Interaction between two discontiguous chain segments from the beta-sheet of Escherichia coli thioredoxin suggests an initiation site for folding

The approach of comparing folding and folding/binding processes is exquisitely poised to narrow down the regions of the sequence that drive protein folding. We have dissected the small single alpha/beta domain of oxidized Escherichia coli thioredoxin (Trx) into three complementary fragments (N, residues 1-37; M, residues 38-73; and C, residues 74-108) to study them in isolation and upon recombination by far-UV CD and NMR spectroscopy. The isolated fragments show a minimum of ellipticity of ca. 197 nm in their far-UV CD spectra without concentration dependence, chemical shifts of H(alpha) that are close to the random coil values, and no medium- and long-range NOE connectivities in their three-dimensional NMR spectra. These fragments behave as disordered monomers. Only the far-UV CD spectra of binary or ternary mixtures that contain N- and C-fragments are different from the sum of their individual spectra, which is indicative of folding and/or binding of these fragments. Indeed, the cross-peaks corresponding to the rather hydrophobic beta(2) and beta(4) regions of the beta-sheet of Trx disappear from the (1)H-(15)N HSQC spectra of isolated labeled N- and C-fragments, respectively, upon addition of the unlabeled complementary fragments. The disappearing cross-peaks indicate interactions between the beta(2) and beta(4) regions, and their reappearance at lower temperatures indicates unfolding and/or dissociation of heteromers that are predominantly held by hydrophobic forces. Our results argue that the folding of Trx begins by zippering two discontiguous and rather hydrophobic chain segments (beta(2) and beta(4)) corresponding to neighboring strands of the native beta-sheet.     

Acid-induced equilibrium folding intermediate of human platelet profilin

The acid-induced unfolding of human platelet profilin (HPP) can be minimally modeled as a three-state process. Equilibrium unfolding studies have been performed on human platelet profilin1 (HPP) and monitored by far-UV circular dichroism, tryptophan fluorescence, ANS binding, and NMR spectroscopy. Far-UV CD measurements obtained by acid titration demonstrate that HPP unfolds via a three-state mechanism (N --> I --> U), with a highly populated intermediate between pH 4 and 5. Approximately 80% of native helical secondary structural content remains at pH 4, as indicated by monitoring the CD signal at 222 nm. The stability (DeltaGH2O) of the native conformation at pH 7.0 (obtained by monitoring the change in tryptophan signal as a function of urea concentration) is 5.56 +/- 0.51 kcal mol-1; however, the DeltaGH2O for the intermediate species at pH 4 is 2.01 +/- 0.47 kcal mol-1. The calculated m-values for the pH 7.0 and pH 4.0 species were 1.64 +/- 0.15 and 1.34 +/- 0.17 kcal mol-1 M-1, respectively, which is an indication that the native and intermediate species are similarly compact. Additionally, translational diffusion measurements obtained by NMR spectroscopy and ANS binding studies are consistent with a globular and compact conformation at both pH 7.0 and 4.0. The pKa values for the two histidine (His) residues located on helix 4 of HPP were determined to be 5.6 and 5.7 pH units. These pKa values coincide with the midpoint of the far-UV CD acid titration curve and suggest that the protonation of one or both His residues may play a role in the formation of the unfolding intermediate. Stable intermediate species populate the 2D 1H-15N HSQC NMR spectra between pH 4 and 5. A number of backbone and side-chain resonances show significant perturbations relative to the native spectrum; however, considerable nativelike tertiary contacts remain. Interestingly, the residues on HPP that are significantly altered at low pH coincide with segments of the G-actin binding surface and poly-l-proline binding interface. The earlier reports that a decrease in pH below 6.0 induces structural alterations in profilin, favoring dissociation of the profilin-actin complex, corresponds with the structural alterations observed in the partially unfolded species. Our findings suggest that a novel mechanism for pH induced disruption of the profilin-G-actin complex involve a nativelike unfolding intermediate of profilin.     

