Intein-mediated ligation and cyclization of expressed proteins

Protein splicing is a posttranslational processing event that releases an internal protein sequence from a protein precursor. During the splicing process the internal protein sequence, termed an intein, embedded in the protein precursor self-catalyzes its excision and the ligation of the flanking protein regions, termed exteins. The dissection of the splicing pathway, which involves the precise cleavage and formation of peptide bonds, and the identification of key catalytic residues at the splice junctions have led to the modulation of the protein splicing process as a protein engineering tool. Novel strategies have been developed to use intein-catalyzed reactions for the production and manipulation of proteins and peptides. These new approaches have broken down the size limitation barrier of chemical synthetic methods and are less technically demanding. The purpose of this article is to describe how to use self-splicing inteins in protein semisynthesis and backbone cyclization. The first two sections of the article provide a brief review of the distinct chemical steps that underlie protein splicing and intein enabled technology.     

Intein-mediated, in vitro and in vivo protein modifications with small molecules

We review intein-mediated approaches for the site-specific modifications of proteins and highlight their applications in (1) the site-specific in vitro and in vivo biotinylation of proteins for protein arrays and (2) the site-specific in vivo labeling of proteins in living cells.     

Converting a marginally hydrophobic soluble protein into a membrane protein

δ-Helices are marginally hydrophobic α-helical segments in soluble proteins that exhibit certain sequence characteristics of transmembrane (TM) helices [Cunningham, F., Rath, A., Johnson, R. M. & Deber, C. M. (2009). Distinctions between hydrophobic helices in globular proteins and TM segments as factors in protein sorting. J. Biol. Chem., 284, 5395-402]. In order to better understand the difference between δ-helices and TM helices, we have studied the insertion of five TM-like δ-helices into dog pancreas microsomal membranes. Using model constructs in which an isolated δ-helix is engineered into a bona fide membrane protein, we find that, for two δ-helices originating from secreted proteins, at least three single-nucleotide mutations are necessary to obtain efficient membrane insertion, whereas one mutation is sufficient in a δ-helix from the cytosolic protein P450BM-3. We further find that only when the entire upstream region of the mutated δ-helix in the intact cytochrome P450BM-3 is deleted does a small fraction of the truncated protein insert into microsomes. Our results suggest that upstream portions of the polypeptide, as well as embedded charged residues, protect δ-helices in globular proteins from being recognized by the signal recognition particle-Sec61 endoplasmic-reticulum-targeting machinery and that δ-helices in secreted proteins are mutationally more distant from TM helices than δ-helices in cytosolic proteins.     

Intein-mediated protein trans-splicing and transgene containment in plastids

Transgenes in plastids are contained by stringent maternal inheritance in most cultivated plant species and their expression yields high levels of protein with bona fide structure. Nevertheless, transfer of plastid genes to the nucleus has been reported, with implications for transgene containment. The significance of these transfers will depend on the likelihood that they will become functional nuclear genes. Recently a novel approach, intein-mediated protein trans-splicing, has been demonstrated promising to yield transgenic plants with greatly reduced risk of genetic outcrossing.     

Intein-mediated protein ligation: harnessing nature's escape artists

Inteins are naturally occurring proteins that are involved in the precise cleavage and formation of peptide bonds in a process known as protein splicing. Genetic engineering has allowed the controllable cleavage of peptide bonds at either the N- or C-terminus of the intein. Inteins displaying controllable cleavage have been used in the isolation of bacterially expressed proteins possessing either a C-terminal thioester or an N-terminal cysteine. The specific placement of these reactive groups has allowed either protein-protein or protein-peptide condensation through a native peptide bond. This review describes the methods used to specifically generate these reactive groups on bacterially expressed proteins and some applications of this technique, known as intein-mediated protein ligation. Furthermore, a versatile two intein (TWIN) system will be described which enables the circularization and polymerization of bacterially expressed proteins or peptides.     

An asparaginyl endopeptidase mediates in vivo protein backbone cyclization

Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event.     

