Accessibility Of Tryptophan Residues Research Articles

Fluorescence and circular dichroism studies on the accessibility of tryptophan residues and unfolding of a jacalin-related α-d-galactose-specific lectin from mulberry (Morus indica)

MLGL (Mulberry Latex Galactose-specific Lectin) is an α-d-galactose binding lectin isolated from the latex of mulberry (Morus indica) tree and contains two tryptophan residues in each of its subunits. The fluorescence emission maximum of native MLGL seen at 326nm shifts to 350nm upon incubation with 6M guanidinium thiocyanate (Gdn.SCN), suggesting that the tryptophans are located inside the hydrophobic core of the protein and become fully exposed upon denaturation. Fluorescence quenching studies revealed that the neutral acrylamide exhibits the highest quenching, with ~33% of total fluorescence in the native protein being quenched at a quencher concentration of 0.5M, whereas iodide (~24%) and cesium (~4%) ions showed significantly lower quenching. With the denatured protein, acrylamide quenching involves both dynamic and static processes as evident from an upward curving Stern-Volmer plot. Time-resolved fluorescence studies showed two lifetime components of 3.7ns and 1.3ns for the native protein, while three lifetime components were observed for the denatured protein. MLGL showed high resistance to urea (up to 8M) and guanidine hydrochloride (up to 6M), whereas treatment with 6M Gdn.SCN completely denatured the protein, via a broad sigmoidal transition with a transition midpoint at ~3.75M. Circular dichroism studies and hemagglutination assays showed that the secondary and tertiary structures as well as lectin activity of MLGL were unaffected up to 70°C. Additionally, pH dependent studies showed that the secondary structure of MLGL is unaltered in the pH range 6.2 to 8.5, but a decrease in lectin activity is observed (~50%) at pH6.2.

Fructose-induced modifications of myoglobin: Change of structure from met (Fe 3+) to oxy (Fe 2+) form

We studied structural modifications of metmyoglobin (Mb) after short-term (6 days) and long-term (30 days) glycation by fructose (fructation). Fructation caused gradual changes in the structure of the protein with respect to increased absorbance at 280 nm, enhanced fluorescence emission (with excitation at 285 nm), increased surface accessible tryptophan residues and reduced α-helix content and change in tertiary structure. However, long-term fructation changed Mb to oxymyoglobin (MbO 2), as demonstrated by different spectroscopic (absorption, fluorescence, circular dichroic and electron paramagnetic resonance) studies and trifluoperazine-induced oxygen release experiment. Fructation appeared to modify Arg139 to arg-pyrimidine, which exhibited antioxidative activity and might be involved in the conversion of met (Fe 3+) to oxy (Fe 2+) form of myoglobin.

Non-enzymatic glycation induces structural modifications of myoglobin

Increased glucose concentration in diabetes mellitus causes glycation of several proteins, leading to changes in their properties. Although glycation-induced functional modification of myoglobin is known, structural modification of the protein has not yet been reported. Here, we have studied glucose-modified structural changes of the heme protein. After in vitro glycation of metmyoglobin (Mb) by glucose at 25 degrees C for 6 days, glycated myoglobin (GMb) and unchanged Mb have been separated by ion exchange (BioRex 70) chromatography, and their properties have been compared. Compared to Mb, GMb exhibits increased absorbance around 280 nm and enhanced fluorescence emission with excitation at 285 nm. Fluorescence quenching experiments of the proteins by acrylamide and KI indicate that more surface accessible tryptophan residues are exposed in GMb. CD spectroscopic study reveals a change in the secondary structure of GMb with decreased alpha-helix content. 1-anilino-naphthaline-8-sulfonate (ANS) binding with Mb and GMb indicates that glycation increases hydrophobicity of the heme protein. GMb appears to be less stable with respect to thermal denaturation and differential calorimetry experiments. Heme-globin linkage becomes weaker in GMb, as shown by spectroscopic and gel electrophoresis experiments. A correlation between glycation-induced structural and functional modifications of the heme protein has been suggested.

