Structural analysis of the PsbQ protein of photosystem II by Fourier transform infrared and circular dichroic spectroscopy and by bioinformatic methods

The structure of PsbQ, one of the three main extrinsic proteins associated with the oxygen-evolving complex (OEC) of higher plants and green algae, is examined by Fourier transform infrared (FTIR) and circular dichroic (CD) spectroscopy and by computational structural prediction methods. This protein, together with two other lumenally bound extrinsic proteins, PsbO and PsbP, is essential for the stability and full activity of the OEC in plants. The FTIR spectra obtained in both H(2)O and D(2)O suggest a mainly alpha-helix structure on the basis of the relative areas of the constituents of the amide I and I' bands. The FTIR quantitative analyses indicate that PsbQ contains about 53% alpha-helix, 7% turns, 14% nonordered structure, and 24% beta-strand plus other beta-type extended structures. CD analyses indicate that PsbQ is a mainly alpha-helix protein (about 64%), presenting a small percentage assigned to beta-strand ( approximately 7%) and a larger amount assigned to turns and nonregular structures ( approximately 29%). Independent of the spectroscopic analyses, computational methods for protein structure prediction of PsbQ were utilized. First, a multiple alignment of 12 sequences of PsbQ was obtained after an extensive search in the public databases for protein and EST sequences. Based on this alignment, computational prediction of the secondary structure and the solvent accessibility suggest the presence of two different structural domains in PsbQ: a major C-terminal domain containing four alpha-helices and a minor N-terminal domain with a poorly defined secondary structure enriched in proline and glycine residues. The search for PsbQ analogues by fold recognition methods, not based on the secondary structure, also indicates that PsbQ is a four alpha-helix protein, most probably folding as an up-down bundle. The results obtained by both the spectroscopic and computational methods are in agreement, all indicating that PsbQ is mainly an alpha protein, and show the value of using both methodologies for protein structure investigation.     

Isolation and characterization of a thermostable beta-xylosidase in the thermophilic bacterium Geobacillus pallidus

The isolation, purification, biochemical and biophysical characterization of the first reported beta-xylosidase from Geobacillus pallidus are described. The protein has an optimum pH close to 8 and an optimum temperature of 70 degrees C. These biochemical properties agree with those obtained by spectroscopic techniques, namely, circular dichroism (CD), infrared (FTIR) and fluorescence measurements. Thermal denaturation, followed by CD and FTIR, showed an apparent thermal denaturation midpoint close to 80 degrees C. The protein was probably a hydrated trimer in solution with, an elongated shape, as shown by gel filtration experiments. FTIR deconvolution spectra indicated that the protein contains a high percentage of alpha-helix (44%) and beta-sheet (40%). The sequencing of the N terminus and the biochemical features indicate that this new member of beta-xylosidases belongs to the GH52 family. Since there are no reported structural studies of any member of this family, our studies provide the first clue for the full conformational characterization of this protein family.     

Plasticity of secondary structure in the N-terminal region of beta-dystroglycan

The secondary structure content of the N-terminal extracellular domain of beta-dystroglycan (a recombinant fragment extending from positions 654 to 750) has been quantitatively determined by means of CD and FTIR spectroscopies. The elements of secondary structure, namely an 8-10 residue long alpha-helix (10%) and two beta-strands (24%) have been assigned to specific amino acid sequences by means of a GOR constrained prediction method. The remaining 66% of the whole sequence is classified as turns or unordered. The temperature dependence of CD and FTIR spectra has been investigated in detail. A reversible, non-cooperative thermal transition is observed with both CD and FTIR spectroscopies up to 95 degrees C. The profile of the transition is typical of the unfolding of isolated peptides and corresponds to the progressive loss of the secondary structure elements of the protein with no evidence for collapsing phenomena, typical of globular proteins, upon heating.     

