetyl-CoA thiolase when grown in media containing normal sulfur-to-selenium ratios. Antibodies raised against the purified enzyme permitted quantitative immunoprecipitation of thiolase from crude cell extracts and thus facilitated the systematic analysis of the effects of wide variation in sulfur-to-selenium ratios on selenium incorporation into the enzyme. The extent of incorporation of selenium into thiolase was found to be dependent on the form of selenium supplied. When [75Se]selenomethionine was the source of selenium, the incorporation of selenium into thiolase was inversely proportional to the level of added methionine. However, similar levels of methionine failed to decrease the incorporation of selenium from selenite. To study the location of selenomethionine and methionine residues in the polypeptide chain of the enzyme, thiolase was prepared from cells cultured in the presence of H2 35SO4 or Na2 75SeO3. The 35S- or 75Se-labeled protein was treated with trypsin and the resulting peptides were isolated by reverse phase high performance liquid chromatography. The peptide maps of the enzyme indicated that selenium was distributed throughout the primary structure in a manner that paralleled methionine. From these studies, it is concluded that selenium occurs in thiolase adventitiously and is not required for any biological function.     

The synthesis of β-galactosidase inEscherichia coli in the presence of Methionine analogues

The effect of methionine analogues, selenomethionine and selenoethionine, on the synthesis of β-galactosidase inEscherichia coli was investigated. It was found that the incorporation of selenomethionine into β-galactosidase results in the formation of a protein exhibiting normal enzyme activity and immunologically cross-reacting with the antiserum against normal β-galactosidase. However, the selenomethionine enzyme was found to be more susceptible to heat, urea and trypsin. On the other hand, an immunologically cross-reacting protein without the enzyme activity was synthesized in the presence of selenoethionine. The results obtained seem to support the idea that the presence of methionine methyl groups is essential for the activity of β-galactosidase, whereas methionine sulphur can be replaced without changing the enzyme activity.     

Tryptophan fluorescence of calmodulin binding domain peptides interacting with calmodulin containing unnatural methionine analogues

The interactions between the abundant methionine residues of the calcium regulatory protein calmodulin (CaM) and several of its binding targets were probed using fluorescence spectroscopy. Tryptophan steady-state fluorescence from peptides encompassing the CaM-binding domains of the target proteins myosin light chain kinase (MLCK), cyclic nucleotide phosphodiesterase (PDE) and caldesmon site A and B (CaD A, CaD B), and the model peptide melittin showed Ca(2+)-dependent blue-shifts in their maximum emission wavelength when complexed with wild-type CaM. Blue-shifts were also observed for complexes in which the CaM methionine residues were replaced by selenomethionine, norleucine and ethionine, and when a quadruple methionine to leucine C-terminal mutant of CaM was studied. Quenching of the tryptophan fluorescence intensity was observed with selenomethionine, but not with norleucine or ethionine substituted protein. Fluorescence quenching studies with added potassium iodide (KI) demonstrate that the non-native proteins limit the solvent accessibility of the Trp in the MLCK peptide to levels close to that of the wild-type CaM-MLCK interaction. Our results show that the methionine residues from CaM are highly sensitive to the target peptide in question, confirming the importance of their role in binding interactions. In addition, we provide evidence that the nature of binding in the CaM-CaD B complex is unique compared with the other complexes studied, as the Trp residue of this peptide remains partially solvent exposed upon binding to CaM.     

Selenomethionine incorporation into a protein by cell-free synthesis

Multi-wavelength anomalous diffraction phasing is especially useful for high-throughput structure determinations. Selenomethionine substituted proteins are commonly used for this purpose. However, the cytotoxicity of selenomethionine drastically reduces the efficiency of its incorporation in in vivo expression systems. In the present study, an improved E. coli cell-free protein synthesis system was used to incorporate selenomethionine into a protein, so that highly efficient incorporation could be achieved. A milligram quantity of selenomethionine-containing Ras was obtained using the cell-free system with dialysis. The mass spectrometry analysis showed that more than 95% of the methionine residues were substituted with selenomethionine. The crystal of this protein grew under the same conditions and had the same unit cell constants as those of the native Ras protein. The three-dimensional structure of this protein, determined by multi-wavelength anomalous diffraction phasing, was almost the same as that of the Ras protein prepared by in vivo expression. Therefore, the cell-free synthesis system could become a powerful protein expression method for high-throughput structure determinations by X-ray crystallography.     

