Building bridges: disulphide bond formation in the cell

Disulphides are often vital for the folding and stability of proteins. Dedicated enzymatic systems have been discovered that catalyse the formation of disulphides in the periplasm of prokaryotes. These discoveries provide compelling evidence for the actual catalysis of protein folding in vivo. Disulphide bond formation in Escherichia coli is catalysed by at least three ‘Dsb’ proteins; DsbA, -B and -C. The DsbA protein has an extremely reactive, oxidizing disulphide which it simply donates directly to other proteins. DsbB is required for the reoxidation of DsbA. DsbC is active in disulphide rearrangements and appears to work synergistically with DsbA. The relative rarity of disulphides in cytoplasmic proteins appears to be dependent upon a disulphide-destruction machine. One pivotal cog in this machine is thioredoxin reductase.     

Importance of post-translational modifications for functionality of a chloroplast-localized carbonic anhydrase (CAH1) in Arabidopsis thaliana

Background

The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells.

Methodology/Principal Findings

Transient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited.

Conclusions/Significance

We show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native protein explain the need for an alternative route to the chloroplast.     

Analysing the origin of long-range interactions in proteins using lattice models

Background  

Long-range communication is very common in proteins but the physical basis of this phenomenon remains unclear. In order to gain insight into this problem, we decided to explore whether long-range interactions exist in lattice models of proteins. Lattice models of proteins have proven to capture some of the basic properties of real proteins and, thus, can be used for elucidating general principles of protein stability and folding.     

On the relevance of non-random polypeptide conformations for protein folding

The possibility that any non-random conformation in reduced bovine pancreatic trypsin inhibitor (BPTI) and ribonuclease A might be significant for folding has been considered, using the experimental data available on forming the first disulphide bond in each. It is a thermodynamic necessity that whatever conformation stabilises a particular disulphide bond be stabilised to the same extent by the presence of the disulphide. The stabilising effects of disulphides are known approximately, so the stability of any non-random conformation found in a one-disulphide intermediate can be estimated in the absence of the disulphide bond. The non-random conformation in the BPTI intermediates is sufficiently labile to indicate that it would be expected to be present in no more than 5% of the reduced BPTI molecules. There is much less non-random conformation apparent in ribonuclease A. Whatever conformations are represented in the bulk of these two reduced proteins cannot favour disulphide formation and further productive folding.     

Identification of thioredoxin h-reducible disulphides in proteomes by differential labelling of cysteines: insight into recognition and regulation of proteins in barley seeds by thioredoxin h

Using thiol-specific fluorescence labelling, over 30 putative target proteins of thioredoxin h with diverse structures and functions have been identified in seeds of barley and other plants. To gain insight at the structural level into the specificity of target protein reduction by thioredoxin h, thioredoxin h-reducible disulphide bonds in individual target proteins are identified using a novel strategy based on differential alkylation of cysteine thiol groups by iodoacetamide and 4-vinylpyridine. This method enables the accessible cysteine side chains in the thiol form (carbamidomethylated) to be distinguished from those inaccessible or disulphide bound form (pyridylethylated) according to the mass difference in the peptide mass maps obtained by matrix-assistend laser desorption/ionisation-time of flight mass spectrometry. Using this approach, in vitro reduction of disulphides in recombinant barley alpha-amylase/subtilisin inhibitor (BASI) by barley thioredoxin h isoform 1 was analysed. Furthermore, the method was coupled with two-dimensional electrophoresis for convenient thioredoxin h-reducible disulphide identification in barley seed extracts without the need for protein purification or production of recombinant proteins. Mass shifts of 15 peptides, induced by treatment with thioredoxin h and differential alkylation, identified specific reduction of nine disulphides in BASI, four alpha-amylase/trypsin inhibitors and a protein of unknown function. Two specific disulphides, located structurally close to the alpha-amylase binding surfaces of BASI and alpha-amylase inhibitor BMAI-1 were demonstrated to be reduced to a particularly high extent. For the first time, specificity of thioredoxin h for particular disulphide bonds is demonstrated, providing a basis to study structural aspects of the recognition mechanism and regulation of target proteins.     

Accessibilities and reactivities of cysteine thiols during refolding of reduced bovine pancreatic trypsin inhibitor

The experimental basis of the pathway of refolding of reduced bovine pancreatic trypsin inhibitor that accompanies disulphide bond formation is explained in the light of a recent suggestion that the inability of certain Cys residues to form disulphide bonds could be explained simply by their thiol groups being inaccessible to disulphide reagents. This explanation is not valid, because part of the experimental evidence for inability to form disulphides is that the Cys residues accumulate as mixed-disulphides with the reagent. That these thiol groups are observed to react normally with the reagent, and with iodoacetic acid, is direct positive proof that they were not inaccessible or otherwise unreactive. The experimentally determined refolding pathway accurately reflects the energetics of the protein folding transitions and is consistent with all general observations of the folding transitions of other small proteins, whether or not disulphide bond formation is involved.     