Alteration of a single tryptophan residue of the cellulose-binding domain blocks secretion of the Erwinia chrysanthemi Cel5 cellulase (ex-EGZ) via the type II system

Cel5 (formerly known as endoglucanase Z) of Erwinia chrysanthemi is secreted by the Out type II pathway. Previous studies have shown that the catalytic domain (CD), linker region (LR) and cellulose-binding domain (CBD) each contain information needed for secretion. The aim of this work was to further investigate the secretion-related information present in the CBD(Cel5). Firstly(, )deleting a surface-exposed flexible loop had no effect on secretion. This indicated that some structural freedom is tolerated by the type II system. Secondly, mutation of a single tryptophan residue, previously shown to be important for binding to cellulose, i.e. Trp43, was found also to impair secretion. This indicated that the flat cellulose-binding surface of CBD(Cel5 )contains secretion-related information. Thirdly, CBD(Cel5) was substituted by the CBD(EGG) of Alteromonas haloplanctis endoglucanase G, yielding a hybrid protein CD(Cel5)-LR(Cel5)-CBD(EGG) that exhibited 90 % identity with Cel5, including the Trp43 residue. The hybrid protein was not secreted. This indicated that the Trp43 residue is necessary but not sufficient for secretion. Here we propose a model in which the secretion of Cel5 involves a transient intramolecular interaction between the cellulose-binding surface of CBD(Cel5) and a region close to the entry into the active site in CD(Cel5). Once secreted, the protein may then open out to allow the cellulose-binding surface of CBD(Cel5 )to interact with the surface of the cellulose substrate. An implication of this model is that protein molecules fold to a specific secretion-competent conformation prior to secretion that is different from the folding state of the secreted species.     

Structure and conformational stability of the enzyme I of Streptomyces coelicolor explored by FTIR and circular dichroism

The bacterial phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS), formed by a cascade of several proteins, couples the translocation and phosphorylation of specific sugars across cell membranes. The structure and thermal stability of the first protein (enzyme I, EI) of the PTS in Streptomyces coelicolor is studied by using far-UV circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) at pH 7.0. The deconvolution of FTIR spectra indicates that the protein is mainly composed by a 35% of alpha-helical structure and 30% of beta-sheet. The thermal denaturation curves, as followed by both techniques, show only a midpoint at 330 K. This thermal denaturation behaviour is different to that observed in other members of the EI family.     

Trifluoroethanol-induced conformational change of tetrameric and monomeric soybean agglutinin: Role of structural organization and implication for protein folding and stability

2,2,2-Trifuoroethanol (TFE)-induced conformational structure change of a β-sheet legume lectin, soybean agglutinin (SBA) has been investigated employing its exclusive structural forms in quaternary (tetramer) and tertiary (monomer) states, by far- and near-UV CD, FTIR, fluorescence, low temperature phosphorescence and chemical modification. Far-UV CD results show that, for SBA tetramer, native atypical β-conformation transforms to a highly α-helical structure, with the helical content reaching 57% in 95% TFE. For SBA monomer, atypical β-sheet first converts to typical β-sheet at low TFE concentration (10%), which then leads to a nonnative α-helix at higher TFE concentration. From temperature-dependent studies (5–60 °C) of TFE perturbation, typical β-sheet structure appears to be less stable than atypical β-sheet and the induced helix entails reduced thermal stability. The heat induced transitions are reversible except for atypical to typical β-sheet conversion. FTIR results reveal a partial α-helix conversion at high protein concentration but with quantitative yield. However, aggregation is detected with FTIR at lower TFE concentration, which disappears in more TFE. Near-UV CD, fluorescence and phosphorescence studies imply the existence of an intermediate with native-like secondary and tertiary structure, which could be related to the dissociation of tetramer to monomer. This has been further supported by concentration dependent far-UV CD studies. Chemical modification with N-bromosuccinimide (NBS) shows that all six tryptophans per monomer are solvent-exposed in the induced α-helical conformation. These results may provide novel and important insights into the perturbed folding problem of SBA in particular, and β-sheet oligomeric proteins in general.     

