Structure and stability of gamma-crystallins. I. Spectroscopic evaluation of secondary and tertiary structure in solution

The three major bovine gamma-crystallin fractions (gamma-II, gamma-III and gamma-IV) are known to have closely related (80-90%) amino acid sequences and three-dimensional folding of the polypeptide backbone. Their chiroptical and emission properties, as measured by circular dichroism (CD) and fluorescence, are now shown to differ distinctly. The far-ultraviolet CD spectra indicate that all three gamma-crystallins have predominantly beta-sheet conformation (45-60%) with only subtle differences in secondary structure. The fluorescence emission maxima of gamma-II, gamma-III and gamma-IV, due to the four tryptophan residues, appear at 324, 329 and 334 nm, respectively, suggesting that tryptophan residues are buried in environments of decreasing hydrophobicity. Corresponding differences in quantum yield may be due to fluorescence quenching by neighboring sulfur-containing residues. Titratable tyrosines are maximal for gamma-III, as manifested from difference absorption spectra at alkaline pH. The near-ultraviolet CD spectra differ in position, magnitude and sign of tryptophan and tyrosine transitions. In addition, a characteristic CD maximum at 235 nm, presumably due to tyrosine-tyrosine exciton interactions, differs in magnitude for each gamma-crystallin. This study shows that the environment and interactions of the aromatic residues of the individual gamma-crystallin fractions are quite different. These variations in tertiary structure may be significant, in terms of stability of gamma-crystallins towards aggregation and denaturation, for understanding lens transparency and cataract formation in general.     

Fluorescence quenching of tryptophan by trifluoroacetamide

Trifluoroacetamide was found to be a good quencher of tryptophan fluorescence, and the quenching was shown to proceed via both a dynamic and a static process. The respective quenching constants were determined by the measurement of the decrease of the fluorescence lifetime in the presence of the quencher. The static and the bimolecular rate quenching constants of N-acetyltryptophanamide are equal to 0.34 1·mol^?1 and 1.9·10⁹ 1·mol^?1·s^?1, respectively. These values indicate that trifluoroacetamide is an efficient quencher of tryptophan fluorescence. This conclusion is also supported by a complete quenching of bovine serum albumin and wheat germ agglutinin fluorescence. In the case of lysozyme, trifluoroacetamide quenches the fluorescence of tryptophan residues which fluoresce with a maximum at 348 nm but not the buried tryptophan residues which fluoresce with a maximum at 333 nm. Trifluoroacetamide quenching of wheat germ agglutinin emission confirms the homogeneity and the high accessibility of emitting tryptophan residues, in agreement with a previous report (Privat, J.P. and Monsigny, M. (1975) Eur. J. Biochem. 60, 555–567). The tryptophan fluorescence decay of wheat germ agglutinin is biexponential even in the presence of the quencher; the static and bimolecular rate quenching constants are equal to 0.22 1·mol^?1 and 092·10⁹ 1·mol^?1·^?1, respectively. In the presence of a specific lectin ligand, the methyldi-N,N′-trifluoroacetyl-β- chitobioside, the quenching of wheat germ agglutinin fluorescence involves a direct contact between tryptophan residues and trifluoroacetamido groups of the ligand and in contrast with the quenching induced by free trifluoroacetamide shows that the tryptophan fluorescence is not fully quenched.     

Spectroscopic studies on human lens crystallins

Human lens crystallins were studied by absorption, circular dichroism and fluorescence spectroscopy. The absorption spectra in the near-ultraviolet region show some differences in intensity, but spectral features are similar, except for the alpha-crystallin, which gives a fine structure due to phenylalanine between 250 and 270 nm. Tryptophan fluorescence and near-ultraviolet circular dichroism indicate that tryptophan residues are more exposed in alpha-crystallin than in either beta- or gamma-crystallin, and that the degree of exposure decreases in the order of alpha less than beta 1 greater than beta 2 greater than beta 3 greater than gamma. The far ultraviolet CD suggests that these proteins exist mainly in a beta-sheet conformation and that the amount does not vary much among them. The greater exposure of the tryptophan residues in the high-molecular-weight crystallins may reflect greater unfolding in their protein domains. Spectroscopic measurements are thus useful in predicting protein tertiary structure in the absence of the complete sequence and X-ray data. The fact that the high-molecular-weight proteins exist in a more unfolded state may render them more vulnerable to exogeneous insults, and these effects may be studied by spectroscopic measurements.     