Schistosoma mansoni: surface membrane stability in vitro and in vivo

The human complement component C3b is known to bind in vitro to the surfaces of all developmental stages of schistosomes as a consequence of complement activation by the alternative pathway. C3b bound to Schistosoma mansoni parasites has now been used in combination with fluorescent labeled antibodies against C3b to label the surfaces of living schistosomes. Binding of complement components and labeled antibodies to adult schistosomes rendered their surface membrane homogeneously fluorescent. At the ultrastructural level, the label was seen as a dense deposit lying on the tegumental membrane. Surface damage was not observed in labeled adults by electron microscopy. Fluorescent schistosomes were cultured in vitro for periods of up to 2 weeks, during which time the parasites remained fully viable and their surface membrane was still fluorescent. The electron dense deposit persisted, and tegumental damage at the electron microscope level was minimal or absent. Consequently, adult schistosomes would seem able to survive in vitro in the absence of rapid and general turnover of their surface membrane. Loss of fluorescence was observed consistently only at the anterior end of the parasite, including the suckers, a finding which indicates that membrane turnover may occur at different rates on different parts of the body. Fluorescent 3-week-old juveniles and 6-day-old lung stage parasites were cultured under the same conditions with similar results: they remained viable and fluorescent for at least 2 weeks. Results with skin schistosomula were different in the sense that many worms died during culture, and those which survived lost large parts of their fluorescent surface. A few of the surviving and fluorescent schistosomula developed the elongate shape typical of lung stage parasites. Fluorescent viable skin schistosomula were injected intravenously into mice and subsequently recovered from the lungs after varying periods. Fluorescence was lost in a patchy way within a few minutes from some individuals and within several hours from most of the worms. These data permit the following conclusions: C3b is a suitable tracer for membrane renewal in all developmental stages of schistosomes. Very slow membrane renewal in vitro and very rapid renewal in vivo are both compatible with parasite survival.     

Quantitative protein stability measurement in vivo

The equilibrium between the native and denatured states of a protein can be key to its function and regulation. Traditionally, the folding equilibrium constant has been measured in vitro using purified protein and simple buffers. However, the biological environment of proteins can differ from these in vitro conditions in ways that could significantly perturb stability. Here, we present the first quantitative comparison between the stability of a protein in vitro and in the cytoplasm of Escherichia coli using amide hydrogen exchange detected by MALDI mass spectrometry (SUPREX). The results indicate that the thermodynamic stability of monomeric lambda repressor within the cell is the same as its stability measured in a simple buffer in vitro. However, when the E. coli are placed in a hyperosmotic environment, the in vivo stability is greatly enhanced. The in vivo SUPREX method provides a general and quantitative way to measure protein stabilities in the cell and will be useful for applications where intracellular stability information provides important biological insights.     

Structure and ligand binding of the soluble domain of a Thermotoga maritima membrane protein of unknown function TM1634

As a part of the Joint Center for Structural Genomics (JCSG) biological targets, the structures of soluble domains of membrane proteins from Thermotoga maritima were pursued. Here, we report the crystal structure of the soluble domain of TM1634, a putative membrane protein of 128 residues (15.1 kDa) and unknown function. The soluble domain of TM1634 is an alpha-helical dimer that contains a single tetratrico peptide repeat (TPR) motif in each monomer where each motif is similar to that found in Tom20. The overall fold, however, is unique and a DALI search does not identify similar folds beyond the 38-residue TPR motif. Two different putative ligand binding sites, in which PEG200 and Co(2+) were located, were identified using crystallography and NMR, respectively.     