Structure of unfolded and refolded recombinant derived [Ala125]interleukin 2

Naturally occurring interleukin 2 (IL-2) contains an odd number (three) of cysteinyl residues and thus is susceptible to the formation of a variety of intramolecular and intermolecular disulfide bonds. The cysteine at residue 125 has been replaced with an alanine residue by site-directed mutagenesis, and hence, this analogue can form only one intrachain disulfide bond. When expressed at high levels in Escherichia coli, this recombinant DNA derived IL-2 analogue is insoluble, reduced, and inactive. The protein was solubilized by denaturants and, after purification, was oxidized to form an intramolecular disulfide bond. Circular dichroism (CD) has been used to investigate the effects of various denaturants on the unfolding-refolding process of the purified, oxidized protein. A similar conformation is obtained when [Ala125]interleukin 2 [IL-2(Ala-125)] is refolded from 6 M guanidine hydrochloride, 8 M urea, or 5% acetic acid. The resultant protein, refolded from these denaturants, is monomeric and has activity comparable to or greater than that reported for naturally derived IL-2. In addition to this form, aggregates, as evidenced from gel filtration, are obtained. The specific activities of these are greatly reduced, and CD spectra indicated that they have much less helical content than the monomeric form of the protein. CD spectra also showed that the tertiary structure of IL-2(Ala-125) is entirely different in the presence of sodium dodecyl sulfate (SDS) from that of the monomeric form in the absence of SDS.     

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.     

Conformation of pepsin and pepsinogen: some aspects of the role of tyrosine residues and the 1-44 segment of pepsinogen on conformational stability

Conformational changes induced in pepsin and pepsinogen by iodination of tyrosine residues and the possible role of lysine residues on conformational stability of pepsinogen are investigated by circular dichroism (CD) studies in solution. At low degrees of iodination (6 I/molecule) the pepsin molecule denatured, with complete loss of β-structure at pH 5.5. Pepsinogen showed greater resistance to conformational change on iodination (10 I/molecule) and about 30% of its ordered structure is retained. In the aromatic region, the tyrosyl CD bands of iodinated pepsin decreased in intensity, indicating a change in the environment of tyrosine residues. A comparison with the CD spectra of expanded structures of pepsin in 6 m guanidine hydrochloride or alkaline solutions (pH 9.75) indicated retention of a significant amount of tertiary structure in iodinated pepsin. Changes in tertiary structures were marginal on iodination of pepsinogen. Less than 1% (residue moles) of poly-l-lysine, a known inhibitor, was found to destabilize the secondary and tertiary structure of pepsin at pH 6.75, although the lysine-rich 1–44 segment of pepsinogen tends to stabilize the conformation of the pepsin chain. This seems to suggest that the inhibitory effects of polylysine on pepsin occur by a mechanism different from that of the activity-limiting effect of the lysine-rich 1–44 segment of pepsinogen.     

Trifluoroethanol-induced unfolding of concanavalin A: equilibrium and time-resolved optical spectroscopic studies

Conformational structure changes in concanavalin A (Con A), a legume lectin protein which is composed of 18 beta-strands, induced by dissolving in 50% trifluoroethanol (TFE) were monitored at neutral and low pH by far- and near-UV circular dichroism (CD), fluorescence, and FTIR under equilibrium conditions. Stopped-flow studies using CD and fluorescence as well as FTIR, at low and high protein concentration, respectively, were carried out to follow the time-dependent conformation changes occurring after rapid mixing of the protein with TFE. Equilibrium CD results show that, upon addition of TFE, low-concentration Con A transforms to a highly alpha-helical conformation at both neutral and low pH. However, at neutral pH under high protein concentration conditions, aggregation and precipitation are eventually detected with FTIR, indicating that a final beta-structure is attained. Stopped-flow fluorescence shows the existence of an unfolding intermediate for pH 2.0 and 4.5, which could be related to the dissociation of the dimer form. However, evidence for an intermediate is not obtained at pH 6.7, where the native protein is a tetramer. Stopped-flow FTIR is consistent with these results, indicating formation of a H(+)-stabilized intermediate alpha-helical conformation before aggregation develops. Con A in TFE provides an example of an intermediate with non-native secondary structure appearing on the unfolding pathway. On the basis of the kinetic results obtained, an unfolding mechanism is proposed and some stable intermediates are identified. In turn, the slow structural change of Con A induced by TFE provides a useful model system for study of protein unfolding due to its accessibility with several spectroscopic and kinetic tools.     

Controlling beta-sheet assembly in genetically engineered silk by enzymatic phosphorylation/dephosphorylation

Enzymatic phosphorylation and dephosphorylation reactions were used to modify a genetically engineered variant of spider dragline silk. The approximately 25 kDa protein was phosphorylated with cyclic AMP-dependent kinase and dephosphorylated with calf intestinal alkaline phosphatase. Phosphorylation inhibited beta-sheet assembly of the protein and enhanced solubility to about 5 mg/mL in water, compared to about 20% of this level upon enzymatic dephosphorylation. The cyclability of the phosphorylation-dephosphorylation system was confirmed by MALDI with a model peptide. Kinetic studies conducted with [gamma-(32)P]ATP illustrate that the phosphorylation reaction proceeds over 6 h. Secondary structure of the phosphorylated and dephosphorylated proteins was determined by CD and FTIR. The results illustrate that an enzymatic phosphorylation event can be used to control the solution structure of a protein like silk, which has a tendency to prematurely precipitate due to the formation of beta-sheets.     