Conformational stability of cytochrome b 5, enhanced green fluorescent protein, and their fusion protein Hmwb5-EGFP

The conformational stabilities of chimeric protein Hmwb(5)-EGFP and its constituents (cytochrome b(5) and enhanced green fluorescent protein) in guanidine hydrochloride solutions are reported in this paper. Intensity of fluorescence of tryptophan residues, intensity of EGFP fluorescence in the visible region, absorbance of cytochrome b(5) heme and EGFP fluorophore, and fluorescence anisotropy were used to follow the unfolding process. Thermodynamic parameters of protein unfolding were obtained using different approaches. The data were analyzed using a two-stage model and a linear extrapolation method. Unfolding of protein molecules was additionally monitored by measuring Stern-Volmer constants for tryptophan fluorescence quenching by acrylamide, cesium, and iodide. The accessibility of tryptophan residues of both components in the fusion molecule is lower than in the separate molecules. The thermodynamic stability of the protein globules in the fusion protein is much lower than in the individual protein molecules in solution, the difference in free energy of unfolding being more considerable for cytochrome b(5) (29 +/- 4 and 13 +/- 2 kJ/mol) than for EGFP (26 +/- 0.9 and 20 +/- 2.7 kJ/mol). The data indicate that artificial protein fusion can greatly affect total structural stability, and in the case of cytochrome b(5) and EGFP it results in decrease in free energy of transition from native to denatured unfolded form and consequently to decrease in thermodynamic stability of protein globules compared to the separate proteins.

Chemoselective Tryptophan Labeling with Rhodium Carbenoids at Mild pH

Significant improvements have been made to a previously reported tryptophan modification method using rhodium carbenoids in aqueous solution, allowing the reaction to proceed at pH 6-7. This technique is based on the discovery that N-(tert-butyl)hydroxylamine promotes indole modification with rhodium carbenoids over a broad pH range (2-7). This methodology was demonstrated on peptide and protein substrates, generally yielding 40-60% conversion with excellent tryptophan chemoselectivity. The solvent accessibility of the indole side chains was found to be a key factor in successful carbenoid addition, as demonstrated by conducting the reaction at temperatures high enough to cause thermal denaturation of the protein substrate. Progress toward the expression of proteins bearing solvent accessible tryptophan residues as reactive handles for modification with rhodium carbenoids is also reported.

Fructose-induced structural and functional modifications of hemoglobin: Implication for oxidative stress in diabetes mellitus

Increased fructose concentration in diabetes mellitus causes fructation of several proteins. Here we have studied fructose-induced modifications of hemoglobin. We have demonstrated structural changes in fructose-modified hemoglobin (Fr-Hb) by enhanced fluorescence emission with excitation at 285 nm, more surface accessible tryptophan residues by using acrylamide, changes in secondary and tertiary structures by CD spectroscopy, and increased thermolability by using differential scanning calorimetry in comparison with those of normal hemoglobin, HbA 0. Release of iron from hemoglobin is directly related with the extent of fructation. H 2O 2-induced iron release from Fr-Hb is significantly higher than that from HbA 0. In the presence of H 2O 2, Fr-Hb degrades arachidonic acid, deoxyribose and plasmid DNA more efficiently than HbA 0, and these processes are significantly inhibited by desferrioxamine or mannitol. Thus increased iron release from Fr-Hb may cause enhanced formation of free radicals and oxidative stress in diabetes. Compared to HbA 0, Fr-Hb exhibits increased carbonyl formation, an index of oxidative modification. Functional modification in Fr-Hb has also been demonstrated by its decreased peroxidase activity and increased esterase activity in comparison with respective HbA 0 activities. Molecular modeling study reveals Lys 7(α), Lys 127(α) and Lys 66(β) to be the probable potential targets for fructation in HbA 0.

Development of indole chemistry to label tryptophan residues in protein for determination of tryptophan surface accessibility

Solvent accessibility can be used to evaluate protein structural models, identify binding sites, and characterize protein conformational changes. The differential modification of amino acids at specific sites enables the accessible surface residues to be identified by mass spectrometry. Tryptophan residues within proteins can be differentially labeled with halocompounds by a photochemical reaction. In this study, tryptophan residues of carbonic anhydrase are reacted with chloroform, 2,2,2-trichloroethanol (TCE), 2,2,2-trichloroacetate (TCA), or 3-bromo-1-propanol (BP) under UV irradiation at 280 nm. The light-driven reactions with chloroform, TCE, TCA, and BP attach a formyl, hydroxyethanone, carboxylic acid, and propanol group, respectively, onto the indole ring of tryptophan. Trypsin and chymotrypsin digests of the modified carbonic anhydrase are used to map accessible tryptophan residues using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Tryptophan reactivity is determined by identifying peptides with tryptophan residues modified with the appropriate label. The reactivity is calculated from the frequency that the modification is identified and a semiquantitative measure of the amount of products formed. Both of these measures of tryptophan reactivity correlate significantly with the accessible surface area of tryptophan residues in carbonic anhydrase determined from the X-ray crystal structure. Therefore the photochemical reaction of halocompounds with tryptophan residues in carbonic anhydrase indicates the degree of solvent accessibility of these residues.

