Threonine Hydroxyl Research Articles

Detection of threonine structural changes upon formation of the M-intermediate of bacteriorhodopsin: evidence for assignment to Thr-89

The behavior of threonine residues in the bacteriorhodopsin (bR) photocycle has been investigated by Fourier transform infrared difference spectroscopy. l-Threonine labeled at the hydroxyl group with 18O ( l-[3- 18O]threonine) was incorporated into bR and the bR→M FTIR difference spectra measured. Bands are assigned to threonine vibrational modes on the basis of 18O induced isotope frequency shifts and normal mode calculations. In the 3500 cm −1 region, a negative band is assigned to the OH stretch of threonine. In the 1125 cm −1 region, a negative band is assigned to a mixed CH 3 rock/CO stretch mode. The frequency of both these bands indicates the presence of at least one hydrogen bonded threonine hydroxyl group in light adapted bR which undergoes a change in structure by formation of the M intermediate. Spectral changes induced by the substitution Thr-89→Asn but not Thr-46→Asn or Asp-96→Asn are consistent with the assignment of these bands to Thr-89. These results along with another related study on the mutant Thr-89→Asn indicate that the active site of bR includes Thr-89 and that its interaction with the retinylidene Schiff base and Asp-85 may play an important role in regulating the color of bacteriorhodopsin and the transfer of a proton to the Schiff base.

Analytical methods for the study of O-GlcNAc glycoproteins and glycopeptides.

Over the past decade, numerous nuclear and cytosolic glycoproteins have been shown to be modified by single N-acetylglucosamine (O-GlcNAc) residues attached to the side chain hydroxyl groups of serine or threonine (, , ). This unique postranslational modification (O-GlcNAcylation), described in lymphocytes by Torres and Hart (), can be found on a wide range of proteins including cytoskeletal proteins (, , , ) nuclear pore proteins (, , , , ), transcription factors (), RNA polymerase II (), oncoproteins (,), and viral proteins (, , ), to name a few. In addition to the O-GlcNAc modification, all of these proteins are known phosphoproteins and many have been reported to be regulated and/or form reversible protein complexes based on their state of phosphorylation. These observations suggest that O-GlcNAcylation also plays a role in the assembly/disassembly of protein complexes and/or the regulation of enzymes, perhaps by acting as an antagonist to phosphorylation. In fact, several studies have shown that phosphorylation and O-GlcNAcylation can occur at the same sites on a given protein (,). For example, a recent study documents that the c-Myc oncoprotein is O-GlcNAcylated at threonine 58, a known site of phosphorylation and mutational hotspot for c-Myc in human Burkitt and AIDS-related lymphomas (). Furthermore, O-GlcNAcylation appears to be a key regulatory modification since it is highly dynamic (,), responsive to external cellular stimuli (), and regulated in a cell cycle-dependent manner (). However, direct evidence for the function of O-GlcNAcylation has been limited, in large part, because of the difficulty and the amount of protein required for mapping sites of glycosylation as a prerequisite to molecular manipulations such as site-directed mutagenesis. The primary method for mapping sites of O-GlcNAcylation requires the enzymatic transfer of a [3H]galactose with bovine milk galactosyltransferase to tag the O-GlcNAc-modified protein, generation of glycopeptides by proteolysis, purification of the glycopeptides by several rounds of high-performance liquid chromatography (HPLC), followed by gas-phase and manual Edman sequencing. Each of these steps is very tedious and often results in significant losses in yield as a result of incomplete proteolysis, poor resolution of the glycopeptides by HPLC, and conservative pooling of HPLC fractions to maximize purity of the glycopeptides. Finally, this method requires a minimum of about 10 pmol of pure, labeled, glycopeptide to generate unambiguous sequence and to identify the site of glycosylation. Therefore, methods to improve recoveries of glycopeptides and increase the sensitivity of the site analysis assays would be a major breakthrough in the study of O-GlcNAcylation.