A characterization of abrin a from the seeds of the Abrus precatorius plant

Abrin A was purified from the seeds of the Abrus precatorius plant and its physical and biological properties were studied. The biological properties of abrin A were found to be similar to the better studied Abrus protein, abrin C, in that it is toxic to cell-free protein synthesis and binds d-galactose.Abrin A contains carbohydrate moieties including both neutral and amine sugars but no metals, similar to the other two Abrus proteins (abrin C and the Abrus agglutinin). Amino acid compositions of the subunits of abrin A indicated that it consists of two different subunits of comparable size. Furthermore, one of the subunits showed microheterogeneity suggesting that abrin A is a mixture of isolectins. A comparative study of abrin A and abrin C based on compositions and tryptic maps reveals them to be closely related. The evidence suggests that the two abrins may have the same mechanisms of toxic action.Far-ultraviolet circular dichroic studies of abrin A show it to contain 47% β-pleated sheet and 10% α-helix, again similar to the other two Abrus proteins.     

Conformational changes of papain induced on interaction with thiol proteinase inhibitors from newborn rat epidermis

The conformational changes of the papain molecular on interaction with two thiol proteinase inhibitors (TPI(1) and TPI(2] from newborn rat epidermis were studied by measuring circular dichroism (CD), the difference absorption spectrum, and the fluorescence spectrum due to tryptophan residues in papain. The far-ultraviolet CD band of papain between 210 and 230 nm was distinctly reduced on interaction with both inhibitors. Also, the near-ultraviolet CD spectrum of TPI(1)-bound papain changed between 285 and 320 nm as well as that of the TPI(2)-bound enzyme. The difference absorption spectrum for TPI(1)-bound papain exhibited two distinct peaks at 276.5 and 282 nm, indicating perturbation of aromatic amino acid residues. The fluorescence intensity of papain was significantly decreased on interaction with both inhibitors, which showed pH-dependency on an ionizable group, with pK values of 8.5 and 7.9 for TPI(1) and TPI(2), respectively. The complex formation of papain with both inhibitors caused a reduction of the susceptibility of a tryptophan residue, probably tryptophan-177, to chemical modification with N-bromosuccinimide. These results suggest that the active site involving histidine-159 in the papain molecule was much influenced by the alteration of the microenvironment of tryptophan-177 as a part of the interaction site for these two thiol proteinase inhibitors.     

Fluorescence polarization as a tool to study lectin-sugar interaction

The binding ofRicinus communis agglutinin andAbrus agglutinin to 4-methylumbelliferyl β-D-galactopyranoside was studied by equilibrium dialysis, fluo-rescence quenching and fluorescence polarization. The number of binding sites and the association constant value obtained by fluorescence polarization for bothRicinus communis agglutinin andAbrus agglutinin are in close agreement with those obtained by the other methods. This indicates the potential of ligand-fluorescence polarization measurements in the investigation of lectin-sugar interactions.     

Structural characterization of myo-inositol monophosphatase from bovine brain by secondary structure prediction,fluorescence, circular dichroism and Raman spectroscopy

Structural aspects of myo-inositol monophosphatase were examined by spectroscopic techniques and empirical prediction methods. The enzyme belongs to the α/β class of proteins, with approx. 33% α-helix and 29% β-sheet, as shown by circular dichroism (CD), Raman spectroscopy and prediction based on the amino-acid sequence. The Raman spectrum also suggests that the three tryptophan residues in myo-inositol monophosphatase are not expose to solvent. This was confirmed by a blue shift of 25 nm in the fluorescence emission spectrum, as compared to tryptophan in water, and by quenching studies with acrylamide. The enzyme shows a transition temperature of 87°C for the CD signal at 222 nm. This remarkable heat stability is not due to the presence of disulfide bonds, since both the Raman spectrum and chemical modification studies clearly indicate that all six cysteine residues are in the reduced state.     