Effects of oligomerization and secondary structure on the surface behavior of pulmonary surfactant proteins SP-B and SP-C

The relationship among protein oligomerization, secondary structure at the interface, and the interfacial behavior was investigated for spread layers of native pulmonary surfactant associated proteins B and C. SP-B and SP-C were isolated either from butanol or chloroform/methanol lipid extracts that were obtained from sheep lung washings. The proteins were separated from other components by gel exclusion chromatography or by high performance liquid chromatography. SDS gel electrophoresis data indicate that the SP-B samples obtained using different solvents showed different oligomerization states of the protein. The CD and FTIR spectra of SP-B isolated from all extracts were consistent with a secondary structure dominated by alpha-helix. The CD and FTIR spectra of the first SP-C corresponded to an alpha-helical secondary structure and the spectra of the second SP-C corresponded to a mixture of alpha-helical and beta-sheet conformation. In contrast, the spectra of the third SP-C corresponded to antiparallel beta-sheets. The interfacial behavior was characterized by surface pressure/area (pi-A) isotherms. Differences in the oligomerization state of SP-B as well as in the secondary structure of SP-C all produce significant differences in the surface pressure/area isotherms. The molecular cross sections determined from the pi-A isotherms and from dynamic cycling experiments were 6 nm(2)/dimer molecule for SP-B and 1.15 nm(2)/molecule for SP-C in alpha-helical conformation and 1.05 nm(2)/molecule for SP-C in beta-sheet conformation. Both the oligomer ratio of SP-B and the secondary structure of SP-C strongly influence organization and behavior of these proteins in monolayer assemblies. In addition, alpha-helix --> beta-sheet conversion of SP-C occurs simply by an increase of the summary protein/lipid concentration in solution.     

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.     

MEARA sequence repeat of human CstF-64 polyadenylation factor is helical in solution. A spectroscopic and calorimetric study.

The primary structure of the human CstF-64 polyadenylation factor contains 12 nearly identical repeats of a consensus motif of five amino acid residues with the sequence MEAR(A/G). No known function has yet been ascribed to this motif; however, according to secondary structure prediction algorithms, it should form a helical structure in solution. To validate this theoretical prediction, we synthesized a 31 amino acid residue peptide (MEARA(6)) containing six repeats of the MEARA sequence and characterized its structure and stability by circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC). No effects of concentration on the CD or DSC properties of MEARA(6) were observed, indicating that the peptide is monomeric in solution at concentrations up to 2 mM. The far UV-CD spectra of MEARA(6) indicates that at a low temperature (1 degrees C) the MEARA(6) peptide has a relatively high helical content (76% at pH 2.0 and 65% at pH 7.0). The effects of pH and ionic strength on the CD spectrum of MEARA(6) suggest that a number of electrostatic interactions (e.g., i, i + 3 Arg/Glu ion pair, charge-dipole interactions) contribute to the stability of the helical structure in this peptide. DSC profiles show that the melting of MEARA(6) helix is accompanied by positive change in the enthalpy. To determine thermodynamic parameters of helix-coil transition from DSC profiles for this peptide, we developed a new, semiempirical procedure based on the calculated function for the heat capacity of the coiled state for a broad temperature range. The application of this approach to the partial molar heat capacity function for MEARA(6) provides the enthalpy change for helix formation calculated per amino acid residue as 3.5 kJ/mol.     