Lower kinetic limit to protein thermal stability: a proposal regarding protein stability in vivo and its relation with misfolding diseases

In vitro thermal denaturation experiments suggest that, because of the possibility of irreversible alterations, thermodynamic stability (i.e., a positive value for the unfolding Gibbs energy) does not guarantee that a protein will remain in the native state during a given timescale. Furthermore, irreversible alterations are more likely to occur in vivo than in vitro because (a) some irreversible processes (e.g., aggregation, "undesirable" interactions with other macromolecular components, and proteolysis) are expected to be fast in the "crowded" cellular environment and (b) in many cases, the relevant timescale in vivo (probably related to the half-life for protein degradation) is expected to be longer than the timescale of the usual in vitro experiments (of the order of minutes). We propose, therefore, that many proteins (in particular, thermophilic proteins and "complex" proteins systems) are designed (by evolution) to have significant kinetic stability when confronted with the destabilizing effect of irreversible alterations. We show that, as long as these alterations occur mainly from non-native states (a Lumry-Eyring scenario), the required kinetic stability may be achieved through the design of a sufficiently high activation barrier for unfolding, which we define as the Gibbs energy barrier that separates the native state from the non-native ensemble (unfolded, partially folded, and misfolded states) in the following generalized Lumry-Eyring model: Native State <--> Non-Native Ensemble --> Irreversibly Denatured Protein. Finally, using familial amyloid polyneuropathy (FAP) as an illustrative example, we discuss the relation between stability and amyloid fibril formation in terms of the above viewpoint, which leads us to the two following tentative suggestions: (a) the hot spot defined by the FAP-associated amyloidogenic mutations of transthyretin reflects the structure of the transition state for unfolding and (b) substances that decrease the in vitro rate of transthyretin unfolding could also be inhibitors of amyloid fibril formation.     

Modulation of function in a minimalist heme-binding membrane protein

De novo designed heme-binding proteins have been used successfully to recapitulate features of natural hemoproteins. This approach has now been extended to membrane-soluble model proteins. Our group designed a functional hemoprotein, ME1, by engineering a bishistidine binding site into a natural membrane protein, glycophorin A (Cordova et al. in J Am Chem Soc 129:512–518, 2007). ME1 binds iron(III) protoporphyrin IX with submicromolar affinity, has a redox potential of −128 mV, and displays peroxidase activity. Here, we show the effect of aromatic residues in modulating the redox potential in the context of a membrane-soluble model system. We designed aromatic interactions with the heme through a single-point mutant, G25F, in which a phenylalanine is designed to dock against the porphyrin ring. This mutation results in roughly tenfold tighter binding to iron(III) protoporphyrin IX (K _d,app = 6.5 × 10⁻⁸ M), and lowers the redox potential of the cofactor to −172 mV. This work demonstrates that specific design features aimed at controlling the properties of bound cofactors can be introduced in a minimalist membrane hemoprotein model. The ability to modulate the redox potential of cofactors embedded in artificial membrane proteins is crucial for the design of electron transfer chains across membranes in functional photosynthetic devices.     

Intein-mediated site-specific conjugation of Quantum Dots to proteins in vivo

We describe an intein based method to site-specifically conjugate Quantum Dots (QDs) to target proteins in vivo. This approach allows the covalent conjugation of any nanostructure and/or nanodevice to any protein and thus the targeting of such material to any intracellular compartment or signalling complex within the cells of the developing embryo. We genetically fused a pleckstrin-homology (PH) domain with the N-terminus half of a split intein (I_N). The C-terminus half (I_C) of the intein was conjugated to QDs in vitro. I_C-QD's and RNA encoding PH-I_N were microinjected into Xenopus embryos. In vivo intein-splicing resulted in fully functional QD-PH conjugates that could be monitored in real time within live embryos. Use of Near Infra Red (NIR)-emitting QDs allowed monitoring of QD-conjugates within the embryo at depths where EGFP is undetectable demonstrating the advantages of QD's for this type of experiment. In conclusion, we have developed a novel in vivo methodology for the site-specific conjugation of QD's and other artificial structures to target proteins in different intracellular compartments and signaling complexes.     