Disulphide bridges in globular proteins

Disulphide bridges in proteins of known sequence, connectivity and structure were studied to search for common features. Their distribution, topology, conformation and conservation were analysed in detail. Several general patterns emerge which to some extent dictate disulphide bridge formation. For example, there is a strong preference for shorter connections, with half-cystines separated by less than 24 residues in 49% of all disulphides. Right- and left-handed disulphides occur equally; the left-handed structures adopt one predominant conformation (symmetric χ₁ = ?60 °, χ₂ = ?80 °, χ₃ = t-90 °). Cystines are generally very well conserved, in contrast to cysteines, with a free —SH group, which mutate rapidly. If a disulphide is not conserved, both cystines are mutated. The role of disulphide bridges in globular proteins is discussed.     

Predicting protein folding pathways at the mesoscopic level based on native interactions between secondary structure elements

Background  

Since experimental determination of protein folding pathways remains difficult, computational techniques are often used to simulate protein folding. Most current techniques to predict protein folding pathways are computationally intensive and are suitable only for small proteins.     

Intramolecular disulphide bond arrangements in nonhomologous proteins

The presence and location of intramolecular disulphide bonds are a key determinant of the structure and function of proteins. Intramolecular disulphide bonds in proteins have previously been analyzed under the assumption that there is no clear relationship between disulphide arrangement and disulphide concentration. To investigate this, a set of sequence nonhomologous protein chains containing one or more intramolecular disulphide bonds was extracted from the Protein Data Bank, and the arrangements of the bonds, Protein Data Bank header, and Structural Characterization of Proteins fold were analyzed as a function of intramolecular disulphide bond concentration. Two populations of intramolecular disulphide bond-containing proteins were identified, with a naturally occurring partition at 25 residues per bond. These populations were named intramolecular disulphide bond-rich and -poor. Benefits of partitioning were illustrated by three results: (1) rich chains most frequently contained three disulphides, explaining the plateaux in extant disulphide frequency distributions; (2) a positive relationship between median chain length and the number of disulphides, only seen when the data were partitioned; and (3) the most common bonding pattern for chains with three disulphide bonds was based on the most common for two, only when the data were partitioned. The two populations had different headers, folds, bond arrangements, and chain lengths. Associations between IDSB concentration, IDSB bonding pattern, loop sizes, SCOP fold, and PDB header were also found. From this, we found that intramolecular disulphide bond-rich and -poor proteins follow different bonding rules, and must be considered separately to generate meaningful models of bond formation.     

Directionality in protein fold prediction

Background  

Ever since the ground-breaking work of Anfinsen et al. in which a denatured protein was found to refold to its native state, it has been frequently stated by the protein fold prediction community that all the information required for protein folding lies in the amino acid sequence. Recent in vitro experiments and in silico computational studies, however, have shown that cotranslation may affect the folding pathway of some proteins, especially those of ancient folds. In this paper aspects of cotranslational folding have been incorporated into a protein structure prediction algorithm by adapting the Rosetta program to fold proteins as the nascent chain elongates. This makes it possible to conduct a pairwise comparison of folding accuracy, by comparing folds created sequentially from each end of the protein.     

Directed evolution of single-chain Fv for cytoplasmic expression using the β-galactosidase complementation assay results in proteins highly susceptible to protease degradation and aggregation

Background  

Antibody fragments are molecules widely used for diagnosis and therapy. A large amount of protein is frequently required for such applications. New approaches using folding reporter enzymes have recently been proposed to increase soluble expression of foreign proteins in Escherichia coli. To date, these methods have only been used to screen for proteins with better folding properties but have never been used to select from a large library of mutants. In this paper we apply one of these methods to select mutations that increase the soluble expression of two antibody fragments in the cytoplasm of E. coli.     

GANDivAWeb: A web server for detecting early folding units ("foldons") from protein 3D structures

Background  

It has long been known that small regions of proteins tend to fold independently and are then stabilized by interactions between these distinct subunits or modules. Such units, also known as autonomous folding units (AFUs) or"foldons" play a key role in protein folding. A knowledge of such early folding units has diverse applications in protein engineering as well as in developing an understanding of the protein folding process. Such AFUs can also be used as model systems in order to study the structural organization of proteins.     

The Critical Role of N- and C-Terminal Contact in Protein Stability and Folding of a Family 10 Xylanase under Extreme Conditions

Background

Stabilization strategies adopted by proteins under extreme conditions are very complex and involve various kinds of interactions. Recent studies have shown that a large proportion of proteins have their N- and C-terminal elements in close contact and suggested they play a role in protein folding and stability. However, the biological significance of this contact remains elusive.

Methodology

In the present study, we investigate the role of N- and C-terminal residue interaction using a family 10 xylanase (BSX) with a TIM-barrel structure that shows stability under high temperature, alkali pH, and protease and SDS treatment. Based on crystal structure, an aromatic cluster was identified that involves Phe4, Trp6 and Tyr343 holding the N- and C-terminus together; this is a unique and important feature of this protein that might be crucial for folding and stability under poly-extreme conditions.