Conformational analysis of peptide fragments derived from the peripheral subunit-binding domain from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus: evidence for nonrandom structure in the unfolded state

There is currently a great deal of interest in the early events in protein folding. Two issues that have generated particular interest are the nature of the unfolded state under native conditions and the role of local interactions in folding. Here, we report the results of a study of a set of peptides derived from a small two-helix protein, the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex. Five peptides of overlapping sequence were prepared, including sequences corresponding to each of the helices and to the region connecting them. The peptides were characterized by CD and, where possible, nmr. A peptide corresponding to the second helix is between 12 and 17% helical at neutral pH. CD also indicates a lower percentage of helical structure in the peptide corresponding to the first alpha-helix, although the values of the alpha-proton chemical shifts suggest some preference for nonrandom structure. Peptides corresponding to the interhelical loop, which in the full domain contains two overlapping beta-turns and a 5-residue 3(10)-helix, are less structured. There is no significant change in the helicity of any of these peptides with pH. To test for fragment complementation, CD spectra of the two peptides derived from each helix and the long connecting peptide were compared to the spectra of each possible pair, as well as to a mixture containing all three. No increase in structure was observed. We complement our peptide studies by characterizing a point mutant, D34V, which disrupts a critical hydrogen bonding network. This mutant is unable to fold and provides a useful model of the denatured state. The mutant is between 9 and 16% helical as judged by CD. The modest amount of helical structure formed in some of the peptide fragments and in the point mutant suggests that the denatured state of the peripheral subunit binding domain is not completely unstructured. This may contribute to the very rapid folding observed for the intact protein.     

Conformational changes upon calcium binding and phosphorylation in a synthetic fragment of calmodulin

We have recently investigated by far-UV circular dichroism (CD) the effects of Ca(2+) binding and the phosphorylation of Ser 81 for the synthetic peptide CaM [54-106] encompassing the Ca(2+)-binding loops II and III and the central alpha helix of calmodulin (CaM) (Arrigoni et al., Biochemistry 2004, 43, 12788-12798). Using computational methods, we studied the changes in the secondary structure implied by these spectra with the aim to investigate the effect of Ca(2+) binding and the functional role of the phosphorylation of Ser 81 in the action of the full-length CaM. Ca(2+) binding induces the nucleation of helical structure by inducing side chain stacking of hydrophobic residues. We further investigated the effect of Ca(2+) binding by using near-UV CD spectroscopy. Molecular dynamics simulations of different fragments containing the central alpha-helix of CaM using various experimentally determined structures of CaM with bound Ca(2+) disclose the structural effects provided by the phosphorylation of Ser 81. This post-translational modification is predicted to alter the secondary structure in its surrounding and also to hinder the physiological bending of the central helix of CaM through an alteration of the hydrogen bond network established by the side chain of residue 81. Using quantum mechanical methods to predict the CD spectra for the frames obtained during the MD simulations, we are able to reproduce the relative experimental intensities in the far-UV CD spectra for our peptides. Similar conformational changes that take place in CaM [54-106] upon Ca(2+) binding and phosphorylation may occur in the full-length CaM.     

Trapping the on-pathway folding intermediate of Im7 at equilibrium

The four-helical protein Im7 folds via a rapidly formed on-pathway intermediate (k(UI)=3000 s(-1) at pH 7.0, 10 degrees C) that contains three (helices I, II and IV) of the four native alpha-helices. The relatively slow (k(IN)=300 s(-1)) conversion of this intermediate into the native structure is driven by the folding and docking of the six residue helix III onto the developing hydrophobic core. Here, we describe the structural properties of four Im7* variants designed to trap the protein in the intermediate state by disrupting the stabilising interactions formed between helix III and the rest of the protein structure. In two of these variants (I54A and L53AI54A), hydrophobic residues within helix III have been mutated to alanine, whilst in the other two mutants the sequence encompassing the native helix III was replaced by a glycine linker, three (H3G3) or six (H3G6) residues in length. All four variants were shown to be monomeric, as judged by analytical ultracentrifugation, and highly helical as measured by far-UV CD. In addition, all the variants denature co-operatively and have a stability (DeltaG(UF)) and buried hydrophobic surface area (M(UF)) similar to those of the on-pathway kinetic intermediate. Structural characterisation of these variants using 1-anilino-8-napthalene sulphonic acid (ANS) binding, near-UV CD and 1D (1)H NMR demonstrate further that the trapped intermediate ensemble is highly structured with little exposed hydrophobic surface area. Interestingly, however, the structural properties of the variants I54A and L53AI54A differ in detail from those of H3G3 and H3G6. In particular, the single tryptophan residue, located near the end of helix IV, and distant from helix III, is in a distinct environment in the two sets of mutants as judged by fluorescence, near-UV CD and the sensitivity of tryptophan fluorescence to iodide quenching. Overall, the results confirm previous kinetic analysis that demonstrated the hierarchical folding of Im7 via an on-pathway intermediate, and show that this species is a highly helical ensemble with a well-formed hydrophobic core. By contrast with the native state, however, the intermediate ensemble is flexible enough to change in response to mutation, its structural properties being tailored by residues in the sequence encompassing the native helix III.     