Energetics of membrane protein folding and stability

The critical role of membrane proteins in a myriad of biological and physiological functions has spawned numerous investigations over the past several decades with the long-term goal of identifying the molecular origins and energetic forces that stabilize these proteins within the membrane. Parallel structural and thermodynamics studies on several systems have provided significant insight regarding the driving forces governing folding, assembly, insertion, and translocation of membrane proteins. The present review surveys families of membrane-associated proteins including α-helical and β-barrel structures, viral surface receptors, and pore-forming toxins, citing representative proteins within each of these classes for further scrutiny in terms of structure-function relationships and global conformational stability. This overview presents seminal findings from pioneering studies on the energetics of membrane protein folding and stability to modern techniques that are exploiting the use of molecular genetics and single molecule studies. An overall consensus regarding the molecular origins of membrane protein stability is that a number of intrinsic properties resemble features of soluble proteins, yet there are distinct energetic differences arising from specific intra- and intermolecular interactions within the membrane. The combined efforts from structural, energetics, and dynamics approaches offer unique insights and improve our fundamental understanding of the driving forces dictating membrane protein folding and stability.     

Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence

Statistical analysis of 12 unstable and 32 stable proteins revealed that there are certain dipeptides, the occurrence of which is significantly different in the unstable proteins compared with those in the stable ones. Based on the impact of these dipeptides on the unstable proteins over the stable ones, a weight value of instability is assigned to each of the dipeptides. For a given protein the summation of these weight values normalized to the length of its sequence helps to distinguish between unstable and stable proteins. Results suggest that the in vivo instability of proteins is possibly determined by the order of certain amino acids in its sequence. An attempt is made to correlate metabolic stability of proteins with features of their primary sequence where weight values of instability for a protein of known sequence could thus be used as an index for predicting its stability characteristics.     

Intein-mediated affinity-fusion purification of the Escherichia coli RecA protein

The RecA protein of Escherichia coli plays important roles in homologous recombination, recombinational DNA repair, and SOS induction. Because its functions are conserved among the phylogenetic kingdoms, RecA investigations have provided a paradigm for understanding these biological processes. The RecA protein has been overproduced in E. coli and purified using a variety of purification schemes requiring multiple, time-intensive steps. The purification schemes share a dependence on appropriate RecA structure and/or function at one or more steps. In this report, we used a modified protein splicing element (intein) and a chitin-binding domain, fused to the C-terminus of RecA, to facilitate a one-step affinity purification of RecA protein without modification of the native protein sequence. Following the single chromatographic step, RecA protein that is greater than 95% physical purity at a concentration of greater than microM was obtained. The protein displays in vitro activities that are identical to those of protein isolated using classical procedures. The purification strategy described here promises to yield mutant RecA proteins in sufficient quantity for rigorous biophysical characterization without dependence on intrinsic RecA function.     

Biotinylation in vivo as a sensitive indicator of protein secretion and membrane protein insertion.

Escherichia coli biotin ligase is a cytoplasmic protein which specifically biotinylates the biotin-accepting domains from a variety of organisms. This in vivo biotinylation can be used as a sensitive signal to study protein secretion and membrane protein insertion. When the biotin-accepting domain from the 1.3S subunit of Propionibacterium shermanii transcarboxylase (PSBT) is translationally fused to the periplasmic proteins alkaline phosphatase and maltose-binding protein, there is little or no biotinylation of PSBT in wild-type E. coli. Inhibition of SecA with sodium azide and mutations in SecB, SecD, and SecF, all of which slow down protein secretion, result in biotinylation of PSBT. When PSBT is fused to the E. coli inner membrane protein MalF, it acts as a topological marker: fusions to cytoplasmic domains of MalF are biotinylated, and fusions to periplasmic domains are generally not biotinylated. If SecA is inhibited by sodium azide or if the SecE in the cell is depleted, then the insertion of the MalF second periplasmic domain is slowed down enough that PSBT fusions in this part of the protein become biotinylated. Compared with other protein fusions that have been used to study protein translocation, PSBT fusions have the advantage that they can be used to study the rate of the insertion process.     

