On the isolation and conformation of bovine β-casein A1

A new method, involving only gentle procedures, is described for the isolation of bovine β-casein. The optical rotatory dispersion (o.r.d.) and circular dichroism (c.d.) of the A¹ variant, so isolated, are determined in the temperature range 2–60°C, at pH 6.9 (25°C) and $\text{I = 0.012}\text{mol l}{}^{\mathrm{?1}}$. Conformational analyses are made of the o.r.d. and c.d. results using the reference c.d. spectra of Brahms and Brahms, the Kronig—Kramers transform, and also of the c.d. results by the method of Provencher and Glöckner. The conformations obtained are compared with those predicted for the amino acid sequence by the methods of Chou and Fasman and of Lim. It is concluded that β-casein contains $\text{9 ± 2\%}$ α-helical structure and $\text{25 ± 6\%}$ β-sheet at 2°C. A structural interpretation is proposed for the effect of increase of temperature on the o.r.d. and c.d., involving an increase in the proportion of β-sheet at the expense of aperiodic structure.     

Variable selection method improves the prediction of protein secondary structure from circular dichroism spectra

A new procedure based on the statistical method of "variable selection" is used to predict the secondary structure of proteins from circular dichroism spectra. Variable selection adds the flexibility found in the Provencher and Gl?ckner method (S. W. Provencher and J. Gl?ckner, 1981, Biochemistry 20, 33-37) to the method of Hennessey and Johnson (J. P. Hennessey and W. C. Johnson, 1981, Biochemistry 20, 1085-1094). Two analytical methods are presented for choosing a solution from the series generated by the Provencher and Gl?ckner method, and this improves the technique. All three methods are compared and it is shown that both the variable selection method and the improved Provencher and Gl?ckner methods have equivalent reliability superior to the original Hennessey and Johnson method. For the new variable selection method, correlation coefficients calculated between X-ray structure and predicted secondary structures for data measured to 178 nm are: 0.97 for alpha-helix, 0.75 for beta-sheet, 0.50 for beta-turn, and 0.89 for other structures. Although the variable selection method improves the analysis of circular dichroism data truncated at 190 nm, data measured to 178 nm gives superior results. It is shown that improving the fit to the measured CD beyond the accuracy of the data can result in poorer analyses.     

Conformational analysis of a snake neurotoxin by prediction from sequence, circular dichroism, and raman spectroscopy.

The secondary structure of the major neurotoxin from the sea snake Lapemis hardwickii was investigated by several methods of conformational analysis: structure prediction, circular dichroism, and laser Raman spectroscopy. From the primary structure, secondary structure prediction yielded two regions of β-sheet structure at residues 1–7 and 41–45. β-Turns were predicted at residues 14–17, 20–23, 30–33, 37–40, and 46–49. From the predictions, the toxin appears to be composed of approximately 20% β-sheet and 33% β-turn. The CD spectrum of the native toxin appears to be a hybrid of model spectra for β-sheet and β-turn proteins. The pH perturbation studies on the toxin observed by CD demonstrated that the toxin is a very stable molecule except at extremely high or low pH values. The Raman data indicated that the toxin contains both antiparallel β-sheet and β-turn structure. Using two methods of secondary structure quantitation from Raman spectra the molecule was calculated to contain 35% β-sheet from one method and 27% from the other. Overall, the various methods demonstrate that the toxin is composed of β-sheet and β-turn structure with little or no α-helix present. From the comparison of these different techniques appreciation can be gained for the necessity of several methods when identifying and quantitating secondary structure.     

