Lysine 58-cleaved beta2-microglobulin is not detectable by 2D electrophoresis in ex vivo amyloid fibrils of two patients affected by dialysis-related amyloidosis

The lysine 58 cleaved and truncated variant of beta(2)-microglobulin (DeltaK58-beta2m) is conformationally unstable and present in the circulation of a large percentage of patients on chronic hemodialysis, suggesting that it could play a role in the beta2-microglobulin (beta2m) amyloid fibrillogenesis associated with dialysis-related amyloidosis (DRA). However, it has yet to be detected in the amyloid deposits of such patients. Here, we extracted amyloid fibrils, without denaturation or additional purification, from different amyloidotic tissues of two unrelated individuals suffering from DRA, and characterized them by high-sensitivity bidimensional gel electrophoresis (2D-PAGE), immunoblotting, MALDI time-of-flight mass spectrometry, and protein sequencing. To confirm whether or not this species could be identified by our proteomic approaches, we mapped its location in 2D-PAGE, in mixtures of pure DeltaK58-beta2m, and extracts of amyloid fibrils from patients, to a discrete region of the gel distinct from other isoforms of beta2m. Using this approach, the two known principal isoforms found in beta2m amyloid were identified, namely, the full-length protein and the truncated species lacking six N-terminal amino acid residues (DeltaN6-beta2m). In contrast, we found no evidence for the presence of DeltaK58-beta2m.     

Characterization of the pressure-induced intermediate and unfolded state of red-shifted green fluorescent protein--a static and kinetic FTIR,UV/VIS and fluorescence spectroscopy study

The green fluorescence proteins (GFP) are widely used as reporters in molecular and cell biology. For their use it in high-pressure microbiology and biotechnology studies, their structural properties, thermodynamic parameters and stability diagrams have to be known. We investigated the pressure stability of the red-shifted green fluorescent protein (rsGFP) using Fourier-transform infrared spectroscopy, fluorescence and UV/Vis spectroscopy. We found that rsGFP does not unfold up to approximately 9kbar at room temperature. Its unique three-dimensional structure is held responsible for the high-pressure stability. At higher temperatures, its secondary structure collapses below 9kbar (e.g. the denaturation pressure at 58 degrees C is 7.8kbar). The analysis of the IR data shows that the pressure-denatured state contains more disordered structures at the expense of a decrease of intramolecular beta-sheets. As indicated by the large volume change of DeltaV degrees (u) approximately -250(+/-50)mlmol(-1) at 58 degrees C, this highly cooperative transition can be interpreted as a collapse of the beta-can structure of rsGFP. For comparison, the temperature-induced unfolding of rsGFP has also been studied. At high temperature (T(m)=78 degrees C), the unfolding resulted in the formation of an aggregated state. Contrary to the pressure-induced unfolding, the temperature-induced unfolding and aggregation of GFP is irreversible. From the FT-IR data, a tentative p,T-stability diagram for the secondary structure collapse of GFP has been obtained. Furthermore, changes in fluorescence and absorptivity were found which are not correlated to the secondary structural changes. The fluorescence and UV/Vis data indicate smaller conformational changes in the chromophore region at much lower pressures ( approximately 4kbar) which are probably accompanied by the penetration of water into the beta-can structure. In order to investigate also the kinetics of this initial step, pressure-jump relaxation experiments were carried out. The partial activation volumes observed indicate that the conformational changes in the chromophore region when passing the transition state are indeed rather small, thus leading to a comparably small volume change of -20 ml mol(-1) only. The use of the chromophore absorption and fluorescence band of rsGFP in using GFP as reporter for gene expression and other microbiological studies under high pressure conditions is thus limited to pressures of about 4kbar, which still exceeds the pressure range relevant for studies in vivo in micro-organisms, including piezophilic bacteria from deep-sea environments.     

Peptide and beta 2-microglobulin regulation of cell surface MHC class I conformation and expression.

