Conformational analysis and design of cross-strand disulfides in antiparallel β-sheets

Cross‐strand disulfides bridge two cysteines in a registered pair of antiparallel β‐strands. A nonredundant data set comprising 5025 polypeptides containing 2311 disulfides was used to study cross‐strand disulfides. Seventy‐six cross‐strand disulfides were found of which 75 and 1 occurred at non‐hydrogen‐bonded (NHB) and hydrogen‐bonded (HB) registered pairs, respectively. Conformational analysis and modeling studies demonstrated that disulfide formation at HB pairs necessarily requires an extremely rare and positive χ¹ value for at least one of the cysteine residues. Disulfides at HB positions also have more unfavorable steric repulsion with the main chain. Thirteen pairs of disulfides were introduced in NHB and HB pairs in four model proteins: leucine binding protein (LBP), leucine, isoleucine, valine binding protein (LIVBP), maltose binding protein (MBP), and Top7. All mutants LIVBP T247C V331C showed disulfide formation either on purification, or on treatment with oxidants. Protein stability in both oxidized and reduced states of all mutants was measured. Relative to wild type, LBP and MBP mutants were destabilized with respect to chemical denaturation, although the sole exposed NHB LBP mutant showed an increase of 3.1°C in T _m . All Top7 mutants were characterized for stability through guanidinium thiocyanate chemical denaturation. Both exposed and two of the three buried NHB mutants were appreciably stabilized. All four HB Top7 mutants were destabilized (ΔΔG ⁰ = ?3.3 to ?6.7 kcal/mol). The data demonstrate that introduction of cross‐strand disulfides at exposed NHB pairs is a robust method of improving protein stability. All four exposed Top7 disulfide mutants showed mild redox activity. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Evolution of proteins formed by β-sheets: I. Plastocyanin and azurin

Plastocyanin and azurin form a family of small copper-containing proteins, active in the electron transport systems of plants and bacteria, respectively. The crystal structures of two members of this family have been determined: poplar leaf plastocyanin and Pseudomonas aeruginosa azurin. Both proteins contain two β-sheets, packed face-to-face. Using computed superpositions of the structures, we have aligned the sequences, identified homologous positions, and studied how the structures have changed as a result of mutations.The residues in the vicinity of the copper-binding site show minimal amino acid substitution and form almost identical structures. Other portions of these proteins are more variable in sequence and in structure. Buried residues tend to maintain their hydrophobic character, but mutations change their volume. The mean variation in volume of homologous buried residues is 54 ų. The differences in size and shape of these buried residues are accommodated by a 3.8 Å shift in relative position of the packed β-sheets. This shift does not affect the copper binding site, because the residues that form this site are in, or adjacent to, just one of the β-sheets.     

Identification and analysis of extended strands and β-sheets in globular proteins

A method to identify β-sheets in globular proteins from extended strands, using only α-carbon positions, has been developed. The strands that form β-sheets are picked up by means of simple distance criteria. The method has been tested by applying it to three proteins with accurately known secondary structures. It has also been applied to ten other proteins wherein only α-carbon coordinates are available, and the list of β-sheets obtained. The following points are worth noting: (i) The sheets identified by the algorithm are found to agree satisfactorily with the reported ones based on backbone hydrogen bonding, wherever this information is available. (ii) β-Strands that do not form parts of any sheet are a common feature of protein structures. (iii) Such isolated β-strands tend to be short. (iv) The conformation corresponding to the preferred right-handed twist of the sheet is overwhelmingly observed in both the sheet-forming and isolated β-strands.     