7S Globulins from Phaseolus vulgaris L.: Impact of Structural Aspects on the Nutritional Quality

7S globulins were extracted from common bean (Phaseolus vulgaris L.) seeds and characterized. SDS–PAGE showed major bands corresponding to the phaseolin subunits (43–53 kDa). An amino acid analysis indicated that, in spite of the limited amounts of sulphur amino acids and tryptophan, the globulins contained very high levels of essential amino acids. The protein solubility profiles of native and denatured (120 °C for 20 min) 7S globulins in water and in 0.5 M NaCl showed that NaCl had a limited effect on increasing the solubility of either the native or denatured proteins. The in vivo small intestinal digestibility of the 7S globulins was 90%, this being decreased to 86% after a thermal treatment. Fourier transform infrared spectroscopy revealed a high content of β-sheet and β-turn structures, together with a contribution at 1687 cm^?1 that was assigned to intramolecular β-sheets. These features are diagnostic of a high propensity to irreversible aggregation that may be related to an adverse effect on the protein quality.     

Basic fibroblast growth factor is a β-rich protein

The conformation of the 153-residue form of human basic fibroblast growth factor (bFGF) was studied with circular dichroism (CD) and sequence prediction methods. The far-UV CD spectrum with a minimum at 202 nm resembled that of an unordered polypeptide/protein or a protein rich in distorted antiparallel β-sheets. Analysis of the CD spectrum by the least-squares method of Changet al. (1978) and the CONTIN program of Provencher and Glöckner (1981) suggested that about one half of the molecule consisted of β-sheet and there was no α-helix. These estimates agreed with the prediction by the sequence method of Garnieret al. (1978) using decision constants based on CD results. bFGF had an unusual CD band at 187 nm, which disappeared upon ionization of Tyr side chains atpH 11.7. It also had another unusual property of irreversibly converting the CD spectrum to a helix-like one with a double minimum at 205 and 215 and a maximum at 189 nm upon heating the solution to above 55°C. The helicity was also enhanced in trifluoroethanol and in sodium dodecyl sulfate. The mutant bFGF in which cysteines 76 and 94 were replaced by serine residues had essentially the same properties as the wild-type.     

Secondary structure of human plasma fibronectin: conformational change induced by calf alveolar heparan sulfates

The quantitative analysis of circular dichroic spectra of native human plasma fibronectin according to the method of Provencher and Gl?ckner [Provencher, S. W., & Gl?ckner, J. (1981) Biochemistry 20, 33-37] indicated the presence of beta-sheet (79%), beta-turn (21%), but no alpha-helix or random coil in the secondary structure. The calf alveolar heparan sulfates induced a change in the conformation of fibronectin: the magnitude of the change depended on the molecular properties of the particular heparan sulfate preparations.     

Small-angle X-ray scattering studies of 7S and 11S globulins from pea (Pisum sativum)

Small-angle X-ray scattering studies have been conducted on solutions of 11S and 7S globulins isolated from peas (Pisum sativum cv. Filby), and the radii of gyration and molecular weights determined. The general features of the scattering curves were similar to those reported for other seed storage proteins.     

Conservation and divergence on plant seed 11S globulins based on crystal structures

The crystal structures of two pro-11S globulins namely: rapeseed procruciferin and pea prolegumin are presented here. We have extensively compared them with the other known structures of plant seed 11S and 7S globulins. In general, the disordered regions in the crystal structures among the 11S globulins correspond to their five variable regions. Variable region III of procruciferin is relatively short and is in a loop conformation. This region is highly disordered in other pro-11S globulin crystals. Local helical and strand variations also occur across the group despite general structure conservation. We showed how these variations may alter specific physicochemical, functional and physiological properties. Aliphatic hydrophobic residues on the molecular surface correlate well with T_m values of the globulins. We also considered other structural features that were reported to influence thermal stability but no definite conclusion was drawn since each factor has additive or subtractive effect. Comparison between proA3B4 and mature A3B4 revealed an increase in r.m.s.d. values near variable regions II and IV. Both regions are on the IE face. Secondary structure based alignment of 11S and 7S globulins revealed 16 identical residues. Based on proA3B4 sequence, Pro60, Gly128, Phe163, Phe208, Leu213, Leu227, Ile237, Pro382, Val404, Pro425 and Val 466 are involved in trimer formation and stabilization. Gly28, Gly74, Asp135, Gly349 and Gly397 are involved in correct globular folding.     

