An X-ray study of the structure and binding properties of iodine-inactivated lysozyme

Beddell, C.R.; Blake, C. C. F.; Oatley, Stuart J.

doi:10.1016/s0022-2836(75)80064-7

Cited by 46 publications

(16 citation statements)

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“…In this case the specific oxidation of Trp-62 induced a shift of the three-dimensional structure which blocked subsite C of the active site cleft such that productive binding is prevented (4). Oxidation of Trp-108 in lysozyme similarly induced a conformational change near the catalytic residue Glu-35 (3,11).…”

Section: Discussionmentioning

confidence: 99%

Influence of acarbose and maltose on the reactivity of individual tryptophanyl residues in glucoamylase from aspergillus niger

Svensson

Clarke

Svendsen

1986

Carlsberg Res. Commun.

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Tryptophanyl residues of A. niger glucoamylase G2 (EC 3.2.1.3) involved in substrate and inhibitor binding have been identified following N-bromosuccinimide (NBS) treatment in the presence and absence of protective ligands. Appropriate proteolytic cleavages of the glucoamylase derivatives enabled isolation of individual peptide fragments containing the 15 tryptophan positions and the extent of tryptophan oxidation was measured employing normal and 2nd derivative UV-spectrophotometry.Trp-52, -14 I, -156, -228, -317, and -335 remained unoxidized while complete oxidation of Trp-6, -28, and -466 and partial oxidation of Trp-170 and -178 was observed after NBS-treatment whether ligands were added or not. were partially converted in uncomplexed glucoamylase while the presence of either the substrate maltose or the inhibitor acarbose prevented the oxidation of these residues. Trp-120, required for maintaining the active catalytic site, was protected by acarbose only, and its oxidation did not prevent ligand binding. The functional roles of Trp-212, -417, -437, and -120 are discussed.NBS-treatment of the acarbose-protected large form ofglucoamylase, G 1, destroyed its unique capacity to adsorb onto starch granules while catalytic activity was preserved towards soluble substrates. This effect could be correlated with the oxidation of Trp-590 and -615 located near the COOH-terminus.The reactivities, probably reflecting the degree of solvent exposure, were also assessed for individual tyrosyl residues in G~.The thermal stability of oxidized, catalytically active GI and G2 was remarkably low as compared to the unmodified forms.Abbreviations: G l and G2 designate the larger and the smaller forms ofglucoamylase from A. niger (29,43); CM-= S-carboxymethyl-; NBS = N-bromosuccinimide; nWox = number (n) of oxidized tryptophanyl residues per enzyme molecule; 2-pe-= 2-pyridylethyl-; RP-HPLC = reversed-phase high performance liquid chromatography; Tris = 2-amino-2(hydroxymethyl)-1,3-propandiol; * signifies glycosylation.Springer-Verlag

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Section: Discussionmentioning

confidence: 99%

Influence of acarbose and maltose on the reactivity of individual tryptophanyl residues in glucoamylase from aspergillus niger

Svensson

Clarke

Svendsen

1986

Carlsberg Res. Commun.

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“…In this experiment, deuterated lysozyme was briefly unfolded in protonated 6.00 M GdmCl solution at FIGURE 1: Hen egg white lysozyme. A CR backbone trace and the histidine, tryptophan, and cystine residues are shown (PDB file 8LYZ; Beddell et al, 1975). 10.0°C. Unfolding and exchange were quenched by an 11-fold dilution with D 2 O buffer.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Unfolding Pathway of Hen Egg White Lysozyme

Laurents

1997

Biochemistry

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After the recent discovery of a ribonuclease A unfolding intermediate Nature 375, 513-515], we investigated the unfolding pathway of hen egg white lysozyme. At pH* 4.00 with D 2 O at 10°C and 6 M guanidinium chloride, unfolding shows a single, slow kinetic phase, with a relaxation time of 3300 s when monitored by circular dichroism (CD). Exchange of the tryptophan indole nitrogen protons shows that buried Trp residues 123, 111, and 108 lose tight packing and become solvent-exposed simultaneously, with a mean relaxation time of 3300 s, similar to the CDmonitored unfolding rate. Unfolding monitored by Trp fluorescence shows, moreover, that 90% of the amplitude change occurs in a slow phase, with a relaxation time of 2400 s. Faster-unfolding phases with minor amplitudes are detected by Trp indole hydrogen exchange and by fluorescence. It is likely that these changes are caused by Trp 62 and Trp 63, active site residues which are not buried in the hydrophobic core. Lysozyme unfolding was further monitored by the histidine 15 C 1 proton, which gives resolved lines for the native and unfolded species in one-dimensional 1 H-NMR spectra. The majority of the unfolding reaction, 70%, occurs in a slow phase with a relaxation time of 3600 s, but there is also a rapid unfolding phase; 30% of the His 15 C 1 proton resonance intensity is found at the unfolded chemical shift within tens of seconds after the start of unfolding. The amplitude of the rapid unfolding phase increases proportionally with the concentration of GdmCl denaturant present. These results show that a partially buried residue of lysozyme, histidine 15, takes part in forming an unfolding intermediate similar A. 92, 9318-9322]; the difference between that study and ours may reside in the greater sensitivity of 19 F to the detection of motional differences.Kinetic studies of the unfolding reactions of globular proteins monitored by CD 1 or fluorescence show that proteins typically unfold in a single kinetic step, N f U, with no detectable intermediates (Schmid, 1992; Mücke & Schmid, 1994). Thus, it was surprising when Kiefhaber et al. (1995) These results are startling. To test the generality of this behavior, we decided to study the unfolding of hen egg white lysozyme. Hen egg white lysozyme is composed of two domains: an R-domain that contains several helices and a -domain comprised largely of -strands (Figure 1). Lysozyme has many desirable characteristics for this work; its NMR assignments are known (Redfield & Dobson, 1988), and its refolding behavior has been studied in depth (Kiefhaber, 1995;Itzhaki et al., 1994;Dobson et al., 1994). Lysozyme unfolds very slowly, making the study of unfolding by 1D 1 H-NMR practical. Five of the protein's six tryptophan indole nitrogen protons give unique lines in 1D 1 H-NMR spectra. Upon denaturation, these lines disappear and the indole protons resonate in a common line at 10.06 ppm. Moreover, when the Trp side chain is exposed to D 2 O, the indole proton is exchanged for a deuteron and the NMR line disapp...