Formation of molten globule-like state during acid denaturation of Aspergillus niger glucoamylase

Acid denaturation of Aspergillus niger glucoamylase was studied using different conformational probes. Both far-UV CD spectral signal (MRE_222 nm) and tryptophan fluorescence remained unchanged in the pH range, 7.0–3.0 but decreased significantly below pH 3.0, whereas ANS fluorescence showed a marked increase below pH 1.5. Maximal changes in MRE_222 nm and ANS fluorescence were noticed at pH 1.0. Acid-denatured state of glucoamylase at pH 1.0 retained a significant amount of secondary structure as reflected from far-UV CD spectra but showed a deformed tertiary structure with significant exposure of nonpolar groups as well as tryptophan residues as revealed by increased ANS fluorescence, decreased tryptophan fluorescence and three-dimensional fluorescence spectral signals and increase in K_sv value in acrylamide quenching experiments. Acid-denatured state showed no significant variation in the CD spectral signal throughout the temperature range, 0–100 °C. However, a late cooperative transition was observed upon GdnHCl treatment, compared to the native enzyme. All these results suggested that the acid-denatured state of glucoamylase at pH 1.0 represented the molten globule-like state.     

Circular dichroism and fluorescence studies of homogeneous antibodies to type III pneumococcal polysaccharide.

The near-ultraviolet circular dichroism (CD) of three homogeneous anti-type III pneumococcal antibodies in the absence and the presence of the specific hexasaccharide ligand was studied. In addition recombinations and hybridizations of H and L chains derived from two of these antibodies were carried out and the CD spectra of bound and free reconstituted IgG molecules were measured. The results indicate that the CD spectra of the native antibodies in the 260-310-nm range are very similar in shape and sign and exhibit a positive band at 285 nm. The homologous reconstituted antibody molecules exhibited CD spectra very similar in shape and sign to those of the native antibody molecules although recombinant molecules are no longer stabilized by interchain disulfide bonds. Upon addition of the hexasaccharide ligand, a significant decrease in amplitude of the CD spectra (18-21%) occurred in all three native antibodies and their Fab fragments as well as in the homologous recombinant molecules. No CD spectral changes could be detected upon interaction of the hapten ligand with the heterologous recombinants. All homogeneous antibodies studied exhibited fluorescence quenching upon oligosaccharide binding and a blue shift of the emission maximum. This property allowed the determination of the binding constant of one selected antibody to be made. Taken together, CD and fluorescence spectroscopic data suggest that oligosaccharide ligands induced detectable conformational changes in the Fab fragment of the antibody.     

Spectroscopic evidence for ligand-induced conformational change in NADP+:isocitrate dehydrogenase

Conformational changes induced by binding of ligands to cytosolic NADP(+)-specific isocitrate dehydrogenase from lactating bovine mammary gland were assessed using circular dichroism and fluorescence techniques. The secondary structure of isocitrate dehydrogenase, as monitored by CD spectra in the far-UV region, is unaltered by enzyme-ligand interactions; in contrast, dramatic changes occur in the near-UV region (270-290 nm) assigned to tyrosine and/or solvent-exposed tryptophan residues. Both the coenzyme analog, 2'-phosphoadenosine 5'-diphosphoribose, and NADPH have an effect on the CD spectrum which is opposite to that produced by metal complexes of either isocitrate or citrate. A CD band at 292 nm assigned to approximately 2 tryptophan residues in a hydrophobic environment is unchanged by binding of substrate or coenzyme. Approximately 30% of the intrinsic fluorescence of isocitrate dehydrogenase, corresponding to approximately 2 tryptophan residues, is not quenched by acrylamide in the absence of 6.3 M guanidine hydrochloride and remains unquenched in the enzyme-substrate complex. The constancy in the proportion of buried and exposed tryptophan residues implicates tyrosine in the observed near-UV CD spectral changes. Since binding of ligands does not influence quaternary structure (Seery, V.L., and Farrell, H. M., Jr. (1989) Arch. Biochem. Biophys. 274, 453-462), activation of isocitrate dehydrogenase may be related to a substrate-induced conformational transition.     