Conformational changes of maize and wheat NADP-malic enzyme studied by quenching of protein native fluorescence

Quenching of tryptophan fluorescence of maize and wheat NADP-malic enzyme by KI and acrylamide was studied after denaturating proteins with guanidine hydrochloride, and subjecting them to different pH values or temperatures. Protein unfolding by guanidine hydrochloride resulted in a red shift of the fluorescence spectrum, providing further support for the motion that several of the tryptophan residues evolved from an apolar to a polar environment. Protein denaturation was accompanied by an increase in the effective dynamic quenching constant values and by loss of the enzyme's activities. Thermal denaturation gave results consistent with the ones observed for chemical denaturation suggesting that a putative intermediate is involved in the denaturation process. Finally, exposure of both enzymes at various pH values allowed us to infer the number of accessible tryptophan residues in the different oligomeric conformations. The results suggest that the aggregation process seems to be different for each enzyme. Thus, as the maize enzyme associated from monomer to tetramer, one tryptophan residue would change from a polar to an apolar environment, while the association of the wheat enzyme would cause that two tryptophan residues to be excluded from quenching. Hitherto, quenching of the tryptophan fluorescence provides a good tool for studying conformational changes of proteins. The future availability of the crystal structures of plant NADP-malic enzymes will offer a good validation point for our model and the technology used.

Role of tryptophan residues of lipoxygenase-1 in activity, structure and stability: chemical modification studies with N-bromosuccinimide

The tryptophan residues of lipoxygenase-1 (LOX1) from soybeans (Glycine max) were modified using N-bromosuccinimide under both native and denaturing conditions. The accessibility of tryptophan residues was pH-dependent. Only one tryptophan residue was accessible at the optimum pH of enzyme activity and with a decrease in pH from 9 to 2, the accessibility increased. Modification of the accessible four tryptophan residues at pH 4.0 under non-denaturing conditions resulted in complete loss of enzyme activity; one tryptophan residue was critical for enzyme activity. Modification of the tryptophan residues did not alter the substrate binding affinity; the presence of the substrate during modification did not alter the extent of modification. Modification of the surface-exposed tryptophans did not affect (i) the conformation or structural integrity but (ii) decreased the stability.

Accessibility of Tryptophan Residues in Immunoglobulin M as an Index of Its Conformational Changeability

Demonstrated herein is the possibility of using the accessibility of tryptophan (Trp) residues in immunoglobulin M (IgM) upon modification with Koshland reagent (2-hydroxy-5-nitrobenzyl bromide) as an index of the conformational changeability of IgM. Of fourteen Trp's in the native IgM (per HL-region) only one appeared to be most accessible, evidently Trp312 in the mu-chain. Irreversible acidic and thermal conformational transitions in IgM increase the number of accessible Trp's approximately two-fold. Following partial enzymatic deglycosylation of IgM, deep scission of mannose in particular, all Trp's become inaccessible. Modification of the most accessible Trp increases 2-3 fold the number of tyrosine residues readily accessible upon nitration with tetranitromethane. Modification of four trp's using spin-label method data causes a sharp reduction of the mobility of the C mu 3 domain and a simultaneous decrease in the solubility of modified IgM.

Regulation and quaternary structural changes in rabbit muscle phosphofructokinase

Subunit assembly plays a significant role in the regulation of rabbit muscle phosphofructokinase (PFK), although conformational changes and post-translational modifications have also been implicated to regulate the enzyme activity. In the absence of high-resolution structural information, the three-dimensional arrangements of subunits in the rabbit muscle PFK in its active and inactive states are not known. Hence, a systematic study is initiated, and phosphorylation of PFK subunit is employed as a probe for the structure-function correlation of the enzyme. The self-association of the phosphorylated and dephosphorylated PFK was monitored by sedimentation velocity at pH 7.0 and 23 degrees C. Results show that both the phosphorylated and dephosphorylated forms of PFK exhibit the same mechanism of assembly. The secondary structures of both forms of PFK were monitored by circular dichroism (CD) as a function of protein concentration ranging from 20 to 2000 micrograms/ml. Results show that there is no detectable difference in the structure under all experimental conditions. The accessibility of tryptophan to solvent was monitored by fluorescence quenching within the same range of protein concentration. Results show that the fluorophores are more accessible to the quencher at higher protein concentrations. Hence, post-translational modification and subunit association do not induce significant structural change in PFK subunit, although the accessibility of tryptophan residues is altered with oligomer formation. Furthermore, sedimentation and CD studies show that the activation of PFK by substrate includes no detectable modification in secondary/tertiary structure but a quaternary structural change, and the local environments of some, if not all, of the tryptophan residues are less accessible to solvent. Hence, the change in sedimentation behavior between the active and inactive tetrameric PFK is due to a rearrangement of subunit-subunit interactions. In order to correlate the physical properties of PFK to the regulatory behavior of enzyme activity, the steady-state kinetics were investigated under the same experimental conditions. In conditions where enhancement of self-association is observed, the kinetic behavior reflects activation of the enzyme. Hence, this correlation between subunit assembly and the regulation of enzyme activity in PFK must reflect an intrinsic property of the muscle enzyme.