Estimation of protein secondary structure and error analysis from circular dichroism spectra

The estimation of protein secondary structure from circular dichroism spectra is described by a multivariate linear model with noise (Gauss-Markoff model). With this formalism the adequacy of the linear model is investigated, paying special attention to the estimation of the error in the secondary structure estimates. It is shown that the linear model is only adequate for the alpha-helix class. Since the failure of the linear model is most likely due to nonlinear effects, a locally linearized model is introduced. This model is combined with the selection of the estimate whose fractions of secondary structure summate to approximately one. Comparing the estimation from the CD spectra with the X-ray data (by using the data set of W.C. Johnson Jr., 1988, Annu. Rev. Biophys. Chem. 17, 145-166) the root mean square residuals are 0.09 (alpha-helix), 0.12 (anti-parallel beta-sheet), 0.08 (parallel beta-sheet), 0.07 (beta-turn), and 0.09 (other). These residuals are somewhat larger than the errors estimated from the locally linearized model. In addition to alpha-helix, in this model the beta-turn and "other" class are estimated adequately. But the estimation of the antiparallel and parallel beta-sheet class remains unsatisfactory. We compared the linear model and the locally linearized model with two other methods (S. W. Provencher and J. Gl?ckner, 1981, Biochemistry 20, 1085-1094; P. Manavalan and W. C. Johnson Jr., 1988, Anal. Biochem. 167, 76-85). The locally linearized model and the Provencher and Gl?ckner method provided the smallest residuals. However, an advantage of the locally linearized model is the estimation of the error in the secondary structure estimates.     

Unfolding and partial refolding of a cellulase from the SDS-denatured state: From β-sheet to α-helix and back

Globular proteins are typically unfolded by SDS to form protein-decorated micelle-like structures. Several proteins have been shown subsequently to refold by addition of the nonionic surfactant octaethylene glycol monododecyl ether (C₁₂E₈). Thus SDS converts β-lactoglobulin, which has mainly β-sheet secondary structure, into a state rich in α-helicality, while addition of C₁₂E₈ leads to refolding and recovery of the original β-sheet structure. Here we extend these studies to the large β-sheet-rich cellulase Cel7b from Humicola insolens whose enzymatic activity provides a very sensitive refolding parameter. The enzymes widespread usage in the detergent industry makes it an obvious model system for protein-surfactant interactions. SDS-unfolding and subsequent refolding using C₁₂E₈ were investigated at pH 4.2 using near- and far-UV circular dichroism (CD), small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), size-exclusion chromatography (SEC) and activity measurements. The Cel7b:SDS complex can be described as a random configuration of 3–4 connected core-shell structures in which the protein is converted to a mainly α-helical secondary structure. Addition of C₁₂E₈ recovers almost all the secondary structure, part of the tertiary structure, about 50% of the activity and dissociates part of the protein population completely from detergent micelles. The lack of complete refolding may be due to charge neutralisation of Cel7b by SDS, kinetically trapping the enzyme into aggregated structures. In support of this, aggregates did not form when C₁₂E₈ was first mixed with Cel7b followed by addition of SDS. Formation of such aggregates may be a general phenomenon hampering quantitative refolding from the SDS-denatured state.     

Electron tunneling in proteins: role of the intervening medium

Analysis of electron-transfer (ET) kinetics data obtained from experiments on Ru-modified proteins (azurin, cytochrome c, myoglobin) and the bacterial photosynthetic reaction center reveals that distant donor-acceptor electronic couplings depend upon the secondary structure of the intervening polypeptide matrix. The β-sheet azurin structure efficiently and isotropically mediates coupling with an exponential distance-decay constant of 1.1?Å^–1. The experimentally derived distance-decay constant of 1.4?Å^–1 for long-range ET in myoglobin and the reaction center suggests that hydrogen-bond couplings are weaker through α helices than across β sheets. The donor-acceptor interactions of systems with comparable tunneling energies fall into two coupling zones: the β zone (bounded by distance-decay constants of 0.9?and 1.15 Å^–1) includes all the β-sheet (azurin) couplings and all but one coupling in cytochrome c; the α zone (boundaries: 1.25 and 1.6?Å^–1) includes less strongly coupled donor-acceptor pairs in myoglobin and the reaction center as well as a relatively weakly coupled pair in cytochrome c.     