A circular dichroism study of sheath contraction in pyocin R1

Pyocin R1, a bacteriocin of Pseudomonas aeruginosa, is a protein particle shaped like a bacteriophage tail composed of a contractile sheath, core, baseplate and tail fibers. Alkaline treatment with sodium carbonate caused sheath contraction without considerable disassembly of other components. Circular dichroism (CD) spectra of pyocin R1 before and after the treatment, and of isolated sheath, were measured in wavelength regions around 220 and 290 nm at neutral pH. The alkaline treatment caused a red shift of the minimum from 208 nm to 212 nm. A marked difference in the CD spectrum was found in the near-ultraviolet region. THe difference is considered to be mainly due to a CD spectra change of tryptophan residues in the sheath subunits.     

Effect of ethanol on the activity and conformation of Penaeus penicillatus acid phosphatase.

The effect of ethanol on the activity of Penaeus penicillatus acid phosphatase has been studied. The results show that ethanol significantly inhibits enzyme activity as a non-competitive inhibitor, with Ki 8.75%. The conformational changes of the enzyme molecule induced by ethanol were followed using fluorescence emission, ultraviolet difference and circular dichroism (CD) spectra. Increasing the ethanol concentration caused the fluorescence emission intensity of the enzyme to increase. The ultraviolet difference spectra of the enzyme denatured with ethanol had two negative peaks at 220 and 278 nm, and a positive peak at 240 nm. Increasing the ethanol concentration produced a small shoulder peak at 287 nm in addition to the increases in the negative magnitudes of the 220 and 278 nm peaks. The changes of the fluorescence and ultraviolet difference spectra reflected the changes of the microenvironments of the tryptophan and tyrosine residues of the enzyme. The CD spectrum changes of the enzyme show that the secondary structure of the enzyme also changed. The results suggest that ethanol is a non-competitive inhibitor and the conformational integrity of the enzyme is essential for its activity.     

Spectral contribution of the individual tryptophan of alphaB-crystallin: a study by site-directed mutagenesis

There are two tryptophan residues in the lens alphaB-crystallin, Trp9 and Trp60. We prepared two Trp --> Phe substituted mutants, W9F and W60F, for use in a spectroscopic study. The two tryptophan residues contribute to Trp fluorescence and near-ultraviolet circular dichroism (UV CD) differently. The major difference in the near-UV CD is the contribution of 1La of Trp: it is positive in W60F but becomes negative in W9F. Further analysis of the near-UV CD shows an increased intensity in the region of 270-280 nm for W60F, suggesting that the Tyr48 is affected by the W60F mutation. It appears that Trp60 is located in a more rigid environment than Trp9, which agrees with a recent structural model in which Trp60 is in a beta-strand.     

Circular dichroism and saccharide-induced conformational transitions of lectins from Ricinus communis.

Conformational studies on lectins from castor beans (Ricinus communis), RCA_I and RCA_II, were performed by using circular dichroism (CD). The CD spectra were similar showing several negative bands at 270–320 nm, a positive region at 230–250 nm, several negative bands at 205–225 nm, and a positive peak at about 195 nm. However, significant differences were observed in the band strength between RCA_I and RCA_II. Lactose, melibiose, and d-fucose induced marked Conformational alterations in RCA_I, whereas weaker effects were produced by d-galactose and l-rhamnose. Saccharide-induced conformational alterations were weaker in RCA_II than in RCA_I, with only lactose and melibiose inducing significant alterations. d-Glucose and 2-acetamido-2-deoxy-d-glucose, which do not inhibit hemagglutination by RCA_I or RCA_II, did not influence lectin conformation. Acetylation of tyrosyl groups with N-acetylimidazole produced changes in the CD bands in the near uv indicating involvement of tyrosine residues. The saccharide effect was most pronounced at 285 nm, a band that was assigned to a tyrosine chromophore. Analysis of the CD bands in the far-uv zone indicated the presence of approximately 50% pleated sheet (β) structure, and 13–15% α-helix in both RCA_I and RCA_II. According to the CD results, the polypeptide chain backbone in the lectins was not affected by the saccharides, whereas significant disorganization occurred in 7 m guanidine-HCl.     