How amino acids control the binding of Cu(II) ions to DNA: I—The role of the hydroxyl group of serine and threonine in fixing the orientation of the complexes

ESR data have been obtained on some hydrated copper(I)) and copper(II) amino acid complexes bound to highly oriented DNA fibers. The complexes bind with random orientation on A-form DNA, while some of them bind stereospecifically on B-form DNA. The hydrated copper(II) ions bind to at least two different sites on B-form DNA at room temperature; one site is so mobile that the orientations of the complex fluctuate dynamically, while the other site fixes the orientation of the bound ions such that the angle between the g  axis and the DNA helical axes becomes ca. 55°. The g  axis of glycine complex on B-form DNA at room temperature is aligned parallel to the fiber axis with large fluctuations in the orientation. L-serine and L-threonine fix the g  axes of some of the complexes parallel to the fiber axes, indicating that the hydroxyl groups work as linkers to bind the complexes stereospecifically to B-form DNA. The hydroxyl groups in D-isomers do not work as L-isomers.

Cyanopeptolins, depsipeptides from cyanobacteria

Summary The wide range of secondary metabolites from cyanobacteria includes cyclic depsipeptides. They can be filed into several classes. Members of class V, the cyanopeptolins, are proposed in this review to be defined as (a) cyanobacterial 19-membered cyclic depsipeptides cyclized by an ester-linkage of the hydroxyl-group of threonine with the carboxy terminus of the C-terminal amino acid of a proposed linear precursor, (b) an unusual 3-amino-6-hydroxy-2-piperidone unit, and (c) a cis -configurated amide linkage between the amino acids in position 3 and 4. The cyanobacterial depsipeptides known so far, matching with this definition, are the cyanopeptolins A, B, C, and D, the aeruginopeptins 228-A, 228-B, 95-A, and 95-B, microcystilide A, the micropeptins A, B, and 90, compound A90720A, and the sulfate-containing cyanopeptolins S and SS. The cyanopeptolins and related depsipeptides from cyanobacteria may serve a biochemical and pharmacological tools, because most of them exhibit protease-inhibitory and/or cytotoxic activities. The structure-function relationships of the serine protease inhibitor A90720A and trypsin is known by a cocrystallization study. There are indications for a possible cyanobacterial origin of cyanopeptolin-like compounds found in marine eukaryotes.

O-linked protein glycosylation structure and function.

There has been a recent resurgence of interest in the post-translational modification of serine and threonine hydroxyl groups by glycosylation, because the resulting O-linked oligosaccharide chains tend to be clustered over short stretches of peptide and hence they can present multivalent carbohydrate antigenic or functional determinants for antibody recognition, mammalian cell adhesion and microorganism binding. Co-operativity can greatly increase the affinity of interactions with antibodies or carbohydrate binding proteins. Thus, in addition to their known importance in bearing tumour associated antigens in the gastrointestinal and respiratory tracts, glycoproteins with O-linked chains have been implicated as ligands or co-receptors for selectins (mammalian carbohydrate binding proteins). Microorganisms may have adopted similar mechanisms for interactions with mammalian cells in infection, by having relatively low affinity ligands (adhesins) for carbohydrate binding, which may bind with higher affinity due to the multivalency of the host ligand and which are complemented by other virulence factors such as interactions with integrin-type molecules. In addition to specific adhesion signals from O-linked carbohydrate chains, multivalent O-glycosylation is involved in determining protein conformation and forming conjugate oligosaccharide-protein antigenic, and possible functional determinants.

High frequency (139.5 GHz) electron paramagnetic resonance spectroscopy of the GTP form of p21 ras with selective 17O labeling of threonine.

Electron paramagnetic resonance spectroscopy at 139.5 GHz has been used to study p21 ras complexed with Mn(II) and guanosine 5'-(beta, gamma-imidotriphosphate), an analog of GTP. The p21 sample studied was selectively labeled with [17O gamma]threonine to a final enrichment of 30%. A Mn(II)-17O hyperfine interaction was observed, but the value of the coupling constant, 0.11 +/- 0.04 mT, is the smallest such value yet reported. Ab initio calculations indicate that this value is consistent with direct coordination of the threonine hydroxyl group and provide an estimate for the Mn(II)-17O bond length of 2.7 A. The measured hyperfine coupling constant and associated bond length starkly contrast with typical values for Mn(II)-17O coordination complexes, namely, approximately 0.25 mT and approximately 2.2 A, respectively. This contrast underscores the peculiar weakness of this Mn(II)-O interaction in p21 and persuasively argues that the nucleotide-induced conformational change, which is known to encompass the region of p21 involving Thr35, is not driven by Mn(II) coordination of the Thr35 hydroxyl group.