Conclusion

A series of mutants was created to disrupt this aromatic cluster formation and study the loss of stability and function under given conditions. While the deletions of Phe4 resulted in loss of stability, removal of Trp6 and Tyr343 affected in vivo folding and activity. Alanine substitution with Phe4, Trp6 and Tyr343 drastically decreased stability under all parameters studied. Importantly, substitution of Phe4 with Trp increased stability in SDS treatment. Mass spectrometry results of limited proteolysis further demonstrated that the Arg344 residue is highly susceptible to trypsin digestion in sensitive mutants such as ΔF4, W6A and Y343A, suggesting again that disruption of the Phe4-Trp6-Tyr343 (F-W-Y) cluster destabilizes the N- and C-terminal interaction. Our results underscore the importance of N- and C-terminal contact through aromatic interactions in protein folding and stability under extreme conditions, and these results may be useful to improve the stability of other proteins under suboptimal conditions.     

Detecting internally symmetric protein structures

Background  

Many functional proteins have a symmetric structure. Most of these are multimeric complexes, which are made of non-symmetric monomers arranged in a symmetric manner. However, there are also a large number of proteins that have a symmetric structure in the monomeric state. These internally symmetric proteins are interesting objects from the point of view of their folding, function, and evolution. Most algorithms that detect the internally symmetric proteins depend on finding repeating units of similar structure and do not use the symmetry information.     

Pairwise covariance adds little to secondary structure prediction but improves the prediction of non-canonical local structure

Background  

Amino acid sequence probability distributions, or profiles, have been used successfully to predict secondary structure and local structure in proteins. Profile models assume the statistical independence of each position in the sequence, but the energetics of protein folding is better captured in a scoring function that is based on pairwise interactions, like a force field.     

ProtSA: a web application for calculating sequence specific protein solvent accessibilities in the unfolded ensemble

Background  

The stability of proteins is governed by the heat capacity, enthalpy and entropy changes of folding, which are strongly correlated to the change in solvent accessible surface area experienced by the polypeptide. While the surface exposed in the folded state can be easily determined, accessibilities for the unfolded state at the atomic level cannot be obtained experimentally and are typically estimated using simplistic models of the unfolded ensemble. A web application providing realistic accessibilities of the unfolded ensemble of a given protein at the atomic level will prove useful.     

Bacterial conjugation: a two-step mechanism for DNA transport

Ten years ago it was thought that disulphide bond formation in prokaryotes occurred spontaneously. Now two pathways involved in disulphide bond formation have been well characterized, the oxidative pathway, which is responsible for the formation of disulphides, and the isomerization pathway, which shuffles incorrectly formed disulphides. Disulphide bonds are donated directly to unfolded polypeptides by the DsbA protein; DsbA is reoxidized by DsbB. DsbB generates disulphides de novo from oxidized quinones. These quinones are reoxidized by the electron transport chain, showing that disulphide bond formation is actually driven by electron transport. Disulphide isomerization requires that incorrect disulphides be attacked using a reduced catalyst, followed by the redonation of the disulphide, allowing alternative disulphide pairing. Two isomerases exist in Escherichia coli, DsbC and DsbG. The membrane protein DsbD maintains these disulphide isomerases in their reduced and thereby active form. DsbD is kept reduced by cytosolic thioredoxin in an NADPH-dependent reaction.     

Evolutionary Pareto-optimization of stably folding peptides

Background  

As a rule, peptides are more flexible and unstructured than proteins with their substantial stabilizing hydrophobic cores. Nevertheless, a few stably folding peptides have been discovered. This raises the question whether there may be more such peptides that are unknown as yet. These molecules could be helpful in basic research and medicine.     

Varicose: a MAGUK required for the maturation and function of <Emphasis Type="Italic">Drosophila</Emphasis> septate junctions

Background  

Scaffolding proteins belonging to the membrane associated guanylate kinase (MAGUK) superfamily function as adapters linking cytoplasmic and cell surface proteins to the cytoskeleton to regulate cell-cell adhesion, cell-cell communication and signal transduction. We characterize here a Drosophila MAGUK member, Varicose (Vari), the homologue of vertebrate scaffolding protein PALS2.     

<Emphasis Type="Italic">In silico</Emphasis> characterisation and chromosomal localisation of human <Emphasis Type="Italic">RRH</Emphasis> (peropsin) – implications for opsin evolution

Background  

The vertebrate opsins are proteins which utilise a retinaldehyde chromophore in their photosensory or photoisomerase roles in the visual/irradiance detection cycle. The majority of the opsins, such as rod and cone opsins, have a very highly conserved gene structure suggesting a common lineage. Exceptions to this are RGR-opsin and melanopsin, whose genes have very different intron insertion positions. The gene structure of another opsin, peropsin (retinal pigment epithelium-derived rhodopsin homologue,RRH) is unknown.