Conformational diversity of acid-denatured cytochrome c studied by a matrix analysis of far-UV CD spectra.

The singular value decomposition (SVD) analysis was applied to a large set of far-ultraviolet circular dichroism (far-UV CD) spectra (100-400 spectra) of horse heart cytochrome c (cyt c). The spectra were collected at pH 1.7-5.0 in (NH4)2SO4, sorbitol and 2,2,2-trifluoroethanol (TFE) solutions. The present purpose is to develop a rigorous matrix method applied to far-UV CD spectra to resolve in details conformational properties of proteins in the non-native (or denatured) regions. The analysis established that three basis spectral components are contained in a data set of difference spectra (referred to the spectrum of the native state) used here. By a further matrix transformation, any observed spectrum could be decomposed into fractions of the native (N), the molten-globule (MG), the highly denatured (D), and the alcohol-induced helical (H) spectral forms. This method could determine fractional transition curves of each conformer as a function of solution conditions, which gave the results consistent with denaturation curves of cyt c monitored by other spectroscopic methods. The results in sorbitol solutions, for example, suggested that the preferential hydration effect of the co-solvent stabilizes the MG conformer of cyt c. This report has found that the systematic SVD analysis of the far-UV CD spectra is a powerful tool for the conformational analysis of the non-native species of a protein when it is suitably supplemented with other experimental methods.     

Accuracy of protein secondary structure determination from circular dichroism spectra based on immunoglobulin examples

Strong contribution of the aromatic amino acid side chain chromophores to the far-UV circular dichroism (CD) spectra substantially distorts a relatively weak CD signal originating from beta sheet, the main type of immunoglobulin secondary structure. In this study we compared the secondary structure calculated from the far-UV CD spectra with the X-ray data for three antibody Fab fragments. Calculations were performed with three different algorithms, using two sets of reference proteins. Low standard deviations between all six estimates indicate stable mathematical solutions. Despite pronounced differences in the shape and amplitude of the CD spectra, we found a strong correlation between CD and X-ray data in the secondary structure for every protein studied. The number and average length of the secondary structure elements estimated from the CD spectra closely resemble those of the X-ray data. Agreement between spectroscopic and crystallographic results demonstrates that modern methods of secondary structure calculation are resilient to distortions of the far-UV CD spectra of immunoglobulins caused by aromatic side chain chromophores.     

Native-like secondary structure of molten globules

The most common evidence for the existence of secondary structure in a globular protein is the presence of a strongly pronounced far-UV circular dichroism (CD) spectrum. Although CD spectra of native proteins are well described and their quantitative analysis is widely used, similar studies for denatured proteins have still to be done. Far-UV CD spectra of nine proteins in the native and the pH-induced molten globule states were acquired and analyzed. Singular value decomposition showed that the spectra of molten globules could be described as a superposition of at least three independent components (most likely alpha-, beta- and irregular structure). A self-consistent procedure of CD spectra analysis revealed the existence of a clear correlation between the shape of the molten globule spectra and the content of secondary structure elements in the corresponding native proteins, as determined from X-ray data. A mathematical expression of this correlation in terms of the Pierson coefficient amounts to the value of 0.9 for both the alpha-helix and the beta-structure. Thus, the secondary structure of proteins in the molten globule state is close to that in the native state.     

Conformational analysis of hemopexin by fourier-transform infrared and circular dichroism spectroscopy

Hemopexin is a serum glyco-protein that binds heme with the highest known affinity of any characterized heme-binding protein and plays an important role in receptormediated cellular heme uptake. Complete understanding of the function of hemopexin will require the elucidation of its molecular structure. Previous analysis of the secondary structure of hemopexin by far-UV circular dichroism (CD) failed due to the unusual positive ellipticity of this protein at 233 nm. In this paper, we present an examination of the structure of hemopexin by both Fourier-transform infrared (FTIR) and circular dichroism spectroscopy. Our studies show that hemopexin contains about 55% β-structure, 15% α-helix, and 20% turns. The two isolated structural domains of hemopexin each have secondary structures similar to hemopexin. Although there are significant tertiary conformational changes indicated by the CD spectra, the overall secondary structure of hemopexin is not affected by binding heme. However, moderate changes in secondary structure do occur when the heme-binding domain of hemopexin associates with heme. In spite of the exceptionally tight binding at neutral pH, heme is released from the bis-histidyl heme–hemopexin complex at pH 5.0. Under this acidic condition, hemopexin maintains the same overall secondary structure as the native protein and is able to resume the heme-binding function and the native structure of the hemeprotein (as indicated by the CD spectra) when returned to neutral pH. We propose that the state of hemopexin identified in vitro at pH 5.0 resembles that of this protein in the acidic environment of the endosomes in vivo when hemopexin releases heme during receptor-mediated endocytosis. © 1994 Wiley-Liss, Inc.     