We have examined the roles of peptide and beta 2-microglobulin (beta 2m) in regulating the conformation and expression level of class I molecules on the cell surface. Using a cell line synthesizing H-2Dd H chain and mouse beta 2m but defective in endogenous peptide loading, we demonstrate the ability of either exogenous peptide or beta 2m alone to increase surface H-2Dd expression at both 25 degrees C and 37 degrees C. Peptide and beta 2m show marked synergy in their abilities to increase surface class I expression, with minimal increases promoted by peptide in the absence of free beta 2m. Low temperature-induced molecules have indistinguishable rates of loss of beta 2m and alpha 1/alpha 2 domain conformational epitopes during culture at 37 degrees C. However, the rate of alpha 3 epitope loss is much slower, indicating a minimum of two steps in class I loss from the cell surface: 1) loss of beta 2m binding to H chain and unfolding of the alpha 1/alpha 2 region; then 2) denaturation, degradation, or internalization of the free H chains possessing alpha 3 epitopes. These data show for the first time that free H chains survive for a finite time on the membrane in a form capable of refolding into alpha 1/alpha 2 epitope positive molecules upon addition of beta 2m and peptide. This refolding in the presence of beta 2m and peptide can explain the reported requirement for both components in sensitizing cells for class I-dependent CTL lysis. It also indicates that such conformational changes in class I molecules are not strictly dependent on either newly synthesized H chains or on intracellular chaperons. The study of H chain-peptide-beta 2m interaction on the cell surface may be relevant to understanding intracellular peptide loading events.     

A native to amyloidogenic transition regulated by a backbone trigger

Many polypeptides can self-associate into linear, aggregated assemblies termed amyloid fibers. High-resolution structural insights into the mechanism of fibrillogenesis are elusive owing to the transient and mixed oligomeric nature of assembly intermediates. Here, we report the conformational changes that initiate fiber formation by beta-2-microglobulin (beta2m) in dialysis-related amyloidosis. Access of beta2m to amyloidogenic conformations is catalyzed by selective binding of divalent cations. The chemical basis of this process was determined to be backbone isomerization of a conserved proline. On the basis of this finding, we designed a beta2m variant that closely adopts this intermediate state. The variant has kinetic, thermodynamic and catalytic properties consistent with its being a fibrillogenic intermediate of wild-type beta2m. Furthermore, it is stable and folded, enabling us to unambiguously determine the initiating conformational changes for amyloid assembly at atomic resolution.     

Co-populated conformational ensembles of beta2-microglobulin uncovered quantitatively by electrospray ionization mass spectrometry

Ordered assembly of monomeric human beta(2)-microglobulin (beta(2)m) into amyloid fibrils is associated with the disorder hemodialysis-related amyloidosis. Previously, we have shown that under acidic conditions (pH <5.0 at 37 degrees C), wild-type beta(2)m assembles spontaneously into fibrils with different morphologies. Under these conditions, beta(2)m populates a number of different conformational states in vitro. However, this equilibrium mixture of conformationally different species is difficult to resolve using ensemble techniques such as nuclear magnetic resonance or circular dichroism. Here we use electrospray ionization mass spectrometry to resolve different species of beta(2)m populated between pH 6.0 and 2.0. We show that by linear deconvolution of the charge state distributions, the extent to which each conformational ensemble is populated throughout the pH range can be determined and quantified. Thus, at pH 3.6, conditions under which short fibrils are produced, the conformational ensemble is dominated by a charge state distribution centered on the 9+ ions. By contrast, under more acidic conditions (pH 2.6), where long straight fibrils are formed, the charge state distribution is dominated by the 10+ and 11+ ions. The data are reinforced by investigations on two variants of beta(2)m (V9A and F30A) that have reduced stability to pH denaturation and show changes in the pH dependence of the charge state distribution that correlate with the decrease in stability measured by tryptophan fluorescence. The data highlight the potential of electrospray ionization mass spectrometry to resolve and quantify complex mixtures of different conformational species, one or more of which may be important in the formation of amyloid.     

Variants of beta-microglobulin cleaved at lysine-58 retain the main conformational features of the native protein but are more conformationally heterogeneous and unstable at physiological temperature

Cleavage of the small amyloidogenic protein beta2-microglobulin after lysine-58 renders it more prone to unfolding and aggregation. This is important for dialysis-related beta2-microglobulin amyloidosis, since elevated levels of cleaved beta2-microglobulin may be found in the circulation of dialysis patients. However, the solution structures of these cleaved beta2-microglobulin variants have not yet been assessed using single-residue techniques. We here use such methods to examine beta2-microglobulin cleaved after lysine-58 and the further processed variant (found in vivo) from which lysine-58 is removed. We find that the solution stability of both variants, especially of beta2-microglobulin from which lysine-58 is removed, is much reduced compared to wild-type beta2-microglobulin and is strongly dependent on temperature and protein concentration. 1H-NMR spectroscopy and amide hydrogen (1H/2H) exchange monitored by MS show that the overall three-dimensional structure of the variants is similar to that of wild-type beta2-microglobulin at subphysiological temperatures. However, deviations do occur, especially in the arrangement of the B, D and E beta-strands close to the D-E loop cleavage site at lysine-58, and the experiments suggest conformational heterogeneity of the two variants. Two-dimensional NMR spectroscopy indicates that this heterogeneity involves an equilibrium between the native-like fold and at least one conformational intermediate resembling intermediates found in other structurally altered beta2-microglobulin molecules. This is the first single-residue resolution study of a specific beta2-microglobulin variant that has been found circulating in dialysis patients. The instability and conformational heterogeneity of this variant suggest its involvement in beta2-microglobulin amyloidogenicity in vivo.     