Specific recognition in the tertiary structure of β-sheets of proteins

The frequency of occurrence of nearest neighbour residue pairs on adjacent antiparallel (β_A) and parallel (β_P) strands is obtained from 30 known protein structures. The specificity of interstrand recognition due to such pairing as a factor in the folding of β-sheets is studied by statistical methods. Residues of sufficiently high count for statistical analysis are treated individually while the rest are combined into small groups of similar size, polarity, and/or genetic exchangeability. The hypothesis of specific recognition between individuals and small groups is contrasted with the alternative hypothesis of non-specific recognition between broad classes (hydrophobia, neutral, polar) of residues. A χ² test of pair correlations favours specific recognition against non-specific recognition with a high level of confidence. The largest and most significant correlations are: Ser/Thr (1.9 ± 0.3), Ile/Val (1.7 ± 0.3) and Lys-Arg/Asp-Gln (1.8 ± 0.3) in β_A, and Ile/Leu (1.9 ± 0.4) in β_P. The pair Gly/Gly never occurs in any β-sheet. The specific residue-pair correlations derived here may be useful in statistical prediction methods of protein tertiary structure.     

Prediction of the packing arrangement of strands in β-sheets of globular proteins

A method is proposed for predicting the adjacency order in which strands pack in a -sheet in a protein, on the basis of its amino acid sequence alone. The method is based on the construction of a predicted contact map for the protein, in which the probability that various residue pairs are close to each other is computed from statistically determined average distances of residue pairs in globular proteins of known structure. Compact regions, i.e., portions of the sequence with many interresidue contacts, are determined on the map by using an objective search procedure. The proximity of strands in a -sheet is predicted from the density of contacts in compact regions associated with each pair of strands. The most probable -sheet structures are those with the highest density of contacts. The method has been tested by computing the probable strand arrangements in a five-strand -sheet in five proteins or protein domains, containing 62–138 residues. Of the theoretically possible 60 strand arrangements, the method selects two to eight arrangements as most probable; i.e., it leads to a large reduction in the number of possibilities. The native strand arrangement is among those predicted for three of the five proteins. For the other two, it would be included in the prediction by a slight relaxation of the cutoff criteria used to analyze the density of contacts.     

Formation of β-sheets in glutamine and alanine tripeptides

The misfolding and aggregation of proteins is associated with many different diseases including the trinucleotide repeat disorders and Prion diseases. We have studied three residue peptides comprising alanine and glutamine in order to understand the short range interactions affecting the formation of β-rich aggregates. Using infrared spectroscopy, we have found that trialanine and triglutamine form significant amounts of β-sheet, but that tripeptides containing alanine and glutamine are only able to form β-sheet if the glutamine side-chains extend outward on both faces of the sheet. From our data, we conclude that different stabilizing interactions are responsible for β-sheet formation in trialanine and triglutamine.     

The role of β-sheets in the structure and assembly of keratins

X-ray diffraction, infrared and electron microscope studies of avian and reptilian keratins, and of stretched wool and hair, have played a central role in the development of models for the β-conformation in proteins. Both α- and β-keratins contain sequences that are predicted to adopt a β-conformation and these are believed to play an important part in the assembly of the filaments and in determining their mechanical properties. Interactions between the small β-sheets in keratins provide a simple mechanism through which shape and chemical complementarity can mediate the assembly of molecules into highly specific structures. Interacting β-sheets in crystalline proteins are often related to one another by diad symmetry and the data available on feather keratin suggest that the filament is assembled from dimers in which the β-sheets are related by a perpendicular diad. The most detailed model currently available is for feather and reptilian keratin but the presence of related β-structural forms in mammalian keratins is also noted.     