Structural determinants of the intrinsic fluorescence emission in notexin and phospholipase A2 enzymes

Fluorescence measurements of the homologous proteins, notexin and PLA₂ enzymes fromNaja naja atra, Naja nigricollis, and Hemachatus haemachatus venoms, showed that the wavelength of maximum emission and the quantum yield of their intrinsic fluorescence emission spectra were different. To verify the factors which affected their fluorescence characteristics, the dynamics of tryptophan residues in those homologous proteins were studied by quenching with acrylamide, iodide, and cesium. The degrees of exposure of tryptophanyl groups in notexin and PLA₂ enzymes assessed by acrylamide quenching were found to be the major factor that determined their fluorescence characteristics. However, the positively charged groups surrounding tryptophan residues of PLA₂ enzymes fromN. naja atra andN. nigricollis venoms might affect the quantum yield of their fluorophores. Tryptophan residues of notexin were in an environment with less fluctuation, which did not allow free diffusion of ionic quencher. This might render its typtophan residues to fluoresce at a shorter wavelength. These results suggested that the structural determinants affecting the intrinsic fluorescence emission of homologous proteins can be easily assessed by quenching studies.     

Ricin structure: the study by the fluorescence quenching method

To elucidate the details of pH-induced conformational transformation of ricin [I] in the region surrounding tryptophan residues, we studied parameters of fluorescence of the native toxin and its isolated A- and B-subunits at pH 4.0, 5.0 and 7.4. The studies were carried out using resolution of fluorescence spectra according to different degree of tryptophan accessibility to ionic (iodide) and non-ionic organic (acrylamide) quenchers. Application of the new method allowed to reveal three classes of tryptophan residues differing in their accessibility to quenchers alpha-residues are accessible neither to ions nor to organic molecules; beta-residues are accessible only to organic molecules; while surface gamma-residues are accessible to both types of quenchers. The fluorescence spectra were assessed for each class of tryptophan residues. The major part of them was shown to be localized in apolar rigid microenvironment. Fluorescence of ricin and especially of its isolated subunits proved to be strongly dependent on the pH value. At pH less than 5 the structure of B-chain loosens, this process being reflected by an increase in accessibility of tryptophan residues to quenchers. In acidic solution at least one out of seven tryptophan residues in the ricin molecule undergoes conformational transformation. Positive charge prevails in the regions surrounding quencher-accessible tryptophan residues. Binding of lactose leads to a slight compactization of the toxin structure that causes, in its turn, short-wave shifts of the fluorescence spectra and reduction of Stern-Volmer constants for intraglobular tryptophan residues.     

Conformational studies on a modified poly-L-tryptophan: Circular dichroism and optical rotatory dispersion of poly-2-(2-nitrophenylsulfenyl)-L-tryptophan and of random copolymers of L-tryptophan and 2-(2-nitrophenylsulfenyl)-L-tryptophan

Optical rotatory dispersion (ORD) and circular dichroism (CD) measurements were carried out on a block copolymer, (γ-ethyl DL -glutamate)₁₆₀ (L -Trp)₃₂, in which the tryptophan sequence has been modified to various extents by using 2-nitrophenylsulfenyl chloride. The CD spectrum of the completely modified copolymer exhibits bands in some of the regions of maximum absorption of the sidechain chromophores. In the peptide absorption region the spectrum is similar to that reported in the literature for polypeptides in the α-helical conformation. When the extent of modification of the tryptophan sequence is progressively reduced, there is a gradual change in the ORD spectra of the copolymers. On the basis of these data the assumption was made that no conformational change occurs on proceeding from the pure unmodified tryptophan sequence to the completely modified sequence. The results are discussed in connection with the study of possible conformational effects arising from selective chemical modification of tryptophan residues in proteins.     

The identification of tryptophan residues responsible for ATP-induced increase in intrinsic fluorescence of myosin subfragment 1