Circular dichroism and conformation of human alpha1-antitrypsin.

The solution conformation of alpha 1-antitrypsin from human blood plasma was studied by the circular dichroism (CD) probe. The CD spectra revealed in this glycoprotein approximately 16-20% of alpha-helix, the rest of the main polypeptide chain possessing the pleated sheet (beta) and the aperiodic structures. The conformation was stable between pH 4.7 and 8.8. Reversible change in conformation was observed at pH 10.3, and more dratic denaturation occurred at pH 11.6. The environment of the side chain chromophores was strongly affected by acid at pH 2.5, whereas the main chain conformation was changed slightly. A drastic change in the CD spectra, indicating denaturation, was observed in 3.5 M guanidine hydrochloride. Sodium dodecyl sulfate was effective in disorganizing the tertiary structure and in enhancing the helix content. The phenylalanine band fine structure was observed in the native protein and also after denaturation with acid, guanidine hydrochloride and sodium dodecyl sulfate.     

Acquisition of native-like interactions in C-terminal fragments of barnase

We have characterised a series of C-terminal fragments of barnase by different biophysical techniques to find out when they acquire secondary and tertiary native-like structure. Fragments B96-110 (which comprises the last 15 residues of the intact protein) up to B37-110 (which involves most of the protein except the two first helices and a loop) were mainly disordered. Only fragment B23-110, which lacks alpha-helix1, showed native-like near and far-UV CD and fluorescence spectra. The intensities of these spectra were lower than those of the full-length protein, which suggests the absence of complete side-chain packing. Urea denaturation followed by fluorescence, far-UV CD and gel-filtration chromatography techniques indicated a co-operative transition only for B23-110. None of the fragments melted co-operatively with temperature. Thus, the formation of secondary and tertiary structure requires most of the polypeptide chain to be present, that is, secondary and tertiary structure are formed in parallel. This agrees with the proposed model for barnase folding, where the residual structure in small fragments is weak and flickering, and it is only consolidated when there are enough tertiary interactions. Thus, the development of structure in the series of C-terminal fragments follows a similar behaviour to that observed in the series of N-terminal fragments of barnase.     

The perturbations of the native state of goat alpha-lactalbumin induced by 1,1'-bis(4-anilino-5-naphthalenesulfonate) are Ca2+-dependent.

In this work we have studied the interaction of the hydrophobic fluorescent probe 1,1'-bis(4-anilino-5-naphthalenesulfonate) (bis-ANS), with the native state of apo- and Ca2+-bound goat alpha-lactalbumin (GLA). In 10 mM Tris-HCl, pH 7.5, at 4 degrees C in 2 mM EGTA as well as at 37 degrees C in 2 mM Ca2+, the native protein is close to its thermal transition. Therefore, it can be expected that in both conditions the protein is equally susceptible to interaction with bis-ANS. Nevertheless, we have observed a number of interesting differences in the interaction of the dye with the apo and Ca2+ form. Native apo-GLA binds two bis-ANS molecules and in the complex with bis-ANS, the far-UV circular dichroism (CD) spectrum of apo-GLA becomes similar to that of the protein in the molten globule state. In contrast, native Ca2+-GLA binds five bis-ANS molecules and the far-UV CD spectrum of native Ca2+-GLA is conserved for the complex. In both cases, the high activation energies observed in kinetic experiments indicate that upon binding, large parts of the protein structure have to be reorganized. The reduced perturbation of the protein structure in the presence of Ca2+ can be attributed to local stabilization effects.     