β‐Elimination for Release of O‐GalNAc‐Linked Oligosaccharides from Glycoproteins and Glycopeptides

This unit describes release of oligosaccharides that are attached to polypeptides through an N-acetylgalactosamine (GalNAc) linkage to the hydroxyl groups of serine or threonine. The beta-elimination procedures described here can be used to recover the oligosaccharide chains (also called glycans) and/or identify the serine or threonine residues involved in the linkage. A beta-elimination method employing sodium borohydride (NaBH4) and alkaline conditions is described, and an alternative method is also presented in which only alkaline conditions are used without a reducing agent. Another alternative protocol uses sodium sulfite.

Mutational analysis of the ganglioside-binding activity of the type II Escherichia coli heat-labile enterotoxin LT-IIb.

The Escherichia coli type II heat-labile enterotoxin LT-IIb IIb consists of a single A polypeptide and five B polypeptides. The A polypeptide is responsible for the toxic activity, and the B polypeptides function to bind the toxin to gangliosides on the surface of the plasma membrane. Previous studies on the related type II enterotoxin LT-IIa demonstrated the importance of threonine (Thr) residues at positions 13, 14, and 34 in the mature B polypeptide for ganglioside GD1bp-binding activity. In this study, we used sitespecific mutagenesis to investigate ganglioside GD1a-binding activity of the B polypeptide of LT-IIb. We determined that Thr-13 and Thr-14 were involved in binding of ganglioside GD1a by the B polypeptides of LT-IIb but that Thr-34 was not essential. Substitution of serine, but not other amino acids, at position 13 or 14 in the B polypeptide of LT-IIb resulted in retention of ganglioside-binding activity equivalent to that of the wild-type enterotoxin, providing strong evidence that the hydroxyl groups of threonine or serine at positions 13 and 14 are important for the ganglioside-binding activity of LT-IIb. Chimeric genes that expressed hybrids of the B polypeptides of LT-IIb and LT-IIa were also constructed, and analysis of the hybrids showed that the specificity of their ganglioside-binding activity was determined by the N-terminal half of the molecule.

Cysteine-containing peptide sequences exhibit facile uncatalyzed transacylation and acyl-CoA-dependent acylation at the lipid bilayer interface.

A variety of simple cysteine-containing lipopeptides, with sequences modeled on those found in naturally occurring S-acylated proteins, undergo spontaneous S-acylation in phospholipid vesicles at physiological pH when either long-chain acyl-CoAs or other S-acylated peptides are added as acyl donors. Fluorescent or radiolabeled lipopeptides with the sequence myristoyl-GCX- (X = G, L, R, T, or V), a motif found to undergo S-acylation in several intracellular regulatory proteins, and the prenylated peptide -SCRC(farnesyl)-OMe, modeled on the carboxyl terminus of p21H-ras, were all found to be suitable acyl acceptors for such uncatalyzed S-acyl transfer reactions at physiological pH. Acylation of these cysteinyl-containing lipopeptides to high stoichiometry was observed, on time scales ranging from a few hours to a few tens of minutes, in vesicles containing relatively low concentrations (< or = mol %) and only a modest molar excess (2.5:1) of the acyl donor species. No evidence was obtained for acyl transfer to peptide serine or threonine hydroxyl groups under the same conditions. These observations may have significant implications both for the design of in vitro studies of the S-acylation of membrane-associated proteins and for our understanding of the mechanisms of S-acylation of these species in vivo.

Measurement of proteolysis in cheese: relationship between phosphotungstic acid-soluble N fraction by Kjeldahl and 2,4,6- trinitrobenzenesulphonic acid-reactive groups in water-soluble N