Conformational properties of nine purified cystathionine β-synthase mutants

Protein misfolding due to missense mutations is a common pathogenic mechanism in cystathionine β-synthase (CBS) deficiency. In our previous studies, we successfully expressed, purified, and characterized nine CBS mutant enzymes containing the following patient mutations: P49L, P78R, A114V, R125Q, E176K, R266K, P422L, I435T, and S466L. These purified mutants exhibited full heme saturation, normal tetrameric assembly, and high catalytic activity. In this work, we used several spectroscopic and proteolytic techniques to provide a more thorough insight into the conformation of these mutant enzymes. Far-UV circular dichroism, fluorescence, and second-derivative UV spectroscopy revealed that the spatial arrangement of these CBS mutants is similar to that of the wild type, although the microenvironment of the chromophores may be slightly altered. Using proteolysis with thermolysin under native conditions, we found that the majority of the studied mutants is more susceptible to cleavage, suggesting their increased local flexibility or propensity for local unfolding. Interestingly, the presence of the CBS allosteric activator, S-adenosylmethionine (AdoMet), increased the rate of cleavage of the wild type and the AdoMet-responsive mutants, while the proteolytic rate of the AdoMet-unresponsive mutants was not significantly changed. Pulse proteolysis analysis suggested that the protein structure of the R125Q and E176K mutants is significantly less stable than that of the wild type and the other mutants. Taken together, the proteolytic data shows that the conformation of the pathogenic mutants is altered despite retained catalytic activity and normal tetrameric assembly. This study demonstrates that the proteolytic techniques are useful tools for the assessment of the biochemical penalty of missense mutations in CBS.     

Evidence of α-helical coiled coils and β-sheets in hornet silk

α-Helical coiled coil and β-sheet complexes are essential structural building elements of silk proteins produced by different species of the Hymenoptera. Beside X-ray scattering at wide and small angles we applied cryo-electron diffraction and microscopy to demonstrate the presence and the details of such structures in silk of the giant hornet Vespa mandarinia japonica. Our studies on the assembly of the fibrous silk proteins and their internal organization in relation to the primary chain structure suggest a 172 Å pitch supercoil consisting of four intertwined alanine-rich α-helical strands. The axial periodicity may adopt even multiples of the pitch value. Coiled coil motifs form the largest portion of the hornet silk structure and are aligned nearly parallel to the cocoon fiber axis in the same way as the membrane-like parts of the cocoon are molecularly orientated in the spinning direction. Supercoils were found to be associated with β-crystals, predominantly localized in the l-serine-rich chain sequences terminating each of the four predominant silk proteins. Such β-sheet blocks are considered resulting from transformation of random coil molecular sequences due to the action of elongational forces during the spinning process.     

13C structuring shifts for the analysis of model β-hairpins and β-sheets in proteins: diagnostic shifts appear only at the cross-strand H-bonded residues

The present studies have shown that ¹³C=O, ¹³C^α and ¹³C^β of H-bonded strand residues in β-hairpins provide additional probes for quantitating the extent of folding in β-hairpins and other β-sheet models. Large differences in the structuring shifts (CSDs) of these ¹³C sites in H-bonded versus non-H-bonded sites are observed: the differences between H-bonded and non-H-bonded sites are greater than 1.2 ppm for all three ¹³C probes. This prompts us to suggest that efforts to determine the extent of hairpin folding from ¹³C shifts should be based exclusively on the observation at the cross-strand H-bonded sites. Furthermore, the statistics suggest the ¹³C′ and ¹³C^β CSDs will provide the best differentiation with 100 %-folded CSD values approaching ?2.6 and +3 ppm, respectively, for the H-bonded sites. These conclusions can be extended to edge-strands of protein β-sheets. Our survey of reported ¹³C shifts in β-proteins indicates that some of the currently employed random coil values need to be adjusted, particularly for ionization-induced effects.     

Electrophoresis of ribonuclease and of β-lactoglobulin: Isoelectric points of proteins

Electrophoretic mobilities were measured for ribonuclease and for β-lactoglobulin as a function of ionic strength and of pH in unbuffered potassium chloride solutions at 25 °C. Mobilities were also observed for ribonuclease in Tris buffer at 25 °. It is noted that the isoelectric points decrease with increasing ionic strength. The mobilities have been compared with the net charge as estimated from ion-binding studies both for the two proteins indicated above and also for bovine serum albumin. It is found that the point of zero net charge as measured by ion binding does not in general coincide with the isoelectric points and furthermore the greater the ionic strength the greater the divergence. It is concluded that not all the changes on the protein are electrophoretically effective. The electrophoretic radii of the proteins have been calculated from a form of the Henry equation; the variation of the radii with ionic strength is consistent with the notion of electrophoretically hidden charges.     