Expression and characterization of the Arabidopsis thaliana 11S globulin family

The 11S globulins are the principal seed storage proteins in a variety of major crop species, including members of the legume and mustard families. They are targets for protein engineering studies attempting to alter the physicochemical properties of seed protein extracts (e.g. soybean) and to improve the nutritional quality of important agricultural crops. A key factor that has limited the success of this approach to date is insufficient accumulation of the engineered protein variants in vivo due to their improper folding and/or reduced stability, compared to the native protein. We have developed the Arabidopsis thaliana 11S proglobulins as a model system to enable studies exploring the factors underlying structural stability in this family of proteins. Yields of 1.5–4 mg/L were achieved for the three A. thaliana 11S proglobulins expressed in the Origami Escherichia coli cell line in super broth media at 20 °C for 16 h and purified via immobilized-metal affinity chromatography. We also demonstrate that differential scanning fluorimetry is an effective and accessible technique to facilitate the screening of variants to enable the successful engineering of 11S seed storage proteins. The relative in vitro stability of the A. thaliana 11S proglobulins (proAtCRU1 > proAtCRU3 > proAtCRU2) is consistent between chemical and thermal denaturation studies.     

Conformational prediction and circular dichroism studies on the lac repressor

Using the protein predictive model of Chou & Fasman (1974b), the secondary structure of the lac repressor has been elucidated from its amino acid sequence of 347 residues. The conformation is predicted to contain 37% α-helix and 35% β-sheet for the repressor, and 29% helix and 41% β-sheet for the trypsin-resistant core (residues 60 to 327). Circular dichroism studies indicate that native lac repressor contains 40% helix and 42% β-sheet, while the core has 16% helix and 54% β-sheet, in general agreement with the predicted conformation. The sharp reduction in helicity for the trypsinized lac repressor could be due to the loss of two long helical regions, 26–45 and 328–344, predicted at both terminals. There are extensive β-sheets predicted in the 215–324 region, which may be responsible for tetrameric stabilization found in both the lac repressor and the core. Residues 17 to 33 were previously predicted by Adler et al. (1972) to be helical and were proposed to bind in the major groove of DNA. However, the present analysis shows that there are two anti-parallel β-sheet regions: 4–7 and 17–24 at the N-terminal as well as 315–318 and 321–324 at the C-terminal of the lac repressor. These β-sheet pairs may assume the twisted “polypeptide double helix” conformation (Carter & Kraut, 1974) and bind to complementary regions in the major groove of DNA. The OH groups of Tyr at the N-terminal and those of Thr and Ser side chains, in both β-sheets at the N and C-terminal ends, could form hydrogen bonds to specific sites on the lac operator. There are 23 reverse β-turns predicted that may control the tertiary folding of the lac repressor, which is essential for operator binding. The behavior of several lac repressor mutants can be satisfactorily explained in terms of polar to non-polar group replacements as well as conformational changes in light of the present predicted model.     

De Novo Designed α-Sheet Peptides Inhibit Functional Amyloid Formation of Streptococcus mutans Biofilms