Free amino groups produced during cheese ripening are used to indicate the extent of cheese proteolysis. Several studies have shown a high correlation between the level of free amino acids and the flavour of Gouda (Aston et al. 1983) or Comté (Grappin &amp; Berdagué, 1989). Measurement of the level of free amino acids seems useful for the investigation of flavour chemistry in cheese (Lemieux et al. 1990). The determination of N fractions is often used to estimate the degree of proteolysis in cheese, but since this procedure is laborious and time consuming several attempts have been made to replace it by more rapid methods (Ardö &amp; Meisel, 1991). Since its introduction by Satake et al. (1960), the 2,4,6-trinitrobenzenesulphonic acid (TNBS) method has been widely used for the determination of free amino groups. Because TNBS does not react with the imino groups of histidine and proline or the hydroxyl groups of tyrosine, serine or threonine, it has been accepted as a selective reagent for primary amino groups (Burger, 1974). Measurement of N by Kjeldahl in the phosphotungstic acid (PTA)–sulphuric acid extract (Gripon et al. 1975) estimates the N of free amino acids and low molecular mass peptides. The purpose of this study was to compare the TNBS and PTA-soluble N methods in order to find out whether the TNBS procedure can replace that of PTA-soluble N in the determination of a cheese proteolysis index.

Threonine 204 of the chaperone protein Hsc70 influences the structure of the active site, but is not essential for ATP hydrolysis.

The chaperone protein Hsc70 is an ATPase of unknown mechanism, although the crystal structure of the 44-kDa ATPase domain has been solved. This structure shows that the hydroxyl of threonine 204 is located close to the gamma-phosphate of ATP, in a position where it might be an intermediate phosphate acceptor in the hydrolysis reaction. We made two point mutations at residue 204 of Hsc70, threonine to valine (T204V) and threonine to glutamic acid (T204E). The wild-type ATPase domain had a Km for ATP of approximately 1 microM; the mutants had Km values of approximately 90 microM. The kcat values for the mutant proteins were also increased. After crystallization, the structures of the T204V and T204E proteins were solved and refined with data to 2.3- and 2.4-A resolution, respectively. The overall tertiary structure of the mutants showed little change from the wild type; however, significant changes were observed in the active site. Analysis of the structures suggested possible reasons for the changes in kinetic constants. Threonine 204 does not seem to be an obligatory intermediate phosphate acceptor in the hydrolysis reaction since the mutants retained appreciable ATPase activity.

Modulation of allosteric interactions in neurophysin induced by succinylation of serine-56 or cleavage of residues 1-8.

Neurophysin is an allosteric protein in which peptide binding and self-association are positively linked. Reaction of neurophysin with succinic anhydride led to a large decrease in peptide affinity assignable to succinylation of a serine or threonine hydroxyl group. To identify the residue involved, acetimidated protein was reacted with [14C]succinic anhydride and the active and inactive components were separated by affinity chromatography. Performic acid oxidation and tryptic and Asp-N mapping of the two components, followed by automated Edman degradation, allowed identification of the critical residue as Ser-56. This residue is not a direct participant in peptide binding and is distant from the subunit interface of the dimer, but it is immediately adjacent to the site of one of the known mutations associated with familial diabetes insipidus. Examination in solution of the peptide affinity of neurophysin succinylated at Ser-56 indicated a binding affinity approximately 1/20th that of the native protein or of protein succinylated at other residues, and a loss of the normal dependence of binding affinity on protein concentration. Under the same buffer conditions, loss of the concentration dependence of binding, in addition to the previously demonstrated loss of binding affinity, also accompanied excision of residues 1-8, an effect attributed to the loss of binding site residue Arg-8. However, in contrast to the effects of succinylation on native neurophysin, only minor effects of succinylation on the binding affinity of the des-1-8 protein were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficient solution phase synthesis and use of multiple O-phosphothreonyl-containing peptides for calcium phosphate binding studies.

The protected phosphothreonine derivative Boc-Thr(PO3Ph2)-OH was prepared in high yield from Boc-Thr-OH by a simple three-step procedure which involved 4-nitrobenzylcarboxyl protection, either phosphorotriester (diphenyl phosphorochloridate) or "phosphite-triester" (diphenyl N,N-diethylphosphoramidite) phosphorylation of the threonine hydroxyl group of Boc-Thr-ONb followed by hydrogenolytic carboxyl deprotection. The three Thr(P)-containing peptides, H-Thr(P)-Glu-Glu-NHMe.TFA, H-Thr(P)-Thr(P)-Glu-Glu-NHMe.TFA and H-Thr(P)-Thr(P)-Thr(P)-Glu-Glu-NHMe.TFA, were prepared in high yield by the use of Boc-Thr(PO3Ph2)-OH in the Boc mode of peptide synthesis (mixed anhydride method) followed by platinum-mediated hydrogenolytic deprotection of the Thr(PO3Ph2)-containing peptides. The use of the phosphopeptides in calcium phosphate binding studies showed that the triple Thr(P)-cluster was a basic structural requirement, since only the pentapeptide was able to bind calcium phosphate efficiently.