Phosphoinositides: regulators of membrane traffic and protein function

Phosphoinositides serve as important spatio-temporal regulators of intracellular trafficking and cell signalling events. In addition to their recognition by specific phosphoinositide binding domains present within cytoplasmic adaptor proteins or membrane integral channels and transporters phosphoinositides may affect membrane transport by eliciting conformational changes within proteins or by regulating enzymatic activities. During adaptor-mediated membrane traffic phosphoinositides form part of coincidence detection systems that aid in targeting pools of specific phosphoinositides to select intracellular transport pathways. In this review, we discuss potential mechanisms for conferring selectivity onto the phosphoinositide code as well as possible avenues for future research.     

FRET-based in vivo screening for protein folding and increased protein stability

Fluorescence resonance energy transfer (FRET) was used to establish a novel in vivo screening system that allows rapid detection of protein folding and protein variants with increased thermodynamic stability in the cytoplasm of Escherichia coli. The system is based on the simultaneous fusion of the green fluorescent protein (GFP) to the C terminus of a protein X of interest, and of blue-fluorescent protein (BFP) to the N terminus of protein X. Efficient FRET from BFP to GFP in the ternary fusion protein is observed in vivo only when protein X is folded and brings BFP and GFP into close proximity, while FRET is lost when BFP and GFP are far apart due to unfolding or intracellular degradation of protein X. The screening system was validated by identification of antibody V(L) intradomains with increased thermodynamic stabilities from expression libraries after random mutagenesis, bacterial cell sorting, and colony screening.     

Interfacial pH modulation of membrane protein function in vivo. Effect of anionic phospholipids.

In yeast cells, the magnitude of the membrane surface potential (phi) is determined to a large extent by the relative amount of anionic phospholipids (Cerbón and Calderón (1990) Biochim. Biophys. Acta 1028, 261-267). When a significant surface potential exists, the pH at the membrane surface (interfacial pH) will be different to that in the bulk suspending medium. We now report that: (1) In cells with higher phi (phosphatidylinositol-rich cells (PI-rich) and phosphatidylserine-rich cells (PS-rich) a 10-times lower proton concentration in the bulk was enough to achieve the maximum transport activity of H(+)-linked transport systems when compared to normal cells. (2) When the phi was reduced by increasing the concentration of cations in the medium, more protons were required to achieve maximum transport, that is, the pH activity curves shifted downwards to a more acidic pH. (3) The magnitude of the downward pH shift was around 2.5-times higher for the more charged membranes. (4) Around 10-times more KCl than MgCl2 was necessary to give an equivalent pH shift, in agreement with their capacity to reduce the phi of artificial bilayers. The interfacial pH calculated from the values of phi indicates that it was 0.4 pH units lower in the anionic phospholipid rich cells as compared to normal cells. The results indicate that membrane surface potential may explain the complex relationship between pH, ionic strength and membrane protein function. Maximum transport activities were found for glutamate at interfacial pH of 4.2-4.8 and were inhibited at interfacial pH = 3.2-3.4, suggesting that surface groups of the carrier proteins with pK values in the region 3.8-4.2 (aspartyl and glutamyl) are involved in binding and/or release of charged substrates.     

In vivo function of a novel Siah protein in Drosophila

The Siah proteins, mammalian homologues of the Drosophila Sina protein, function as E3 ubiquitin ligase enzymes and target a wide range of cellular proteins for degradation. Here, I investigate the in vivo function of the fly protein, Sina-Homologue (SinaH), which is highly similar to Sina. Flies that completely lack SinaH are viable and in combination with a mutation in the gene, Ebi, show an extra dorsal central bristle phenotype. I also show that SinaH and Ebi can interact with each other both in vivo and in vitro suggesting that they act in the same physical complex. Flies that lack both Sina and Sina-Homologue were also created and show visible eye and bristle phenotypes, which can be explained by an inability to degrade the neuronal repressor, Tramtrack. I find no evidence for redundancy in the function of Sina and SinaH.