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“…The loss of a single disulfide bond was reported not to give major three-dimensional structural change (11,12) but to dramatically decrease the conformational stability (13 (15,16). These intramolecular bridges were shown not to cause structural constraint; therefore, they keep a similar native structure with enhancing protein stability (17,18). Using these derivatives, we evaluated the influence of the conformational stability of HEL on the steps of the antigen presentation pathway.…”

Section: An Antigen-specific Cd4mentioning

confidence: 99%

“…On the other hand, HEL was stabilized by cross-linking between Lys 1 and His 15 with alkyl chain, 1-15CL-HEL (14), and by crosslinking between Glu 35 and Trp 108 through an ester bond, 35-108CL-HEL (15, 16). These intramolecular bridges were shown not to cause structural constraint; therefore, they keep a similar native structure with enhancing protein stability (17,18). Using these derivatives, we evaluated the influence of the conformational stability of HEL on the steps of the antigen presentation pathway.…”

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confidence: 99%

Depression of T-cell Epitope Generation by Stabilizing Hen Lysozyme

So¹,

Ito²,

Koga³

et al. 1997

Journal of Biological Chemistry

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Conformational stability of proteins is an important factor that determines their resistance/susceptibility to proteolytic digestion. Intracellular proteolysis is the key step in antigen presentation events for protein antigens; hence, it is likely that increasing protein stability reduces the antigenicity of proteins. We prepared three hen egg white lysozyme derivatives possessing different stabilities by chemical modification to clarify the relationship between conformational stability and the antigenicity of the protein. One of the derivatives was conformationally unstabilized by removing one intramolecular disulfide bond, whereas the two others were stabilized by the addition of an intramolecular crosslink. The antigenicity of these derivatives was evaluated using hen egg white lysozyme-specific T-cell hybridoma cells and a B-lymphoma cell line, A20, as antigen-presenting cells. With an increase in conformational stability, the T-cell response decreased. However, the reduction was not derived from the inefficiency of internalization to A20 cells nor the alteration of antigenicity by chemical modifications. Moreover, from analyses of their susceptibility to proteolysis and the kinetics of presentation of the T-cell epitope, it was confirmed that increasing protein stability led to the depression of T-cell epitope generation by increasing resistance to proteolysis. These results have an important implication in devising a new strategy for manipulating T-cell response by the stability of protein antigen. An antigen-specific CD4ϩ T-cell recognizes an antigen-derived peptide that is mounted on a major histocompatibility complex class II molecule on a cell surface of antigen-presenting cells, via its antigen receptor (1, 2). Therefore, the conformation of protein antigens is unlikely to have any role in the step of T-cell recognition. Prior to T-cell activation, however, antigen processing is necessary for a protein antigen to stimulate the specific T-cells; this processing consists of multiple steps of cellular events, i.e. internalization of proteins by antigen-presenting cells, reduction of the disulfide bond and unfolding of proteins, enzymatic digestion, and assembly of the generated peptides with major histocompatibility complex class II molecules (3, 4). Proteases preferentially digest proteins in an unfolded state rather than those in a folded state (5-7); thus, the unfolding may be a crucial step for intracellular antigen processing. In this context, we can expect that depression of protein unfolding by increasing protein stability would reduce the antigenicity of proteins for T-cells.Several reports have demonstrated a relationship between increased antigenicity and the decreased stability of proteins (8 -10). However, the influence of protein stability on the antigenicity remains unknown. To address this issue, we prepared three derivatives of hen egg white lysozyme (HEL) 1 possessing different conformational stabilities (see Table I). A three-disulfide derivative of HEL, S-carboxymethylated HEL at Cys ...

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An X-ray study of the structure and binding properties of iodine-inactivated lysozyme

Cited by 46 publications

References 19 publications

Influence of acarbose and maltose on the reactivity of individual tryptophanyl residues in glucoamylase from aspergillus niger

Influence of acarbose and maltose on the reactivity of individual tryptophanyl residues in glucoamylase from aspergillus niger

Characterization of the Unfolding Pathway of Hen Egg White Lysozyme

Depression of T-cell Epitope Generation by Stabilizing Hen Lysozyme

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