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.     

Aromatic side-chain cluster of biotin binding site of avidin allows circular dichroism spectroscopic investigation of its ligand binding properties

Promiscuous ligand binding by hen egg-white avidin has been demonstrated and studied by using circular dichroism (CD) spectroscopy complemented by molecular docking calculations. It has been shown that the biotin-binding pocket of avidin is able to accommodate a wide variety of chemical compounds including therapeutic drugs (e.g., thalidomide, NSAIDs, antihistamines), natural compounds (bilirubin, myristic acid), and synthetic agents (xanthenone dyes). The cluster of aromatic residues located at the biotin-binding pocket renders the intrinsic CD spectrum of avidin sensitive to ligand binding that results in the increase of the vibronic components of the (1) L(b) transition of the Trp residues. Extrinsic (induced) CD bands measured with chemically diverse avidin ligands are generated by intramolecular coupled oscillator (e.g., bilirubin) or by intermolecular ligand-Trp exciton coupling mechanism [e.g., 2-(4'-hydroxyazobenzene)-benzoic acid (HABA)]. Among the compounds of which avidin-binding affinity constants have been calculated, two novel high-affinity ligands, flufenamic acid and an enzyme inhibitor thiazole derivative have been identified (K(d) ≈ 1 μM). Avidin binding mode of the ligand molecules has been discussed in the light of docking results. The induced CD profile of the thiazole derivative has been correlated with the stereochemistry of its docked conformation. The important role in the ligand binding of a polar side-chain cluster at the bottom of the biotin-binding cavity as well as the analogous avidin-binding mode of HABA and fenamic acid type NSAIDs have been proposed.     

Changes of tyrosine and tryptophan residues in human hemoglobin by oxygen binding: near- and far-UV circular dichroism of isolated chains and recombined hemoglobin

In order to assign the circular dichroism (CD) spectral change in the region between 280 and 300 nm of human adult hemoglobin (Hb A) upon the quaternary structure transition induced by oxygen binding, the near- and far-UV CD spectra of the isolated chains and the recombined hemoglobin were examined. Deoxygenation made the negative CD band at 290 nm of oxy-alpha chain deeper. On the other hand, positive CD bands of oxy-beta chain at the 280 to approximately 300 nm became negative upon deoxygenation. These changes were interpreted as being due to environmental alterations of tyrosine (Tyr) and/or tryptophan (Trp) perturbed by tertiary structural changes from the oxy to deoxy form in isolated chains, referring to the CD spectra of model compounds. From the difference between CD bands of the arithmetic mean of deoxy isolated chains and the CD band of deoxyHb tetramer, the contribution of tertiary structural change to the negative CD band of deoxyHb A at 287 nm was estimated to be 50%. This finding has revealed that the net contribution of quaternary structure transition to the negative band is 50%. In far-UV CD spectra, the environmental changes of aromatic residues upon the quaternary structure transition were also detected as a negative band at 225 nm.     

A natively unfolded toxin domain uses its receptor as a folding template

Natively unfolded proteins range from molten globules to disordered coils. They are abundant in eukaryotic genomes and commonly involved in molecular interactions. The essential N-terminal translocation domains of colicin toxins from Escherichia coli are disordered bacterial proteins that bind at least one protein of the Tol or Ton family. The colicin N translocation domain (ColN-(1-90)), which binds to the C-terminal domain of TolA (TolA-(296-421)), shows a disordered far-UV CD spectrum, no near-UV CD signal, and non-cooperative thermal unfolding. As expected, TolA-(296-421) displays both secondary structure in far-UV CD and tertiary structure in near-UV CD. Furthermore it shows a cooperative unfolding transition at 65 degrees C. CD spectra of the 1:1 complex show both increased secondary structure and colicin N-specific near-UV CD signals. A new cooperative thermal transition at 35 degrees C is followed by the unchanged unfolding behavior of TolA-(296-421). Fluorescence and surface plasmon resonance confirm that the new unfolding transition accompanies dissociation of ColN-(1-90). Hence upon binding the disordered structure of ColN-(1-90) converts to a cooperatively folded domain without altering the TolA-(296-421) structure.     

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.     