Streptococcus mutans is a bacterial species that predominates in the oral microbiome. S. mutans binds to the tooth surface, metabolizes sugars and produces acid, leading to cavity formation. S. mutans can also cause infectious endocarditis. Recent evidence suggests that S. mutans biofilms contain amyloid fibrils. Amyloids are insoluble fibrillar protein aggregates, and bacteria use functional amyloids to improve robustness of their biofilms. While the functional amyloids in bacteria such as Escherichia coli and Staphylococcus aureus have been heavily investigated, little is known about the mechanism of S. mutans amyloid formation. Previous results from our laboratory with the amyloidogenic proteins and peptides from the aforementioned bacteria and other mammalian amyloid systems suggest that amyloid formation progresses via an intermediate that adopts a unique secondary structure—α-sheet. De novo designed peptides with alternating l- and d-amino acid also adopt an α-sheet secondary structure and inhibit amyloid formation by binding to soluble oligomeric species during amyloidogenesis. Inhibition of fibrillization by α-sheet peptides suggests the presence of α-sheet during amyloid formation. To investigate the mechanism of functional amyloid formation in S. mutans, α-sheet peptides were compared to epigallocatechin gallate for their ability to inhibit fibril formation in S. mutans. Inhibition was demonstrated in a biofilm plate assay and on hydroxyapatite surfaces both in S. mutans alone and in bacteria from human saliva. The observed inhibition suggests that an α-sheet mediated mechanism may be operative during functional amyloid formation.     

Engineering the thermostability of a xylanase from Aspergillus oryzae by an enhancement of the interactions between the N-terminus extension and the β-sheet A2 of the enzyme

A mesophilic Aspergillus oryzae xylanase (AoXyn11A) belongs to glycoside hydrolase family 11. Hydrogen bonds and a disulfide bridge were introduced between the N-terminus extension and the β-sheet A2 of AoXyn11A, which were located in the corresponding region of a hyperthermostable xylanase. The mutants were designated as AoXyn11A^C5 and AoXyn11A^C5–C32, respectively. The thermostabilities of AoXyn11A and the mutants were assessed by the molecular dynamics simulations. After being incubated at 55 °C for 30 min, AoXyn11A^C5–C32 retained 49 % of its original activity, AoXyn11A^C5 retained 12 % and AoXyn11A retained 3 %. The interactions between the N-terminus extension and the β-sheet A2 were analyzed in depth: there was enhancement of the interactions between the N-terminus extension and the β-sheet A2 of AoXyn11A that improved its thermostability.     

Use of graph theory for secondary structure recognition and sequential assignment in heteronuclear (13C, 15N) NMR spectra: Application to HU protein from Bacillus stearothermophilus

A computer-assisted procedure, based upon a branch of mathematics known as graph theory, has been developed to recognize secondary structure elements in proteins from their corresponding nuclear Overhauser effect spectroscopy (NOESY)-type spectra and to carry out their sequential assignment. In the method, NOE connectivity templates characteristic of regular secondary structures are identified in the spectra. Resonance assignment is then achieved by connecting these NOE patterns of secondary structure together, and thereby matching connected spin systems to specific parts of the primary sequence. The range of NOE-graph templates of secondary structure motifs, incorporating α-helices and β-strand motifs, has been examined for reliability and extent of secondary structure identification in a data base composed of the high resolution structures of 20 proteins. The analysis identified several robust NOE-graph templates and supports the implementation of an ordered search strategy. The method, known as SERENDIPITY, has been applied to the analysis of nuclear Overhauser effect data from a three-dimensional time-shared nuclear Overhauser effect spectroscopy (¹³C, ¹⁵N) heteronuclear single quantum correlation spectrum of the (α + β) type protein HU from Bacillus stearothermophilus. The arrangement of the elucidated elements of secondary structure is very similar to that of the x-ray and nmr structures of HU. In addition, our analysis revealed a pattern of interstrand nuclear Overhauser effect in the β-arm region (residues 53–76) of HU, which suggest irregularities, not reported in the x-ray structure, in both strands of the β-arm at Ala57 and Pro72, respectively. At these residues, both strands of the β-arm appear to flip inside out before continuing as a regular antiparallel β-sheet. © 1996 John Wiley & Sons, Inc.     