Thermodynamic Effects of Noncoded and Coded Methionine Substitutions in Calmodulin

The methionine residues in the calcium (Ca²⁺) regulatory protein calmodulin (CaM) are structurally and functionally important. They are buried within the N- and C-domains of apo-CaM but become solvent-exposed in Ca²⁺-CaM, where they interact with numerous target proteins. Previous structural studies have shown that methionine substitutions to the noncoded amino acids selenomethionine, ethionine, or norleucine, or mutation to leucine do not impact the main chain structure of CaM. Here we used differential scanning calorimetry to show that these substitutions enhance the stability of both domains, with the largest increase in melting temperature (19-26°C) achieved with leucine or norleucine in the apo-C-domain. Nuclear magnetic resonance spectroscopy experiments also revealed the loss of a slow conformational exchange process in the Leu-substituted apo-C-domain. In addition, isothermal titration calorimetry experiments revealed considerable changes in the enthalpy and entropy of target binding to apo-CaM and Ca²⁺-CaM, but the free energy of binding was largely unaffected due to enthalpy-entropy compensation. Collectively, these results demonstrate that noncoded and coded methionine substitutions can be accommodated in CaM because of the structural plasticity of the protein. However, adjustments in side-chain packing and dynamics lead to significant differences in protein stability and the thermodynamics of target binding.     

Failure of selenomethionine residues in albumin and immunoglobulin G to protect against peroxynitrite.

Selenomethionine has been suggested to protect against peroxynitrite by quenching it in vivo. Selenomethionine is distributed randomly in the methionine pool. Albumin and IgG were purified from plasma of a human being before and after 28 days of supplementation with 400 microg selenium/day as selenomethionine. The albumin contained 1 selenium atom, presumably as selenomethionine, per 8000 methionine residues before supplementation and 1 per 2800 after supplementation. Although this ratio suggested that selenomethionine would not have as great an effect in quenching peroxynitrite as would methionine, direct testing of the albumin and IgG fractions was carried out to assess the ability of these proteins to prevent peroxynitrite oxidation of dihydrorhodamine 123 to rhodamine 123. The ability of the albumin preparations to resist nitration of tyrosine residues was also assessed. The high-selenomethionine preparations of the proteins had no greater effect in quenching the peroxynitrite than did the normal-selenomethionine preparations. These results do not support the proposal that selenomethionine in proteins contributes to in vivo protection against peroxynitrite.     

Global incorporation of norleucine in place of methionine in cytochrome P450 BM-3 heme domain increases peroxygenase activity

In this study we have replaced all 13 methionine residues in the cytochrome P450 BM-3 heme domain (463 amino acids) with the isosteric methionine analog norleucine. This experiment has provided a means of testing the functional limits of globally incorporating into an enzyme an unnatural amino acid in place of its natural analog, and also an efficient way to test whether inactivation during peroxide-driven P450 catalysis involves methionine oxidation. Although there was no increase in the stability of the P450 under standard reaction conditions (in 10 mM hydrogen peroxide), complete substitution with norleucine resulted in nearly two-fold-increased peroxygenase activity. Thermostability was significantly reduced. The fact that the enzyme can tolerate such extensive amino acid replacement suggests that we can engineer enzymes with unique chemical properties via incorporation of unnatural amino acids while retaining or improving catalytic properties. This system also provides a platform for directing enzyme evolution using an extended set of protein building blocks.     

Interactions of Methionine and Selenomethionine with Methionine Adenosyltransferase and Ethylene-generating Systems

Since selenomethionine appears to be a better precursor of ethylene in senescing flower tissue of Ipomoea tricolor and in indole acetic acid-treated pea stem sections than is methionine (Konze JR, N Schilling, H Kende 1978 Plant Physiol 62: 397-401), we compared the effectiveness of selenomethionine and methionine to participate in reactions which may be connected to ethylene biosynthesis. Evidence is presented that selenomethionine is also a better substrate of methionine adenosyltransferase (ATP: methionine S-adenosyltransferase, EC 2.5.1.6) from I. tricolor, the V_max for selenomethionine being twice as high as that for methionine. The affinity of the enzyme is higher for methionine than for selenomethionine, however. Methionine added to flower tissue together with selenomethionine inhibits the enhancement of ethylene synthesis by the seleno analog. Likewise, methionine reduces the high, selenomethionine-dependent reaction rates of methionine adenosyltransferase from I. tricolor flower tissue. On the other hand, selenomethionine is less effective as an ethylene precursor than is methionine in model systems involving oxidation by free radicals. It was concluded that activation of methionine by methionine adenosyltransferase and formation of S-adenosylmethionine are more likely to be involved in ethylene biosynthesis than is oxidation of methionine by free radicals.     