Changes in structure and stability of calbindin-D(28K) upon calcium binding

Calbindin-D(28K) is a biologically important protein required for normal neural function and for the transport of calcium in epithelial cells of the intestine and kidney. We have used fluorescence and circular dichroism (CD) spectroscopy to characterize the effects of calcium binding on the structure and stability of calbindin. Ca(2+) titration monitored by fluorescence spectroscopy reveals the presence of two classes of calcium-binding sites with association constants approximately 10(7.5) and approximately 10(8.9)M(-1). CD spectra in the far-UV spectral range show minor changes upon Ca(2+) titration, implying that the secondary structure of calbindin-D(28K) is not greatly affected. On the basis of the CD spectra in the near-UV spectral range, we conclude that the tertiary structure is more sensitive to Ca(2+) addition. The most significant change occurs between pCa 7.0 and pCa 8.0. The variations in the protein thermostability are correlated with those in the near-UV CD spectra. The enthalpy changes upon heat denaturation of calbindin in the apo-state are characteristic of proteins containing several weakly interacting domains with similar thermodynamical properties. Thus, calcium binding by calbindin-D(28K) largely affects the local structure around the aromatic residues and the thermal stability of the protein; the changes in the secondary structure are insignificant.     

Characterization of a molten globule state of bovine carbonic anhydrase III: loss of asymmetrical environment of the aromatic residues has a profound effect on both the near- and far-UV CD spectrum

Bovine muscle carbonic anhydrase (isoenzyme III; BCAIII) exhibited a three-state unfolding process at equilibrium upon denaturation in guanidine hydrochloride (GuHCl). The stable folding intermediate appeared to be of molten globule type. The stability towards GuHCl in terms of mid-point concentrations of denaturation were very similar for BCAIII and human CAII (HCAII). It was further demonstrated that the aromatic amino acid residues contributed significantly to the circular dichroism (CD) spectrum in the far-UV wavelength region during the native-->molten globule state transition. Thus, the ellipiticity change at 218 nm was shown to monitor the loss of tertiary interactions of aromatic side chains at the first unfolding transition as well as the rupture of secondary structure at the second unfolding transition. Similar aromatic contributions to the far-UV CD spectrum, but with varying magnitudes, were also noted for BCAII and HCAII, further emphasizing that interference of aromatic residues should not be neglected at wavelengths that normally are assigned to secondary structural changes.     

Preischemic infusion of alpha-human atrial natriuretic peptide elicits myoprotective effects against ischemia reperfusion in isolated rat hearts

In order to study the role of membrane proteins in bilirubin (BR) binding phenomenon, selective removal of membrane proteins was carried out using various reagents, namely, ethylenediamine tetraacetic acid (EDTA), sodium hydroxide (NaOH), 3,5-diiodosalicylic acid, lithium salt (LIS), dimethylmaleic anhydride (DMMA), sodium iodide (NaI), o-phenanthroline-cupric sulfate (CuP) and phenanthroline-cupric sulfate containing 2-mercaptoethanol (CuP-mercaptoethanol). Effects of these treatments on the conformation and BR binding properties of the membrane were studied using circular dichroism (CD) spectroscopy as well as estimation of membrane-bound BR by diazotised-color reaction. Though a significant amount of protein (ranging from 23–69%) was lost from the membranes upon these treatments, only a small decrease (3–13%) was observed in BR binding, being maximum with NaOH-treated membranes. However, DMMA and NaI treatments produced a little increase in BR binding. Conformation of the membrane was retained to a significant extent as indicated by far-UV CD spectra upon these treatments except in DMMA and NaI treatments which resulted in the perturbation in CD spectra. Taken together, these results suggest that membrane proteins play little role in BR binding, rather act as barriers in BR binding phenomenon.     

Interaction between proteins and cationic gemini surfactant

Surface tension, fluorescence, and circular dichroism (CD) methods have been used to investigate the interaction between cationic gemini surfactant 1,2-ethane bis(dimethyldodecylammonium bromide) (C12C2C12) and proteins including bovine serum albumin (BSA) and gelatin. Surface tension measurements show that the complexes of gelatin--C12C2C12 form more easily than that of BSA--C12C2C12. Addition of C12C2C12 has a different effect not only on the polarity of the microenvironment in BSA and gelatin systems but also on their fluorescence spectra. It can be seen from far-UV CD spectra that the alpha-helical network of BSA is disrupted and its content decreases from 41.7% to 27.6% while the random coil content of gelatin increases from 53.0% to 55.9% with increasing C12C2C12 concentration. The results from near-UV CD spectra show that the binding of C12C2C12 induces changes of the microenvironment around the aromatic amino acid residues and disulfide bonds of BSA at high C12C2C12 concentrations.     