ATP binding to myosin subfragment 1 (S1) induces an increase in tryptophan fluorescence. Chymotryptic rabbit skeletal S1 has 5 tryptophan residues (Trp113, 131, 440, 510 and 595), and therefore the identification of tryptophan residues perturbed by ATP is quite complex. To solve this problem we resolved the complex fluorescence spectra into log-normal and decay-associated components, and carried out the structural analysis of the microenvironment of each tryptophan in S1. The decomposition of fluorescence spectra of S1 and S1-ATP complex revealed 3 components with maxima at ca. 318, 331 and 339-342 nm. The comparison of structural parameters of microenvironment of 5 tryptophan residues with the same parameters of single-tryptophan-containing proteins with well identified fluorescence properties applying statistical method of cluster analysis, enabled us to assign Trp595 to 318 nm, Trp440 to 331 nm, and Trp 13, 131 and 510 to 342 nm spectral components. ATP induced an almost equal increase in the intensities of the intermediate (331 nm) and long-wavelength (342 nm) components, and a small decrease in the short component (318 nm). The increase in the intermediate component fluorescence most likely results from an immobilization of some quenching groups (Met437, Met441 and/or Arg444) in the environment of Trp440. The increase in the intensity and a blue shift of the long component might be associated with conformational changes in the vicinity of Trp510. However, these conclusions can not be extended directly to the other types of myosins due to the diversity in the tryptophan content and their microenvironments.     

Comparison of CD spectra in the aromatic region on a series of variant proteins substituted at a unique position of tryptophan synthase alpha-subunit

CD spectra in the aromatic region of a series of the mutant alpha-subunits of tryptophan synthase from Escherichia coli, substituted at position 49 buried in the interior of the molecule, were measured at pH 7.0 and 25 degrees C. These measurements were taken to gain information on conformational change produced by single amino acid substitutions. The CD spectra of the mutant proteins, substituted by Tyr or Trp residue in place of Glu residue at position 49, showed more intense positive bands due to one additional Tyr or Trp residue at position 49. The CD spectra of other mutant proteins also differed from that of the wild-type protein, despite the fact that the substituted residues at position 49 were not aromatic. Using the spectrum of the wild-type protein (Glu49) as a standard, the spectra of the other mutants were classified into three major groups. For 10 mutant proteins substituted by Ile, Ala, Leu, Met, Val, Cys, Pro, Ser, His, or Gly, their CD values of bands (due to Tyr residues) decreased in comparison with those of the wild-type protein. The mutant protein substituted by Phe also belonged to this group. These substituted amino acid residues are more hydrophobic than the original residue, Glu. In the second group, three mutant proteins were substituted by Lys, Gln, or Asn, and the CD values of tyrosyl bands increased compared to those of the wild-type proteins. These residues are polar. In the third group, the CD values of tyrosyl bands of two mutant proteins substituted by Asp or Thr were similar to those of the wild-type protein, except for one band at 276.5 nm. These results suggested that the changes in the CD spectra for the mutant proteins were affected by the hydrophobicity of the residues at position 49.     

Conformation similarities of the globular and tailed forms of acetylcholinesterase from Torpedo california

The conformation of the globular dimer (G₂), the tailed asymmetric dodecamer (A₁₂, also containing some tailed octamer A₈) and the globular tetramer (G₄, prepared by removing the collagen-like tail from A₁₂) of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) was studied by circular dichroism (CD) in the ultraviolet region. The G₂ and G₄ forms had similar conformation with about 40% α-helix, 35% β-sheets and 4% β-turns; the tailed form had a lower helicity (about 34%) and β-form (about 25%) content probably because of the presence of the tail whose CD spectrum resembles that of an unordered form, but it had about the same amount of β-turns as the other two forms. All three forms also had similar CD spectra in the near-ultraviolet region due to their non-peptide chromophores. The pH, thermal and urea denaturation of the three acetylcholinesterase forms was also similar to each other. The pH-dependency of both the enzymatic activity and CD intensity of the three forms showed bell-shaped curves with a plateau at pH 7–8. The activity was completely lost at pH below 5 or above 10, but the corresponding CD spectra retained 70–80% of the original magnitudes. Thermal denaturation of the three forms at pH 7.5 showed a conformational transition and loss of activity between 30 and 40°C, but the CD intensity of the helical band at 222 nm was reduced by only 20–30%. Urea denaturation of the three form began at 1 M urea; it was protein concentration- and time-dependent. Again, the activity disappeared faster than the decreasing CD intensity. Thus, the overall conformation of the three acetylcholinesterase forms appears to be relatively stable, but their active site is easily perturbed by changing the environment. The loss of activity correlated well with the disapperance of the CD band of tryptophan(s) in the near-ultraviolet region, suggesting that the Trp residue(s) might be at or near the active center of the enzyme.