A selenomethionine-containing azurin from an auxotroph of Pseudomonas aeruginosa

The production and spectroscopic properties of an L-selenomethionine-containing homolog of Pseudomonas aeruginosa azurin are described. The amino acid substitution was carried out by developing an L-methionine-dependent bacterial strain from a fully functional ATCC culture. Uptake studies monitored using L-[75Se]methionine indicated that L-selenomethionine was incorporated into the protein synthetic pathway of Pseudomonas bacteria in a manner analogous to L-methionine. Several batches of bacteria were grown, and one sample of isolated and purified selenoazurin (azurin in which methionine was substituted by selenomethionine) was found (by neutron activation analysis) to contain 5.2 +/- 0.8 seleniums/copper. Correspondingly, a residual 0.35 methionines, relative to 6.0 in the native protein, were found by amino acid analysis in this azurin sample. The redox potential and extinction coefficient of this selenoazurin were found to be 333 +/- 1 mV (pH 7.0, I = 0.22) and 5855 +/- 160 M-1 cm-1 at 626 +/- 1 nm, respectively. Visible electronic, CD, and EPR spectra are reported and Gaussian curve fitting to the former spectrum allowed assignment of the selenomethionine Se----Cu(II) transition to a band found at 18034 cm-1, based upon an observed 450 cm-1 shift to the red from the analogous band position in the native protein. The data are consistent with a relatively more covalent copper site stabilizing the reduced, Cu(I), form in the selenoprotein. A role for the methionine as a modulator of the blue copper site redox potential by metal----ligand back bonding from Cu(I) is discussed in terms of a ligand sphere which limits the valence change at copper to much less than 1 during a redox cycle.     

Oxidation of buried cysteines is slow and an insignificant factor in the structural destabilization of staphylococcal nuclease caused by H<Subscript>2</Subscript>O<Subscript>2</Subscript> exposure

Summary. The oxidation of buried cysteine or methionine residues can destroy the enzyme activity of a protein by disrupting structure. Engineering in such an oxidatively triggered switch for enzyme activity would only be useful if the effects of substitution are relatively minor, while the effects of the oxidized side chain upon structure are significant and the oxidation relatively easy. To assess the feasibility of this strategy for controlling enzyme activity, the effects of such substitutions and their oxidation were studied in a well characterized model protein, staphylococcal nuclease. Stability and enzyme activity of the oxidized proteins was assessed and compared to the stability and enzyme activity of the unoxidized proteins. Cysteines were found to be generally well tolerated in buried positions but these mutants were not more destabilized than wild-type when oxidized. This shows that buried cysteines are difficult enough to oxidize that this is not likely to be a useful protein engineering strategy or a commonly used regulatory modification. Similar effects were observed for methionine.     

Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn

Susceptibility of methionine residues to oxidation is a significant issue of protein therapeutics. Methionine oxidation may limit the product's clinical efficacy or stability. We have studied kinetics of methionine oxidation in the Fc portion of the human IgG2 and its impact on the interaction with FcRn and Protein A. Our results confirm previously published observations for IgG1 that two analogous solvent‐exposed methionine residues in IgG2, Met 252 and Met 428, oxidize more readily than the other methionine residue, Met 358, which is buried inside the Fc. Met 397, which is not present in IgG1 but in IgG2, oxidizes at similar rate as Met 358. Oxidation of two labile methionines, Met 252 and Met 428, weakens the binding of the intact antibody with Protein A and FcRn, two natural protein binding partners. Both of these binding partners share the same binding site on the Fc. Additionally, our results shows that Protein A may serve as a convenient and inexpensive surrogate for FcRn binding measurements.