Conformational stability of the penicillin G acylase fromKluyvera citrophila

Summary The mature penicillin G acylase fromKluyvera citrophila was examined by circular dichroism (CD). The far-UV CD spectrum at neutral pH revealed 11% alpha-helix, 44% beta-sheet, 11% beta-turn and 34% random coil. Far-UV and near-UV CD spectra showed that the enzyme presented a high conformational stability under different conditions of pH and salt concentration. The predictive model of Chou and Fasman indicated the presence of several beta-segments that could be arranged in antiparallel beta-sheets, which might explain the structural stability. The near-UV CD spectrum in the presence of penicillin G sulfoxide showed that the binding of this inhibitor to the enzyme resulted in modification of the dichroism of several aromatic residues.     

Spectroscopic characterization of the EF-hand domain of phospholipase C delta1: identification of a lipid interacting domain

The interaction of the isolated EF-hand domain of phospholipase C delta1 with arachidonic acid (AA) was characterized using circular dichroism (CD) and fluorescence spectroscopy. The far-UV CD spectral changes indicate that AA binds to the EF domain. The near-UV CD spectra suggest that the orientations of aromatic residues in the peptide are affected when AA binds to the protein. The fluorescence of the single intrinsic tryptophan located in EF1 was enhanced by the addition of dodecylmaltoside (DDM) and AA suggesting that this region of the protein is involved in hydrophobic interactions. In the presence of a low concentration of DDM it was found that AA induced a change in fluorescence resonance energy transfer, which is indicative of a conformational change. The lipid induced conformational change may play a role in calcium binding because the isolated EF-hand domain did not bind Ca2+ in the absence of lipids, but Ca2+-dependent changes in the intrinsic tryptophan emission were observed when free fatty acids were present. These studies identify specific EF-hand domains as allosteric regulatory domains that require hydrophobic ligands such as lipids.     

Structural changes in factor VIIa induced by Ca2+ and tissue factor studied using circular dichroism spectroscopy.

Factor VIIa (fVIIa) is composed of four discrete domains, a gamma-carboxyglutamic acid (Gla)-containing domain, two epidermal growth factor (EGF)-like domains, and a serine protease domain, all of which appear to be involved, to different extents, in an optimal interaction with tissue factor (TF). All except the second EGF-like domain contain at least one Ca2+ binding site and many properties of fVIIa, e.g., TF and phospholipid binding and amidolytic activity, are Ca(2+)-dependent. A CD study was performed to characterize and locate the conformational changes in fVIIa induced by Ca2+ and TF binding. In addition to intact fVIIa, derivatives lacking the Gla domain or the protease domain were used. Assignment of the Ca(2+)-induced changes in the far-UV region of the fVIIa spectrum to the Gla domain could be made by comparing the CD spectra obtained with these fVIIa derivatives. The changes primarily appeared to reflect a Ca(2+)-induced ordering of alpha-helices existing in the apo state of fVIIa. This was corroborated by models of the apo and Ca2+ forms of fVIIa, obtained as difference spectra between fVIIa derivatives, were very similar to those of isolated Gla peptides from other vitamin K-dependent plasma proteins. The near-UV CD spectrum of fVIIa was dominated by aromatic residues residing in the protease domain and specific bands affected by Ca2+ were indicative of tertiary structural alterations. The formation of a fVIIa:TF complex led to secondary structural changes that appeared to be restricted to the catalytic domain, possibly shedding light on the mechanism by which TF induces an enhancement of fVIIa catalytic activity.     

Molten globule-like state of human serum albumin at low pH.