Analysis and prediction of the packing of α-helices against a β-sheet in the tertiary structure of globular proteins

The packing of α-helices and β-sheets in six $\text{α}\text{β}$ proteins (e.g. flavodoxin) has been analysed. The results provide the basis for a computer algorithm to predict the tertiary structure of an $\text{α}\text{β}$ protein from its amino acid sequence and actual assignment of secondary structure.The packing of an individual α-helix against a β-sheet generally involves two adjacent ± 4 rows of non-polar residues on the α-helix at the positions i, i + 4, i + 8, i + 1, i + 5, i + 9. The pattern of interacting β-sheet residues results from the twisted nature of the sheet surface and the attendant rotation of the side-chains. At a more detailed level, four of the α-helical residues (i + 1, i + 4, i + 5 and i + 8) form a diamond that surrounds one particular β-sheet residue, generally isoleucine, leucine or valine. In general, the α-helix sits 10 Å above the sheet and lies parallel to the strand direction.The prediction follows a combinational approach. First, a list of possible β-sheet structures (10⁶ to 10¹⁴) is constructed by the generation of all β-sheet topologies and β-strand alignments. This list is reduced by constraints on topology and the location of non-polar residues to mediate the sheet/helix packing, and then rank-ordered on the extent of hydrogen bonding. This algorithm was uniformly applied to 16 $\text{α}\text{β}$ domains in 13 proteins. For every structure, one member of the reduced list was close to the crystal structure; the root-mean-square deviation between equivalenced C^α atoms averaged 5.6 Å for 100 residues. For the $\text{α}\text{β}$ proteins with pure parallel β-sheets, the total number of structures comparable to or better than the native in terms of hydrogen bonds was between 1 and 148. For proteins with mixed β-sheets, the worst case is glyceraldehyde-3-phosphate dehydrogenase, where as many as 3800 structures would have to be sampled. The evolutionary significance of these results as well as the potential use of a combinatorial approach to the protein folding problem are discussed.     

Secondary structure perturbations in salt-induced protein precipitates

The secondary structure implications of precipitation induced by a chaotropic salt, KSCN, and a structure stabilizing salt, Na₂SO₄, were studied for twelve different proteins. α-helix and β-sheet content of precipitate and native structures were estimated from the analysis of amide I band Raman spectra. A statistical analysis of the estimated perturbations in the secondary structure contents indicated that the most significant event is the formation of β-sheet structures with a concomitant loss of α-helix on precipitation with KSCN. The conformational changes for each protein were also analyzed with respect to elements of primary, secondary and tertiary structure existing in the native protein; primary structure was quantified by the fractions of hydrophobic and charged amino acids, secondary structure by x-ray estimates of α-helix and β-sheet contents of native proteins and tertiary structure by the dipole moment and solvent-accessible surface area. For the KSCN precipitates, factors affecting β-sheet content included the fraction of charged amino acids in the primary sequence and the surface area. Changes in α-helix content were influenced by the initial helical content and the dipole moment. The enhanced β-sheet contents of precipitates observed in this work parallel protein structural changes occurring in other aggregative phenomena.     

Seed Storage Proteins in Solanaceae and Cucurbitaceae Species

Electrophoretic analyses of non-reduced and reduced seed storage proteins from Solanaceae and Cucurbitaceae species and cultivars were performed. High molecular disulfide bonded complexes between intermediary subunits of 11S globulins previously detected in Capsicum annuum cultivars, were found in Solanum melongena cultivars as well. The data obtained might be used for further elucidation of peculiarities of the 11S globulins in dicotyledonous plants.     

Conformational structure of bombesin as studied by vibrational and circular dichroism spectroscopy

Raman and Fourier transform infrared (FTIR) spectroscopies and circular dichroism (CD) have been applied to investigate the secondary structure of bombesin in the solid state and in phosphate buffer solution (pH 3.8). At concentrations around 10⁻⁵ M, circular dichroism reveals that bombesin exists as an irregular or disordered conformation. However, the secondary structure of the peptide appears to be a mixture of disordered structure and intermolecular β-sheets in 0.01 M sodium phosphate buffer when the peptide concentrations are higher than around 6.5 mM. The tendency of bombesin to form aggregated β-sheet species seems to be originated mainly in the sequence of the residues 7–14, as supported by the Raman spectra and β-sheet propensities (P_β) of the amino-acid residues. It is the hydrophobic force of this amino-acid sequence, and not a salt bridge effect, that is the factor responsible for the formation of peptide aggregates.