Oligonucleotides and Nucleotide-Peptides. LIV. The Synthesis and Hydrolytic Properties of Adenylyl-(5′→N∊)-lysylpeptides — Model Compounds of the AMP-RNA Ligase Covalent Complex

Abstract RNA ligase active center model compounds-adenylyl-(5′N∊)-lysylpeptides were synthesized. The stability of these compounds in aqueous solutions was studied and it was shown, that the carboxyl group of lysine and glutamic acid or hydroxyl group of threonine have no effect upon the hydrolytic mechanism of the adenylyl-(5′N∊)-lysylpeptides. This led to conclusion, that the hydrolysis of the AMP-RNA ligase complex is dependant upon other amino acid functional groups, which may be located next to the phosphoamide center, as a result of tertiary protein structure.

Purification and characterization of a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase specific for glycosylation of threonine residues.

A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase from porcine submaxillary glands was purified to electrophoretic homogeneity. IgG prepared from antisera against the pure enzyme immunoprecipitated the transferase in Triton X-100 extracts of submaxillary glands. The submaxillary transferase is a membrane-bound enzyme in contrast to the pure bovine colostrum enzyme, which is soluble in the absence of detergents. Both transferases have similar properties but also differ significantly. Examination of the acceptor substrate specificity of the submaxillary gland transferase showed that it specifically transferred N-acetylgalactosamine from UDP-GalNAc to the hydroxyl group of threonine and was devoid of transferase activity toward serine-containing peptides. These results imply that more than one transferase is involved in forming the GalNAc-threonine and the GalNAc-serine linkages found in O-linked oligosaccharides in glycoproteins. The amino acid sequence adjacent to glycosylated threonine residues may influence the rate of glycosylation by the pure transferase. For example, the second threonine residue in the sequence, Thr-Thr, appears to be glycosylated about twice as fast as the first and more rapidly than single, isolated threonine residues. However, no unique consensus sequence for glycosylation of threonine residues is evident, and any accessible threonine residue appears to be a potential acceptor substrate.

The folding of an enzyme: II. Substructure of barnase and the contribution of different interactions to protein stability

Barnase is described anatomically in terms of its substructures and their mode of packing. The surface area of hydrophobic residues buried on formation and packing of the structural elements has been calculated. Changes in stability have been measured for 64 mutations, 41 constructed in this study, strategically located over the protein. The purpose is to provide: (1) information on the magnitudes of changes in stabilization energy for mutations of residues that are important in maintaining the structure; and (2) probes for the folding pathway to be used in subsequent studies. The majority of mutations delete functional moieties of side-chains or make isosteric changes. The energetics of the interactions are variable and context-dependent. The following general conclusions may be drawn, however, from this study about the classes of interactions that stabilize the protein. (1) Truncation of buried hydrophobic side-chains has, in general, the greatest effect on stability. For fully buried residues, this averages at 1.5 kcal mol −1 per methylene group with a standard deviation of ±0.6 kcal mol −1. Truncation of partly exposed leucine, isoleucine or valine residues that are in the range of 50 to 80 Å 2 of solvent-accessible area (30 to 50 % of the total solvent-accessible area on a Gly-X-Gly tripeptide, i.e. those packed against the surface) has a smaller, but relatively constant effect on stability, at 0.81 kcal mol −1 per methylene group with a statistical standard deviation of±0.18 kcal mol −1. (2) There is a very poor correlation between hydrophobic surface area buried and the free energy change for an extensive data set of hydrophobic mutants. The best correlation is found to be between the free energy change and the number of methylene groups within a 6 Å radius of the hydrophobic groups deleted. (3) Burial of the hydroxyl group of threonine in a pocket that is intended for a γ-methyl group of valine costs 2.5 kcal mol −1, in the range expected for the loss of two hydrogen bonds. In extension of previous studies, it is found that: (1) disruption of surface ion pairs or salt bridges lowers stability by only small amounts (0.3 to 1 kcal mol −1) but disruption of buried pairs, by larger amounts (>3 kcal mol −1); (2) removal of the hydrogen bonding partner of an uncharged group loses 0.5 to 2 kcal mol −1 (the energy of the hydrogen bond plus associated van der Waals' interactions); and (3) removal of the partner of a charged group has similar effects to the disruption of salt bridges.