Accessibility of tryptophan residues in Na,K-ATPase.

The accessibility of the tryptophans in dog kidney Na,K-ATPase was studied with the technique of quenching by acrylamide. By use of a modified Stern-Volmer equation, fa, the effective fraction of tryptophans most exposed to quencher, and Ka, the effective quenching constant, were calculated. The direct Stern-Volmer plots are nonlinear under nondenaturing conditions, indicating that the tryptophan residues are unequally accessible to quencher. Modified Stern-Volmer plots revealed marked differences in the exposure of tryptophans in the E1 and E2 states. In the presence of Na or ADP, ligands that stabilize E1, these plots curve downward, indicating that the in addition to buried (unquenched) tryptophans, there is a heterogeneous class of tryptophans. In the presence of K or ouabain, conditions that favor E2, the modified Stern-Volmer plots are linear, consistent with a homogeneous population of tryptophans. Treatment with chymotrypsin to block the E1 to E2 transition results in a new set of quenching parameters which are unchanged with Na or K. Even after detergent denaturation (1% sodium dodecyl sulfate for 30 min), Stern-Volmer plots are nonlinear, and a significant fraction of tryptophan residues remain inaccessible to quencher. Denaturation with urea or guanidine HCl plus dithiothreitol increases the fraction of quenchable fluorescence even more, but still a small fraction, about 7-13%, is buried. The observed changes in exposure of the tryptophan residues would seem to account for the differences in intrinsic fluorescence seen on adding K and Na to Na,K-ATPase. The present results provide new evidence that a significant rearrangement of amino acid residues results from the E1 to E2 transition. Furthermore, a region of the molecule is inaccessible even after denaturation; this may correspond to highly hydrophobic stretches that are normally buried in the membrane.

Analysis of the membrane effects on gangliosides

Gangliosides interact with human serum albumin, inducing conformational changes in its globule. Gangliosides bring about an increase in the binding constant of the fluorescent probe 1-anilinonapthalene-8-sulfonate (ANS) with model phospholipid membranes, a decrease in the rate of pyrene diffusion in liposomes. Gangliosides raise the fluorescence intensity of ANS bound with the erythrocytic membrane as the binding constant of the probe-membrane increases, the fluorescence of tryptophanils of membranous proteins gets intensified, the fluorescence maximum of ANS and tryptophan residues shifts to the short-wave area, and the accessibility of tryptophan residues for NO-3 decreases. At the same time there is a reduction in the efficacy of energy migration from tryptophan to ANS and NAD X H2. The data obtained attest to the capability of gangliosides of influencing the conformation of both the lipid and protein phases of biological membranes.

Fluorescence properties of lipoprotein B and apolipoprotein B

The fluorescence properties of apolipoprotein B (ApoB) in various media, including aqueous solutions of three different pH, 6 m urea, 6 m guanidine-HCl and native lipoprotein B (LP-B) particles have been compared by measuring the accessibility of trytophan side chains to iodide ions. The modified Stern-Volmer plots ( FΔF vs. 1/[KI]) for LP-B demonstrate heterogeneity of quenching rates at pH 9.0, with a total accessibility of fluorescence to iodide of 43%. At pH 7.3, the total accessibility of LP-B fluorescence to iodide is only 20%. Quenching at pH 2.7 follows a pure Stern-Volmer mechanism. A straight line at this pH intercepting y-axis at 1.0 indicates 100% accessibility of tryptophan residues in LP-B. These results suggest that there are at least three different groups of tryptophan residues present per intact LP-B particle and that each group is situated in a different environment. One group, showing an enhanced quenching rate, is probably near the charged domain; another group, showing a slower quenching rate, is in a relatively hindered environment, and a third group is probably buried in a more hydrophobic environment, inaccessible to iodide at neutral or high pH. But at pH 2.7, all tryptophan residues appear to become situated closer to the surface of the LP-B particle. For isolated ApoB at pH 7.3 and 9.0 in aqueous buffer, about 30% of the fluorescence is relatively easily accessible; another 40% is less easily accessible and the remaining 30% is inaccessible to iodide. These inaccessible tryptophan residues are most likely located in a more hydrophobic matrix and probably in the β-pleated sheet region of ApoB. Similarly to LP-B at pH 2.7, all of the tryptophan residues of ApoB are exposed to the aqueous surface except that one third of them are quenched at a faster rate than the rest. At pH 7.3, in the presence of urea or guanidine-HCl, all of the fluorescence of ApoB is exposed to the aqueous surface, suggesting the presence of random and nonrigid conformation in these media. These results suggest that the conformation of ApoB in aqueous media is pH sensitive. This is true whether the ApoB is present in intact LP-B or as the isolated apolipoprotein. Furthermore, upon removal of lipids from LP-B and passing the ApoB into a denaturing environment, the apolipoprotein loses its ordered structure. When passing ApoB from denaturing agents back to aqueous buffers of neutral or basic pH. ApoB is able to reorient itself to gain an ordered structure, not necessarily identical to that in LP-B, but parallel to it.