Molecular dynamics simulations to investigate the domain swapping mechanism of human cystatin C

Human cystatin C (HCC), one of the amyloidgenic proteins, has been proved to form a dimeric structure via a domain swapping process and then cause amyloid deposits in the brains of patients suffering from Alzheimer's disease. HCC monomer consists of a core with a five-stranded antiparallel beta-sheet (beta region) wrapped around a central helix. The connectivity of these secondary structures is: (N)-beta1-alpha-beta2-L1-beta3-AS-beta4-L2-beta5-(C). In this study, various molecular dynamics simulations were conducted to investigate the conformational changes of the monomeric HCC at different temperatures (300 and 500 K) and pH levels (2, 4, and 7) to gain insight into the domain swapping mechanism. The results show that high temperature (500 K) and low pH (pH 2) will trigger the domain swapping process of HCC. We further proposed that the domain swapping mechanism of HCC follows four steps: (1) the alpha-helix moves away from the beta region; (2) the contacts between beta2 and beta3-AS disappear; (3) the beta2-L1-beta3 hairpin unfolds following the so-called "zip-up" mechanism; and finally (4) the HCC dimer is formed. Our study shows that high temperature can accelerate the unfolding of HCC and the departure of the alpha-helix from the beta-region, especially at low pH value. This is attributed to the fact that that low pH results in the protonation of the side chains of Asp, Glu, and His residues, which further disrupts the following four salt-bridge interactions stabilizing the alpha-beta interface of the native structure: Asp15-Arg53 (beta1-beta2), Glu21/20-Lys54 (helix-beta2), Asp40-Arg70 (helix-AS), and His43-Asp81 (beta2-AS).     

From chance to frequent encounters: origins of beta2-microglobulin fibrillogenesis

It is generally accepted that amyloid formation requires partial, but not complete unfolding of a polypeptide chain. Amyloid formation by beta-2 microglobulin (beta2m), however, readily occurs under strongly native conditions provided that there is exposure to specific transition metal cations. In this review, we discuss transition metal catalyzed conformational changes in several amyloidogenic systems including prion protein, Alzheimer's and Parkinson's diseases. For some systems, including beta2m from dialysis related amyloidosis (DRA), catalysis overcomes an entropic barrier to protein aggregation. Recent data suggest that beta2m samples conformations that are under thermodynamic control, resulting in local or partial unfolding under native conditions. Furthermore, exposure to transition metal cations stabilizes these partially unfolded states and promotes the formation of small oligomers, whose structures are simultaneously near-native and amyloid-like. By serving as a tether, Cu(2+) enables the encounter of amyloidogenic conformations to occur on time scales which are significantly more rapid than would occur between freely diffusing monomeric protein. Once amyloid formation occurs, the requirement for Cu(2+) is lost. We assert that beta2m amyloid fiber formation at neutral pH may be facilitated by rearrangements catalyzed by the transient and pair wise tethering of beta2m at the blood/dialysate interface present during therapeutic hemodialysis.     

Main-chain dominated amyloid structures demonstrated by the effect of high pressure

It has been suggested that, while the globular native forms of proteins are a side-chain-dominated compact structure evolved by pursuing a unique fold with optimal packing of amino acid residues, amyloid fibrils are a main-chain-dominated structure with an extensive hydrogen bond network. To address this issue, the effects of hydrostatic pressure on amyloid fibrils of beta2-microglobulin (beta2-m), involved in dialysis-related amyloidosis, were studied. A systematic analysis at various pressures and concentrations of guanidine hydrochloride conducted by monitoring thioflavin T fluorescence, light-scattering, and tryptophan fluorescence revealed contrasting conformational changes occurring consecutively: first, a pressure-induced reorganization of fibrils and then a pressure-induced unfolding. The changes in volume as well as the observed structural changes indicate that the beta2-m amyloid fibrils under ambient pressure are less tightly packed with a larger number of cavities, consistent with the main-chain-dominated amyloid structure. Moreover, the amyloid structure without optimal packing will enable various isoforms to form, suggesting the structural basis of multiple forms of amyloid fibrils in contrast to the unique native-fold.     