THR246 mutations decrease substrate inhibition in lactate dehydrogenase

Threonine 246 in Bacillus stearothermophilus L-lactate dehydrogenase has been changed to valine, serine, and alanine by site-directed mutagenesis. Kinetic analyses show a decrease in substrate inhibition for pyruvate reduction with the T246S mutant and virtual elimination of substrate inhibition for the T246A and T246V mutants. The results indicate that the absence of substrate inhibition in the 246A/V-catalyzed reactions is due to the elimination of a key hydrogen bond between the hydroxyl group of threonine and pyruvate in the wild-type complex that is an important contributor in the formation of the abortive enzyme-NAD +-pyruvate complex responsible for substrate inhibition.

Studies on the uronic acid-containing glycoproteins of Fusarium sp. M7-1: I. Isolation and some properties of the glycoproteins.

An acidic heteropolysaccharide preparation derived from the mycelium of Fusarium sp. M7-1 was fractionated into two fractions, precipitable and nonprecipitable, by treatment with cetyltrimethylammonium bromide (Cetavlon). These two fractions were further purified to apparent homogeneity on ultracentrifugation by treatment with charcoal and gel filtration chromatographies. Two glycoproteins, precipitable GP I and nonprecipitable GP II, were obtained. The molecular weights of GP I and GP II were estimated to be about 8.8 x 10(4) and 3.7 x 10(4), respectively, on gel filtration chromatography. Both GP I and GP II contained a high proportion of serine and threonine. Treatment of GP I and GP II with alkaline solution resulted in an increase in absorbance at 240 nm. Alkaline borohydride treatment markedly decreased the number of seryl and threonyl residues and resulted in an increase in alanine and the formation of 2-aminobutyric acid. It also resulted in release of low and high molecular weight carbohydrate chains. From these results, we conclude that both GP I and GP II are glycoproteins with carbohydrate chains attached to the protein moiety through O-glycosidic linkages to the hydroxyl group of serine and/or threonine.

Mutating protein kinase cAMP-binding sites into cGMP-binding sites. Mechanism of cGMP selectivity.

The cAMP-dependent protein kinase contains two different cAMP-binding sites referred to as the slow and fast sites. Mutation of Ala-334 to a threonine in the slow site of the bovine type I regulatory subunit created a site with marked increase in cGMP affinity without changing cAMP affinity (Shabb, J. B., Ng. L., Corbin, J. D. (1990) J. Biol. Chem. 265, 16031-16034). The corresponding fast site residue (Ala-210) was changed to a threonine by oligonucleotide-directed mutagenesis, and a double mutant containing a threonine in each site was also made. Holoenzymes were formed from native catalytic subunit and each recombinant regulatory subunit. The fast site mutant holoenzyme exhibited an improved cGMP activation constant and an impaired cAMP activation constant. The double mutant cGMP/cAMP selectivity was 200-fold greater than that of wild-type holoenzyme, making it as responsive to cGMP as native cGMP-dependent protein kinase. The increased intrinsic binding energies of mutated sites for cGMP were 2.7-3.0 kcal mol-1, consistent with the presence of an extra hydrogen bond. Cyclic nucleotide analog studies implied that this hydrogen bond was between the threonine hydroxyl and the 2-amino of cGMP. Comparisons of amino acid sequences and cyclic nucleotide specificities suggested that the Ala/Thr difference may also impart cAMP/cGMP binding selectivity to related proteins such as cyclic nucleotide-gated ion channels.

Solid‐phase peptide synthesis without side‐chain hydroxyl protection of threonine

A peptide containing four threonine residues was synthesised by the solid-phase method using fluorenyl-methoxycarbonylamino acid reactive esters or coupling by preactivation with 1-hydroxybenzotriazole and Castro's reagent. In two separate experiments the synthesis was carried out with or without protection of the side-chain hydroxyl group of threonine as the tert.-butyl ether. Comparison of the crude peptides after deprotection and detachment from the synthesis resin suggests that side-chain protection of threonine is unnecessary under the synthetic conditions employed.