Human serum albumin (HSA), under conditions of low pH, is known to exist in two isomeric forms, the F form at around pH 4.0 and the E form below 3.0. We studied its conformation in the acid-denatured E form using far-UV and near-UV CD, binding of a hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (ANS), thermal transition by far-UV and near-UV CD, tryptophan fluorescence, quenching of tryptophan fluorescence using a neutral quencher, acrylamide and viscosity measurements. The results show that HSA at pH 2.0 is characterized by a significant amount of secondary structure, as evident from far-UV CD spectra. The near-UV CD spectra showed a profound loss of tertiary structure. A marked increase in ANS fluorescence signified extensive solvent exposure of non-polar clusters. The temperature-dependence of both near-UV and far-UV CD signals did not exhibit a co-operative thermal transition. The intrinsic fluorescence and acrylamide quenching of the lone tryptophan residue, Trp214, showed that, in the acid-denatured state, it is buried in the interior in a non-polar environment. Intrinsic viscosity measurements showed that the acid-denatured state is relatively compact compared with that of the denatured state in 7 M guanidine hydrochloride. These results suggest that HSA at pH 2.0 represents the molten globule state, which has been shown previously for a number of proteins under mild denaturing conditions.     

Cloning, overexpression, purification, and spectroscopic characterization of human S100P.

The calcium-binding protein S100P has been found to be associated with human prostate cancer. We have overexpressed S100P in Escherichia coli using a T7 expression system. A rapid two-step procedure for the isolation of overexpressed S100P leads to a preparation of >95% pure protein with a yield of approximately 150 mg per liter of culture. The structural integrity of recombinant S100P was analyzed using CD and fluorescence spectroscopic techniques. The far-UV CD shows that secondary structure of recombinant S100P consists predominantly of a-helical structure. Both near-UV CD and tyrosine fluorescence spectra show that aromatic residues are involved in the formation of a specific, well packed structure, indicating that the recombinant S100P protein adopts a compact folded conformation. Ca2+ has a profound effect on S100P structure. Near-UV CD and fluorescence intensity of both internal (tyrosine) and external (ANS) probes suggest significant structural rearrangements in the tertiary structure of the molecule. The similarity of far-UV CD spectrum of S100P in the presence and in the absence of Ca2+ suggests that Ca2+ binding has only minor effects on secondary structure.     

Calcium-dependent conformational rearrangements and protein stability in chicken annexin A5

The conformational rearrangements that take place after calcium binding in chicken annexin A5 and a mutant lacking residues 3-10 were analyzed, in parallel with human annexin A5, by circular dichroism (CD), infrared spectroscopy (IR), and differential scanning calorimetry. Human and chicken annexins present a slightly different shape in the far-UV CD and IR spectra, but the main secondary-structure features are quite similar (70-80% alpha-helix). However, thermal stability of human annexin is significantly lower than its chicken counterpart (approximately 8 degrees C) and equivalent to the chicken N-terminally truncated form. The N-terminal extension contributes greatly to stabilize the overall annexin A5 structure. Infrared spectroscopy reveals the presence of two populations of alpha-helical structures, the canonical alpha-helices (approximately 1650 cm(-1)) and another, at a lower wavenumber (approximately 1634 cm(-1)), probably arising from helix-helix interactions or solvated alpha-helices. Saturation with calcium induces: alterations in the environment of the unique tryptophan residue of the recombinant proteins, as detected by near-UV CD spectroscopy; more compact tertiary structures that could account for the higher thermal stabilities (8 to 12 degrees C), this effect being higher for human annexin; and an increase in canonical alpha-helix percentage by a rearrangement of nonperiodical structure or 3(10) helices together with a variation in helix-helix interactions, as shown by amide I curve-fitting and 2D-IR.     

Optical spectroscopic characterization of single tryptophan mutants of chicken skeletal troponin C: evidence for interdomain interaction.

The effects of metal ion binding on the optical spectroscopic properties and temperature stability of two single tryptophan mutants of chicken skeletal TnC, F78W and F154W, have been examined. The absence of tyrosine and other tryptophan residues allowed the unambiguous assignment of the spectral signal from the introduced Trp residue. Changes in the molar ellipticity values in the far-UV CD spectra of the mutant proteins on metal ion binding were similar to those of wild-type TnC suggesting that the introduction of the Trp residue had no effect on the total secondary structure content. The fluorescence and near-UV absorbance data reveal that, in the apo state, Trp-78 is buried while Trp-154 is exposed to solvent. Additionally, the highly resolved (1)L(b) band of Trp-78 seen in the near-UV absorbance and CD spectra of the apo state of F78W suggest that this residue is likely in a rigid molecular environment. In the calcium-saturated state, Trp-154 becomes buried while the solvent accessibility of Trp-78 increases. The fluorescence emission and near-UV CD of Trp-78 in the N-terminal domain were sensitive to calcium binding at the C-terminal domain sites. Measurements of the temperature stability reveal that events occurring in the N-terminal domain affect the stability of the C-terminal domain and vice versa. This, coupled with the titration data, strongly suggests that there are interactions between the N- and C-terminal domains of TnC.     