Influences of geometrical and mechanical properties of bone tissues in mandible behaviour – experimental and numerical predictions

The properties and geometry of bone in the mandible play a key role in mandible behaviour during a person’s lifetime, and attention needs to be paid to the influence of bone properties. We analysed the effect of bone geometry, size and bone properties in mandible behaviour, experimenting on cadaveric mandibles and FE models. The study was developed using the geometry of a cadaveric mandible without teeth. Three models of cadaveric condyles were experimentally tested with instrumented with four rosettes, and a condyle reaction of 300 N. Four finite element models were considered to validate the experiments and analyse mandible behaviour. One numeric model was simulated with 10 muscles in a quasi-static condition. The experimental results present different condyle stiffness’s, of 448, 215 and 254 N/mm. The values presented in the rosettes are influenced by bone geometry and bone thickness; maximum value was ?600 με in rosette #4, and the maximum strain difference between mandibles was 111%. The numerical results show that bone density decreases and strain distribution increases in the thinner mandible regions. Nevertheless, the global behaviour of the structure remains similar, but presents different strain magnitudes. The study shows the need to take into account bone characteristics and their evolutions in order to improve implant design and fixation throughout the patient life. The change in bone stiffness promotes a change in maximum strain distribution with same global behaviour.     

Expression in E. coli and purification of the fibrillogenic fusion proteins TTR-sfGFP and β2M-sfGFP

The possibility of obtaining recombinant fibrillogenic fusion proteins such as transthyretin (TTR) and β2-microglobulin (β2M) with a superfolder green fluorescent protein (sfGFP) was studied. According to the literature data, sfGFP is resistant to denaturating influences, does not aggregate during renaturation, possesses improved kinetic characteristics of folding, and folds well when fused to different polypeptides. The corresponding DNA constructs for expression in Escherichia coli were created. It could be shown that during expression of these constructs in E. coli, soluble forms of the fusion proteins are synthesized. Efficient isolation of the fusion proteins was performed with the help of nickel-affinity chromatography. For this purpose a polyhistidine sequence (6-His-tag) was incorporated into the C-terminus of the sfGFP. We could show that the purified fusion proteins contained full-size sequences of the most amyloidogenic TTR variant, TTR(L55P) and β2M, and also sfGFP possessing fluorescent properties. In the course of fibrillogenesis both fusion proteins demonstrated their ability to form fibrils that were clearly detectable by atomic force microscopy. Furthermore, with the help of confocal microscopy we were able to reveal structures (exhibiting fluorescence) that are formed during fibrillogenesis. Thus, the use of sfGFP has made it possible to avoid formation of inclusion bodies (IB) during the synthesis of recombinant fusion proteins and to obtain soluble forms of TTR(L55P) and β2M that are suitable for further studies.     

Bcl-2 proteins in diabetes: mitochondrial pathways of β-cell death and dysfunction

Diabetes is a metabolic disease affecting nearly 300 million individuals worldwide. Both types of diabetes (1 and 2) are characterized by loss of functional pancreatic β-cell mass causing different degrees of insulin deficiency. The Bcl-2 family has a double-edged effect in diabetes. These proteins are crucial controllers of the mitochondrial pathway of β-cell apoptosis induced by pro-inflammatory cytokines or lipotoxicity. In parallel, some Bcl-2 members also regulate glucose metabolism and β-cell function. In this review, we describe the role of Bcl-2 proteins in β-cell homeostasis and death. We focus on how these proteins interact, their contribution to the crosstalk between endoplasmic reticulum stress and mitochondrial permeabilization, their context-dependent usage following different pro-apoptotic stimuli, and their role in β-cell physiology.     