Structural properties of semenogelin I

The zinc-binding protein semenogelin I is the major structural component of the gelatinous coagulum that is formed in freshly ejaculated semen. Semenogelin I is a rapidly evolving protein with a primary structure that consists of six repetitive units, each comprising approximately 60 amino acid residues. We studied the secondary and tertiary structure of semenogelin I by circular dichroism (CD) spectroscopy and Trp fluorescence emission spectroscopy. Fitting to the far-UV CD data indicated that the molecule comprises 5-10% alpha-helix and 20-30% beta-sheet formations. The far-UV spectrum of semenogelin I is clearly temperature dependent in the studied range 5-90 degrees C, and the signal at 222 nm increased with increasing temperature. The presence of Zn(2+) did not change the secondary structure revealed by the far-UV CD spectrum, whereas it did alter the near-UV CD spectrum, which implies that rearrangements occurred on the tertiary structure level. The conformational change induced in semenogelin I by the binding of Zn(2+) may contribute to the ability of this protein to form a gel.     

Interaction of insulin with a triblock copolymer of PEG-(fumaric-sebacic acids)-PEG: thermodynamic and spectroscopic studies

A comparative study on the interaction of (PEG-co-P(FA/SC)-co-PEG) triblock copolymer with bovine and human insulins was carried out using isothermal titration calorimetry (ITC), circular dichroism (CD), and fluorescence spectroscopy. ITC data show that the copolymer has a low affinity for both proteins, with an association constant of about 7-9 x 10(3) M (-1). Results also show that binding is enthalpically driven, and disfavored by conformational entropy. CD spectroscopy studies reveal a small increase in the helical content and a decrease in beta-structure as well as random coil in both proteins. Acrylamide quenching experiments display reduced accessibility of tyrosines, while intrinsic fluorescence spectra show lower tyrosine emission. Furthermore, thermal unfolding experiments, studied by far-UV CD at 222 and 217 nm, demonstrate that upon interaction with the copolymer helix structure becomes less stable while the stability of beta-structure remains unchanged. Altogether, these observations indicate that (PEG-co-P(FA/SC)-co-PEG) triblock copolymer has similar effect(s) on both proteins.     

Stable substructures of eightfold beta alpha-barrel proteins: fragment complementation of phosphoribosylanthranilate isomerase.

The (beta alpha)8 (or "TIM")-barrel protein phosphoribosylanthranilate isomerase from Saccharomyces cerevisiae was cleaved between the sixth and seventh beta alpha module to test the capacity of the resulting fragments to adopt native format autonomously. The fragments, which were expressed from separate coding sequences, were soluble and monomeric. The amino-terminal fragment p1 was compact, possessed an almost nativelike far-UV but a strongly reduced near-UV CD spectrum, and unfolded cooperativity with guanidinium chloride. In contrast, the carboxyl-terminal fragment p2 was less compact than fragment p1, possessed only a weak far-UV and no detectable near-UV CD spectrum, and unfolded noncooperatively. The fragments assembled stoichiometrically to a complex with Kd = 0.2 microM, which was enzymically almost fully active. The rate of assembly was limited by a first-order process, probably the isomerization of the carboxyl-terminal fragment p2 to an assembly-competent structure. These results support a folding mechanism that comprises an intermediate with the first six beta alpha units folded in roughly native format and the last two beta alpha units partially unfolded. The similar behavior of the analogous fragments of the alpha subunit of tryptophan synthease supports the hypothesis that these two (beta alpha)8-barrel proteins have evolved from a common ancestor.     

Protein folding intermediates of invasin protein IbeA from Escherichia coli

IbeA of Escherichia coli K1 was cloned, expressed and purified as a His(6)-tag fusion protein. The purified fusion protein inhibited E. coli K1 invasion of human brain microvascular endothelial cells and was heat-modifiable. The structural and functional aspects, along with equilibrium unfolding of IbeA, were studied in solution. The far-UV CD spectrum of IbeA at pH 7.0 has a strong negative peak at 215 nm, indicating the existence of beta-sheet-like structure. The acidic unfolding curve of IbeA at pH 2.0 shows the existence of a partially unfolded molecule (molten globule-like structure) with beta-sheet-like structure and displays strong 8-anilino-2-naphthyl sulfonic acid (ANS) binding. The pH dependent intrinsic fluorescence of IbeA was biphasic. At pH 2.0, IbeA exists in a partially unfolded state with characteristics of a molten globule-like state, and the protein is in extended beta-sheet conformation and exhibits strong ANS binding. Guanidine hydrochloride denaturation of IbeA in the molten globule-like state is noncooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two domains (possibly) in the molecular structure of IbeA, with differential unfolding stabilities. Furthermore, tryptophan quenching studies suggested the exposure of aromatic residues to solvent in this state. Acid denatured unfolding of IbeA monitored by far-UV CD is non-cooperative with two transitions at pH 3.0-1.5 and 1.5-0.5. At lower pH, IbeA unfolds to the acid-unfolded state, and a further decrease in pH to 2.0 drives the protein to the A state. The presence of 0.5 m KCl in the solvent composition directs the transition to the A state by bypassing the acid-unfolded state. Additional guanidine hydrochloride induced conformational changes in IbeA from the native to the A-state, as monitored by near- and far-UV CD and ANS-fluorescence.     