A possible receptor-binding function for the N-terminus of connective tissue activating peptide III

Connective tissue activating peptide III (CTAP-III) is an 85-residue peptide which has been purified from platelets and shown to possess mitogenic activity toward a variety of fibroblastic cell lines. beta-Thromboglobulin (beta TG) is an 81-residue peptide which is derived from CTAP-III by cleavage of the N-terminal tetrapeptide Asn-Leu-Ala-Lys which results in the loss of mitogenic activity. The near-UV CD spectra for the two proteins indicated that the conformations as well as the electronic environments of the two disulfide bonds, and also of the single aromatic tyrosine residue, were similar in CTAP-III and beta TG. However, differences in the far-UV CD spectra of these proteins indicated a substantial decrease in alpha-helical content for beta TG (29%) as compared to CTAP-III (38%). Structure prediction analysis also suggested that the longer N-terminal segment of CTAP-III may form an alpha-helix. The N-terminal region of beta TG, which lacks this tetrapeptide, was predicted to be in an unordered, or possibly a turn, conformation. This predicted structural difference appears to be due to the high helix-forming potential of the N-terminal tetrapeptide Asn-Leu-Ala-Lys in CTAP-III. These results suggest a possible structural role for the N-terminal region of CTAP-III in the expression of the biologic activities of this protein. On the basis of these studies, a reasonable hypothesis to account for the difference in mitogenic activity between beta TG and CTAP-III is that the N-terminal region must be helical for receptor binding to occur.     

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.     

The binding of copper ions to glycine-rich proteins (GRPs) from Cicer arietinum

Cicer arietinum GRP1 and GRP2 are rich in glycine interposed with histidine and tyrosine. In order to study whether or not these proteins bind Cu(2+), circular dichroism (CD) and nuclear magnetic resonance (NMR) were measured for three synthetic peptides corresponding to sections of the protein's sequences including 1, N(1)Y(2)G(3)H(4)G(5)G(6)G(7)N(8)Y(9)G(10)N(11), where all peptides were chemically blocked with an acetyl group at the N-terminus and an -NH(2) group at the C-terminus. The visible CD spectra for 1 showed a positive peak near 590 nm not at pH 6.0 but pH 7.4 in the presence of copper ions. The Cu(2+) binding induced a drastic change in the far-UV CD spectra, showing the occurrence of large conformation changes. In the 2D TOCSY NMR spectra at pH 7.4, the addition of small amounts of CuSO(4) caused a significant broadening of proton resonances of not only His4 but also Gly5, Asn8 and Asn11. CD titration experiment suggested that NYGHGGGNYGN including one repeat unit comprises the fundamental Cu(2+) binding unit.     

Significance of the extra C-terminal tail of CaLP, a novel calmodulin-like protein involved in oyster calcium metabolism

Oyster (Pinctada fucata) calmodulin-like protein (CaLP), containing a C-terminally extra hydrophilic tail (150D–161K), is a novel protein involved in the regulation of oyster calcium metabolism. To investigate the importance of the extra fragment to the Ca²⁺/Mg²⁺-dependent conformational changes in the intact CaLP molecule and the interactions between CaLP and its target proteins, a truncated CaLP mutant (M-CaLP) devoid of the extended C-terminus was constructed and overexpressed in Escherichia coli. The conformational characteristics of M-CaLP were studied by CD and fluorescence spectroscopy and compared with those of the oyster CaM and CaLP. The far-UV CD results reveal that the extra tail has a strong effect on the Ca²⁺-induced, but a relatively weak effect on the Mg²⁺-induced conformational changes in CaLP. However, upon Ca²⁺ or Mg²⁺ binding, only slight changes for intrinsic phenylalanine and tyrosine fluorescence spectra between M-CaLP and CaLP are observed. Our results also indicate that the extra tail can significantly decrease the exposure of the hydrophobic patches in CaLP. Additionally, affinity chromatography demonstrates that the target binding of CaLP is greatly influenced by its additional tail. All our results implicate that the extra tail may play some important roles in the interactions between CaLP and its targets in vivo.