Temperature-induced conformational changes in amyloid beta(1-40) peptide investigated by simultaneous FT-IR microspectroscopy with thermal system

Temperature-dependent secondary structures of the amyloid beta(1-40) peptide in the solid state were studied by simultaneous Fourier transform infrared/differential scanning calorimetry (FT-IR/DSC) microspectroscopic system with the heating-cooling-reheating cycle. The result indicates that a thermal transition temperature at 45 degrees C was easily obtained from the three-dimensional plot of the transmission FT-IR spectra as a function of temperature. Furthermore, the thermal-dependent conformational transformations, due to denaturation and aggregation, of solid amyloid beta(1-40) were mainly evidenced by reducing the compositions from 37 to 20-24% for alpha-helical and random coil structures but increasing the components from 27 to 45% for intermolecular beta-sheet structures. Thermal-irreversible behavior and a poor thermal stability of solid amyloid beta(1-40) were also observed from the poor restoration of the secondary conformational changes in the heated sample.     

A β2-microglobulin cleavage variant fibrillates at near-physiological pH

β₂-microglobulin (β₂m) deposits as amyloid in dialysis-related amyloidosis (DRA), predominantly in joints. The molecular mechanisms underlying the amyloidogenicity of β₂m are still largely unknown. In vitro, acidic conditions, pH < 4.5, induce amyloid fibrillation of native β₂m within several days. Here, we show that amyloid fibrils are generated in less than an hour when a cleavage variant of β₂m—found in the circulation of many dialysis patients—is exposed to pH levels (pH 6.6) occurring in joints during inflammation. Aggregation and fibrillation, including seeding effects with intact, native β₂m were studied by Thioflavin T fluorescence spectroscopy, turbidimetry, capillary electrophoresis, and electron microscopy. We conclude that a biologically relevant variant of β₂m is amyloidogenic at slightly acidic pH. Also, only a very small amount of preformed fibrils of this variant is required to induce fibrillation of native β₂m. This may explain the apparent lack of detectable amounts of the variant β₂m in extracts of amyloid from DRA patients.     

Thermal and conformational stability of seed coat soybean peroxidase

Soybean peroxidase (SBP) obtained from the soybean seed coats belongs to class III of the plant peroxidase superfamily. Detailed circular dichroism and steady state fluorescence studies have been carried out to monitor thermal as well as denaturant-induced unfolding of SBP and apo-SBP. Melting of secondary and tertiary structures of SBP occurs with characteristic transition midpoints, T(m), of 86 and 83.5 degrees C, respectively, at neutral pH. Removal of heme resulted in greatly decreased thermal stability of the protein (T(m) = 38 degrees C). The deltaG degrees (H2O) determined from guanidine hydrochloride-induced denaturation at 25 degrees C and at neutral pH is 43.3 kJ mol(-1) for SBP and 9.0 kJ mol(-1) for apo-SBP. Comparison with the reported unfolding data of the homologous enzyme, horseradish peroxidase (HRP-C), showed that SBP exhibits significantly high thermal and conformational stability. We show that this enhanced structural stability of SBP relative to HRP-C arises due to the unique nature of their heme binding. A stronger heme-apoprotein affinity probably due to the interaction between Met37 and the C8 heme vinyl substituent contributes to the unusually high structural stability of SBP.     

Association of heat-induced conformational change with activity loss of Rubisco.

Circular dichroism (CD), fluorescence, and differential scanning calorimetry (DSC) were used to investigate the thermal conformational change associated with the activity loss of spinach Rubisco. CD and intrinsic fluorescence demonstrated a three stage thermal unfolding of Rubisco. At 25-45 degrees C, the secondary structure did not change but the tertiary and/or quaternary structure changed obviously with increased temperature. In 45-60 degrees C, the secondary structure showed much change with increased temperature and the tertiary and/or quaternary structure changed much faster. Over 60 degrees C, whole conformation changed abruptly with increased temperature and finally unfolded completely. DSC, CD and activity assays after annealing showed that the conformational change and the activity loss of Rubisco were completely reversible if the heating temperature was below 45 degrees C, partly reversible between 45 and 60 degrees C, and irreversible beyond 60 degrees C.     