The family 52 beta-xylosidase from Geobacillus stearothermophilus is a dimer: structural and biophysical characterization of a glycoside hydrolase

Xylans are the most abundant polysaccharides forming the plant cell wall hemicelluloses, and they are degraded, among other proteins, by beta-xylosidase enzymes. In this work, the structural and biophysical properties of the family 52 beta-xylosidase from Geobacillus stearothermophilus, XynB2, are described. Size exclusion chromatography, analytical centrifugation, ITC, CD, fluorescence (steady state and ANS-binding) and FTIR were used to obtain the structure, the oligomerization state and the conformational changes of XynB2, as pH, chemical denaturants or temperature were modified. This report describes the first extensive conformational characterization of a family 52 beta-xylosidase. The active protein was a highly hydrated dimer, whose active site was formed by the two protomers, and it probably involved aromatic residues. At low pH, the protein was not active and it populated a monomeric molten-globule-like species, which had a conformational transition with a pK(a) of approximately 4.0. Thermal and chemical-denaturations of the native protein showed hysteresis behaviour. The protein at physiological pH was formed by alpha-helix (30%) and beta-sheet (30%), as shown by CD and FTIR. Comparison with other xylosidases of the same family indicates that the percentages of secondary structure seem to be conserved among the members of the family.     

RNA-dependent folding and stabilization of C5 protein during assembly of the E. coli RNase P holoenzyme

The pre-tRNA processing enzyme ribonuclease P is a ribonucleoprotein. In Escherichia coli assembly of the holoenzyme involves binding of the small (119 amino acid residue) C5 protein to the much larger (377 nucleotide) P RNA subunit. The RNA subunit makes the majority of contacts to the pre-tRNA substrate and contains the active site; however, binding of C5 stabilizes P RNA folding and contributes to high affinity substrate binding. Here, we show that RNase P ribonucleoprotein assembly also influences the folding of C5 protein. Thermal melting studies demonstrate that the free protein population is a mixture of folded and unfolded conformations under conditions where it assembles quantitatively with the RNA subunit. Changes in the intrinsic fluorescence of a unique tryptophan residue located in the folded core of C5 provide further evidence for an RNA-dependent conformational change during RNase P assembly. Comparisons of the CD spectra of the free RNA and protein subunits with that of the holoenzyme provide evidence for changes in P RNA structure in the presence of C5 as indicated by previous studies. Importantly, monitoring the temperature dependence of the CD signal in regions of the holoenzyme spectra that are dominated by protein or RNA structure permitted analysis of the thermal melting of the individual subunits within the ribonucleoprotein. These analyses reveal a significantly higher Tm for C5 when bound to P RNA and show that unfolding of the protein and RNA are coupled. These data provide evidence for a general mechanism in which the favorable free energy for formation of the RNA-protein complex offsets the unfavorable free energy of structural rearrangements in the RNA and protein subunits.     

Establishing isostructural metal substitution in metalloproteins using 1H NMR, circular dichroism, and Fourier transform infrared spectroscopy.

Far-UV CD, 1H-NMR, and Fourier transform infrared (FTIR) spectroscopy are three of the most commonly used methods for the determination of protein secondary structure composition. These methods are compared and evaluated as a means of establishing isostructural metal substitution in metalloproteins, using the crystallographically defined rubredoxin from Desulfovibrio gigas and its well-characterized cadmium derivative as a model system. It is concluded that analysis of the FTIR spectrum of the protein amide I resonance represents the most facile and generally applicable method of determining whether the overall structure of a metalloprotein has been altered upon metal reconstitution. This technique requires relatively little biological material (ca. 300 micrograms total protein) and, unlike either CD or 1H-NMR spectroscopy, is unaffected by the presence of different metal ions, thus allowing the direct comparison of FTIR spectra before and after metal substitution.     

Conformational analysis of PKI(5-22)amide, the active inhibitory fragment of the inhibitor protein of the cyclic AMP-dependent protein kinase.

Fourier-transform i.r. spectroscopy, 1H-n.m.r. spectroscopy and X-ray scattering were used to study the conformation and shape of the peptide PKI(5-22)amide, which contains the active site of the inhibitor protein of the cyclic AMP-dependent protein kinase [Cheng, Van Pattern, Smith & Walsh (1985) Biochem. J. 231, 655-661]. The X-ray-scattering solution studies show that the peptide has a compact structure with Rg 0.9 nm (9.0 A) and a linear maximum dimension of 2.5 nm (25A). Compatible with this, Fourier-transform i.r. and n.m.r. determinations indicate that the peptide contains approx. 26% alpha-helix located in the N-terminal one-third of the molecule. This region contains the phenylalanine residue that is one essential recognition determinant for high-affinity binding to the protein kinase catalytic site.