Fluorescence quenching, a tool for probing conformational changes in glycogen phosphorylase.

This study shows that conformational changes in glycogen phosphorylase are accompanied by changes in the accessibility of tryptophan residues and of the coenzyme, pyridoxal phosphate, to the surrounding aqueous medium. The accessibility was estimated by determining the extent to which iodide can quench the fluorescence emission of these moieties by colliding with them, since iodide cannot collide with a buried chromophore and hence cannot quench its fluorescence. Rabbit muscle phosphorylase b, its apoform, and phosphorylase a exhibit differences in the number of exposed tryptophans, while the phosphorylase b forms from rabbit skeletal muscle and pig heart also show differences.Differences are also observed in the accessibility of the coenzyme in different forms of the enzyme. The quenching rate constant, a measure of accessibility, differs for phosphorylases a and b, and this constant is affected differently by ligand binding to the two forms. While the allosteric inhibitors, ATP and glucose 6-phosphate, render the pyridoxal phosphate moiety of phosphorylase b more accessible, the activator, AMP, and substrate, glucose 1-phosphate, together cause it to be totally inaccessible to fluorescence quenching by iodide. AMP and glucose 1-phosphate appear to mediate a conformational change which buries the coenzyme. While pyridoxal phosphate is necessary for catalytic activity, one may conclude from these experiments that its ring structure is unlikely to participate directly in the catalytic mechanism.

The modification of the tryptophan residues of bovine α-lactabumin with 2-hydroxy-5-nitrobenzyl bromide and with dimethyl(2-hydroxy-5-nitrobenzyl)sulphonium bromide II. Effect on the specifier protein activity

The effects on the function and conformation of α-lactalbumin after reaction with 2-hydroxy-5-nitrobenzyl bromide and its water soluble derivative dimethyl-(2-hydroxy-5-nitrobenzyl)sulphonium bromide have been studied. Three of the four tryptophan residues of α-lactalbumin (Trp 26, Trp 104 and Trp 118) were accessible to the tryptophan reagents and the reaction mixtures were complex. The labelling process could not be made more selective by changing the experimental conditions and to determine the importance of each of the accessible tryptophan residues an attempt was made to separate α-lactalbumin labelled uniquely at Trp 26, Trp 104 or Trp 118. A partial separation was possible by ion-exchange chromatography and three preparations thus isolated were subjected to kinetic and conformational studies. The results obtained suggest that when Trp 26, Trp 104 or Trp 118 is alkylated with the 2-hydroxy-5-nitrobenzyl group, α-lactalbumin loses its specifier protein activity. The labelling of tryptophan did not cause a gross conformational change but a minor change may have occurred.

Spectroscopic studies on the conformation of native and denatured glycinin.

Optical rotatory dispersion (ORD) studies and infrared spectra in deuterium oxide indicate that native glycinin exhibits mainly a β‐conformation structure. Ultraviolet difference spectra showed that urea ami guanidine hydrochloride at increasing concentrations cause progressive exposure of tyrosine and tryptophan residues. Ultraviolet difference spectroscopy was also used in following the course of acid‐induced denaturation of glycinin. It was observed that acid denaturation starts approximately at pH 3.50 reaching a maximum at pH 2.0. Data obtained by solvent perturbation of native glycinin using perturbants exhibiting mean diameters of 2.0, 4.4, and 7.2 A indicate that approximately 34 to 37 residues (45%of the total) are accessible to these perturbants whereas the accessibility of tryptophan residues is decreased with increasing mean diameters of the perturbant.