Conformational unfolding studies of three-disulfide mutants of bovine pancreatic ribonuclease A and the coupling of proline isomerization to disulfide redox reactions

The equilibrium stability and conformational unfolding kinetics of the [C40A, C95A] and [C65S, C72S] mutants of bovine pancreatic ribonuclease A (RNase A) have been studied. These mutants are analogues of two nativelike intermediates, des[40-95] and des[65-72], whose formation is rate-limiting for oxidative folding and reductive unfolding at 25 degrees C and pH 8.0. Upon addition of guanidine hydrochloride, both mutants exhibit a fast conformational unfolding phase when monitored by absorbance and fluorescence, as well as a slow phase detected only by fluorescence which corresponds to the isomerizations of Pro93 and Pro114. The amplitudes of the slow phase indicate that the two prolines, Pro93 and Pro114, are fully cis in the folded state of the mutants and furthermore that the 40-95 disulfide bond is not responsible for the quenching of Tyr92 fluorescence observed in the slow unfolding phase, contrary to an earlier proposal [Rehage, A., and Schmid, F. X. (1982) Biochemistry 21, 1499-1505]. The ratio of the kinetic unfolding m value to the equilibrium m value indicates that the transition state for conformational unfolding in the mutants exposes little solvent-accessible area, as in the wild-type protein, indicating that the unfolding pathway is not dramatically altered by the reduction of the 40-95 or 65-72 disulfide bond. The stabilities of the folded mutants are compared to that of wild-type RNase A. These stabilities indicate that the reduction of des[40-95] to the 2S species is rate-limited by global conformational unfolding, whereas that of des[65-72] is rate-limited by local conformational unfolding. The isomerization of Pro93 may be rate-limiting for the reduction of the 40-95 disulfide bond in the native protein and in the des[65-72] intermediate.     

Structural stability and unfolding properties of thermostable bacterial alpha-amylases: a comparative study of homologous enzymes

In a comparative investigation on two thermostable alpha-amylases [Bacillus amyloliquefaciens (BAA), T(m) = 86 degrees C and Bacillus licheniformis (BLA), T(m) = 101 degrees C], we studied thermal and guanidine hydrochloride (GndHCl)-induced unfolding using fluorescence and CD spectroscopy, as well as dynamic light scattering. Depletion of calcium from specific ion-binding sites in the protein structures reduces the melting temperature tremendously for both alpha-amylases. The reduction is nearly the same for both enzymes, namely, in the order of 50 degrees C. Thus, the difference in thermostability between BLA and BAA (DeltaT(m) approximately 15 degrees C) is related to intrinsic properties of the respective protein structures themselves and is not related to the strength of ion binding. The thermal unfolding of both proteins is characterized by a full disappearance of secondary structure elements and by a concurrent expansion of the 3D structure. GndHCl-induced unfolding also yields a fully vanishing secondary structure but with more expanded 3D structures. Both alpha-amylases remain much more compact upon thermal unfolding as compared to the fully unfolded state induced by chemical denaturants. Such rather compact thermal unfolded structures lower the conformational entropy change during the unfolding transition, which principally can contribute to an increased thermal stability. Structural flexibilities of both enzymes, as measured with tryptophan fluorescence quenching, are almost identical for both enzymes in the native states, as well as in the unfolded states. Furthermore, we do not observe any difference in the temperature dependence of the structural flexibilities between BLA and BAA. These results indicate that conformational dynamics on the time scale of our studies seem not to be related to thermal stability or to thermal adaptation.     

Heat-induced changes in the conformation of alpha- and beta-crystallins: unique thermal stability of alpha-crystallin

Of the crystallin proteins of the lens, the principal subunit of the beta-crystallin, beta B2 (beta Bp), has been considered to be the only heat-stable protein because it does not precipitate upon heating. In our recent investigations, however, we have found that the alpha-crystallin from bovine lenses is not only heat stable but also does not denature at temperatures up to 100 degrees C. Using circular dichroism and fluorescence to monitor the conformational changes of alpha- and beta B2-crystallins upon heating, we found that alpha-crystallin maintains a high degree of structure, whereas the beta B2-crystallin shows a reversible sigmoidal order-disorder transition at about 58 degrees C.     

Impact of the tryptophan residues of Humicola lanuginosa lipase on its thermal stability.