Selenocysteine in proteins—properties and biotechnological use

Selenocysteine (Sec), the 21st amino acid, exists naturally in all kingdoms of life as the defining entity of selenoproteins. Sec is a cysteine (Cys) residue analogue with a selenium-containing selenol group in place of the sulfur-containing thiol group in Cys. The selenium atom gives Sec quite different properties from Cys. The most obvious difference is the lower pK_a of Sec, and Sec is also a stronger nucleophile than Cys. Proteins naturally containing Sec are often enzymes, employing the reactivity of the Sec residue during the catalytic cycle and therefore Sec is normally essential for their catalytic efficiencies. Other unique features of Sec, not shared by any of the other 20 common amino acids, derive from the atomic weight and chemical properties of selenium and the particular occurrence and properties of its stable and radioactive isotopes. Sec is, moreover, incorporated into proteins by an expansion of the genetic code as the translation of selenoproteins involves the decoding of a UGA codon, otherwise being a termination codon. In this review, we will describe the different unique properties of Sec and we will discuss the prerequisites for selenoprotein production as well as the possible use of Sec introduction into proteins for biotechnological applications. These include residue-specific radiolabeling with gamma or positron emitters, the use of Sec as a reactive handle for electophilic probes introducing fluorescence or other peptide conjugates, as the basis for affinity purification of recombinant proteins, the trapping of folding intermediates, improved phasing in X-ray crystallography, introduction of ⁷⁷Se for NMR spectroscopy, or, finally, the analysis or tailoring of enzymatic reactions involving thiol or oxidoreductase (redox) selenolate chemistry.     

Conformational Dynamics and Functional Implications of Phosphorylated β-Arrestins

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Thermodynamic and structural analysis of homodimeric proteins: model of β-lactoglobulin

The energetics of protein homo-oligomerization was analyzed in detail with the application of a general thermodynamic model. We have studied the thermodynamic aspects of protein-protein interaction employing β-lactoglobulin A from bovine milk at pH=6.7 where the protein is mainly in its dimeric form. We performed differential calorimetric scans at different total protein concentration and the resulting thermograms were analyzed with the thermodynamic model for oligomeric proteins previously developed. The thermodynamic model employed, allowed the prediction of the sign of the enthalpy of dimerization, the analysis of complex calorimetric profiles without transitions baselines subtraction and the obtainment of the thermodynamic parameters from the unfolding and the association processes and the compared with association parameters obtained with Isothermal Titration Calorimetry performed at different temperatures. The dissociation and unfolding reactions were also monitored by Fourier-transform infrared spectroscopy and the results indicated that the dimer of β-lactoglobulin (N(2)) reversibly dissociates into monomeric units (N) which are structurally distinguishable by changes in their infrared absorbance spectra upon heating. Hence, it is proposed that β-lactoglobulin follows the conformational path induced by temperature:N(2)?2N?2D. The general model was validated with these results indicating that it can be employed in the study of the thermodynamics of other homo-oligomeric protein systems.     

Enzymes of Trichomonas foetus. Separation and properties of two β-galactosidases

The beta-galactosidase activity in extracts of Trichomonas foetus is separable into two fractions by gel filtration on Sephadex G-200. When o-nitrophenyl beta-d-galactoside is used as substrate the first fraction to be eluted, beta-galactosidase 1, has 50 times the activity (units per mg of protein) of the crude preparation. This fraction is activated by Mn(2+) and Co(2+) and inhibited by Hg(2+) and EDTA. In the presence of Mn(2+) the pH optimum for the hydrolysis of o-nitrophenyl beta-d-galactoside or lactose is 5.8-6.0. beta-Galactosidase 1 is an exoglycosidase that releases beta-linked galactose joined to aliphatic and various carbohydrate aglycones. Hydrolysis is prevented, however, by a substituent on either the subterminal sugar or the terminal non-reducing beta-galactosyl residue in an oligosaccharide. The second fraction, beta-galactosidase 2, is not activated by metal ions or inhibited by EDTA and has a broad pH optimum from 4.5 to 6.0.