Conformational analysis of a set of peptides corresponding to the entire primary sequence of the N-terminal domain of the ribosomal protein L9: evidence for stable native-like secondary structure in the unfolded state

There is considerable interest in the structure of the denatured state and in the role local interactions play in protein stability and protein folding. Studies of peptide fragments provide one method to assess local conformational preferences which may be present in the denatured state under native-like conditions. A set of peptides corresponding to the individual elements of secondary structure derived from the N-terminal domain of the ribosomal protein L9 have been synthesized. This small 56 residue protein adopts a mixed alpha-beta topology and has been shown to fold rapidly in an apparent two-state fashion. The conformational preferences of each peptide have been analyzed by proton nuclear magnetic resonance spectroscopy and circular dichroism spectroscopy. Peptides corresponding to each of the three beta-stands and to the first alpha-helix are unstructured as judged by CD and NMR. In contrast, a peptide corresponding to the C-terminal helix is remarkably structured. This 17 residue peptide is 53 % helical at pH 5.4, 4 degrees C. Two-dimensional NMR studies demonstrate that the helical structure is distributed approximately uniformly throughout the peptide, although there is some evidence for fraying at the C terminus. Detailed analysis of the NMR spectra indicate that the helix is stabilized, in part, by a native N-capping interaction involving Thr40. A mutant peptide which lacks Thr40 is only 32 % helical. pH and ionic strength-dependent studies suggested that charge charge interactions make only a modest net contribution to the stability of the peptide. The protein contains a trans proline peptide bond located at the first position of the C-terminal helix. NMR analysis of the helical peptide and of a smaller peptide containing the proline residue indicates that only a small amount of cis proline isomer (8 %) is likely to be populated in the unfolded state.     

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K12. Carboxymethylation of the enzyme: threonine binding and inhibition are functionally dissociable.

The inactivation of the aspartokinase I-homoserine dehydrogenase I by iodoacetic acid and the effect on the sensitivity to its inhibitor, L-threonine, were examined. Both aspartokinase and homoserine dehydrogenase inactivation, as well as the dehydrogenase desensitization toward L-threonine occur as a pseudo-first order process. During its inactivation, the aspartokinase remains sensitive to L-threonine. At 50% inactivation, the inhibition curve of the aspartokinase by L-threonine displays homotropic cooperative effects. This alkylated protein retains eight binding sites for L-threonine. During the carboxymethylation, the protein remains in the tetrameric form until half of the kinase activity is lost. At the end of the inactivation aggregate forms and dimers appear.     

Circular dichroism studies on the alpha-D-galactopyranosyl binding lectin isolated from the seeds of Bandeiraea simplicifolia.

The conformation of the alpha-D-galactopyranosyl binding lectin isolated from Bandeiraea simplicifolia seeds has been investigated over a broad range of pH in the presence of various solvents by circular dichroism (CD) spectroscopy in the region 200-300 nm. Analyses of the spectra obtained on the native protein show the lectin to contain a considerable proportion of beta structure (30-40%). The native conformation was found to be largely insensitive to changes in pH, but was influenced by sodium dodecyl sulfate or trifluoroethanol. Alterations in conformation in the presence of these agents were reflected in the CD spectra and show the presence of alpha helix under these conditions. These changes in conformation are accompanied by a loss in polysaccharide-precipitating activity. The protein is irreversibly denatured in 8 M urea. Neither removal of the intrinsic calcium ions from the protein nor addition of methyl alpha-D-galactopyranoside induces any appreciable change in the CD spectra of the protein although the former treatment abolishes the polysaccharide-precipitating capacity of the lectin. The conformational data obtained in the present study are compared with data available from conformational studies of other lectins and leads to the hypothesis that most lectins probably contain beta structure as the predominant conformational feature.