Thermal stability of wild type Humicola lanuginosa lipase (wt HLL) and its two mutants, W89L and the single Trp mutant W89m (W117F, W221H, and W260H), were compared. Differential scanning calorimetry revealed unfolding of HLL at T(d)=74.4 degrees C whereas for W89L and W89m this endotherm was decreased to 68.6 and 62 degrees C, respectively, demonstrating significant contribution of the above Trp residues to the structural stability of HLL. Fluorescence emission spectra revealed the average microenvironment of Trps of wt HLL and W89L to become more hydrophilic at elevated temperatures whereas the opposite was true for W89m. These changes in steady-state emission were sharp, with midpoints (T(m)) at approx. 70.5, 61.0, and 65.5 degrees C for wt HLL, W89L, and W89m, respectively. Both steady-state and time resolved fluorescence spectroscopy further indicated that upon increasing temperature, the local movements of tryptophan(s) in these lipases were first attenuated. However, faster mobilities became evident when the unfolding temperatures (T(m)) were exceeded, and the lipases became less compact as indicated by the increased hydrodynamic radii. Even at high temperatures (up to 85 degrees C) a significant extent of tertiary and secondary structure was revealed by circular dichroism. Activity measurements are in agreement with increased amplitudes of conformational fluctuations of HLL with temperature. Our results also indicate that the thermal unfolding of these lipases is not a two-state process but involves intermediate states. Interestingly, a heating and cooling cycle enhanced the activity of the lipases, suggesting the protein to be trapped in an intermediate, higher energy state. The present data show that the mutations, especially W89L in the lid, contribute significantly to the stability, structure and activity of HLL.     

A test of the relationship between sequence and structure in proteins: excision of the heme binding site in apocytochrome b5.

The water-soluble domain of rat hepatic holocytochrome b5 is an alphabeta protein containing elements of secondary structure in the sequence beta1-alpha1-beta4-beta3-alpha2-alpha3-beta5- alpha4-alpha5-beta2-alpha6. The heme group is enclosed by four helices, a2, a3, a4, and a5. To test the hypothesis that a small b hemoprotein can be constructed in two parts, one forming the heme site, the other an organizing scaffold, a protein fragment corresponding to beta1-alpha1-beta4-beta3-lambda-beta2-alpha6 was prepared, where lambda is a seven-residue linker bypassing the heme binding site. The fragment ("abridged b5") was found to contain alpha and beta secondary structure by circular dichroism spectroscopy and tertiary structure by Trp fluorescence emission spectroscopy. NMR data revealed a species with spectral properties similar to those of the full-length apoprotein. This folded form is in slow equilibrium on the chemical shift time scale with other less folded species. Thermal denaturation, as monitored by circular dichroism, absorption, and fluorescence spectroscopy, as well as size-exclusion chromatography-fast protein liquid chromatography (SEC-FPLC), confirmed the coexistence of at least two distinct conformational ensembles. It was concluded that the protein fragment is capable of adopting a specific fold likely related to that of cytochrome b5, but does not achieve high thermodynamic stability and cooperativity. Abridged b5 demonstrates that the spliced sequence contains the information necessary to fold the protein. It suggests that the dominating influence to restrict the conformational space searched by the chain is structural propensities at a local level rather than internal packing. The sequence also holds the properties necessary to generate a barrier to unfolding.     

Tracking local conformational changes of ribonuclease A using picosecond time-resolved fluorescence of the six tyrosine residues

The six tyrosine residues of ribonuclease A (RNase A) are used as individual intrinsic probes for tracking local conformational changes during unfolding. The fluorescence decays of RNase A are well described by sums of three exponentials with decay times (tau(1) = 1.7 ns, tau(2) = 180 ps, and tau(3) = 30 ps) and preexponential coefficients (A(1) = 1, A(2) = 1, and A(3) = 4) at pH 7, 25 degrees C. The decay times are controlled by photo-induced electron transfer from individual tyrosine residues to the nearest disulphide (-SS-), bridge, which is distance (R) dependent. We assign tau(1) to Tyr-76 (R = 12.8 A), tau(2) to Tyr-115 (R = 6.9 A), and tau(3) to Tyr-25, Tyr-73, Tyr-92, and Tyr-97 (all four at R = 5.5 +/- 0.3 A) at 23 degrees C. On the basis of this assignment, the results show that, upon thermal or chemical unfolding only Tyr-25, Tyr-92, and Tyr-76 undergo significant displacement from their nearest -SS- bridge. Despite reporting on different regions of the protein, the concordance between the transition temperatures, T(m), obtained from Tyr-76 (T(m) = 59.2 degrees C) and Tyr-25 and Tyr-92 (T(m) = 58.2 degrees C) suggests a single unfolding event in this temperature range that affects all these regions similarly.