Are turns required for the folding of ribonuclease T1?

Ribonuclease T1 (RNase T1) is a small, globular protein of 104 amino acids for which extensive thermodynamic and structural information is known. To assess the specific influence of variations in amino acid sequence on the mechanism for protein folding, circularly permuted variants of RNase T1 were constructed and characterized in terms of catalytic activity and thermodynamic stability. The disulfide bond connecting Cys-2 and Cys-10 was removed by mutation of these residues to alanine (C2, 10A) to avoid potential steric problems imposed by the circular permutations. The original amino-terminus and carboxyl-terminus of the mutant (C2, 10A) were subsequently joined with a tripeptide linker to accommodate a reverse turn and new termini were introduced throughout the primary sequence in regions of solvent-exposed loops at Ser-35 (cp35S1), Asp-49 (cp49D1), Gly-70 (cp70G1), and Ser-96 (cp96S1). These circularly permuted RNase T1 mutants retained 35-100% of the original catalytic activity for the hydrolysis of guanylyl(3'-->5')cytidine, suggesting that the overall tertiary fold of these mutants is very similar to that of wild-type protein. Chemical denaturation curves indicated thermodynamic stabilities at pH 5.0 of 5.7, 2.9, 2.6, and 4.6 kcal/mol for cp35S1, cp49D1, cp70G1, and cp96S1, respectively, compared to a value of 10.1 kcal/mol for wild-type RNase T1 and 6.4 kcal/mol for (C2, 10A) T1. A fifth set of circularly permuted variants was attempted with new termini positioned in a tight beta-turn between Glu-82 and Gln-85. New termini were inserted at Asn-83 (cp83N1), Asn-84 (cp84N1), and Gln-85 (cp85Q1). No detectable amount of protein was ever produced for any of the mutations in this region, suggesting that this turn may be critical for the proper folding and/or thermodynamic stability of RNase T1.     

Circularly permuted monomeric red fluorescent proteins with new termini in the ��-sheet

Circularly permuted fluorescent proteins (FPs) have a growing number of uses in live cell fluorescence biosensing applications. Most notably, they enable the construction of single fluorescent protein‐based biosensors for Ca²⁺ and other analytes of interest. Circularly permuted FPs are also of great utility in the optimization of fluorescence resonance energy transfer (FRET)‐based biosensors by providing a means for varying the critical dipole–dipole orientation. We have previously reported on our efforts to create circularly permuted variants of a monomeric red FP (RFP) known as mCherry. In our previous work, we had identified six distinct locations within mCherry that tolerated the insertion of a short peptide sequence. Creation of circularly permuted variants with new termini at the locations corresponding to the sites of insertion led to the discovery of three permuted variants that retained no more than 18% of the brightness of mCherry. We now report the extensive directed evolution of the variant with new termini at position 193 of the protein sequence for improved fluorescent brightness. The resulting variant, known as cp193g7, has 61% of the intrinsic brightness of mCherry and was found to be highly tolerant of circular permutation at other locations within the sequence. We have exploited this property to engineer an expanded series of circularly permuted variants with new termini located along the length of the 10th β‐strand of mCherry. These new variants may ultimately prove useful for the creation of single FP‐based Ca²⁺ biosensors.     

Random circular permutation of DsbA reveals segments that are essential for protein folding and stability

One of the key questions in protein folding is whether polypeptide chains require unique nucleation sites to fold to the native state. In order to identify possible essential polypeptide segments for folding, we have performed a complete circular permutation analysis of a protein in which the natural termini are in close proximity. As a model system, we used the disulfide oxidoreductase DsbA from Escherichia coli, a monomeric protein of 189 amino acid residues. To introduce new termini at all possible positions in its polypeptide chain, we generated a library of randomly circularly permuted dsbA genes and screened for active circularly permuted variants in vivo. A total of 51 different active variants were identified. The new termini were distributed over about 70 % of the polypeptide chain, with the majority of them occurring within regular secondary structures. New termini were not found in approximately 30 % of the DsbA sequence which essentially correspond to four alpha-helices of DsbA. Introduction of new termini into these "forbidden segments" by directed mutagenesis yielded proteins with altered overall folds and strongly reduced catalytic activities. In contrast, all active variants analysed so far show structural and catalytic properties comparable with those of DsbA wild-type. We suggest that random circular permutation allows identification of contiguous structural elements in a protein that are essential for folding and stability.     

Construction of a dual chain pseudotetrameric chicken avidin by combining two circularly permuted avidins

Two distinct circularly permuted forms of chicken avidin were designed with the aim of constructing a fusion avidin containing two biotin-binding sites in one polypeptide. The old N and C termini of wild-type avidin were connected to each other via a glycine/serine-rich linker, and the new termini were introduced into two different loops. This enabled the creation of the desired fusion construct using a short linker peptide between the two different circularly permuted subunits. The circularly permuted avidins (circularly permuted avidin 5 --> 4 and circularly permuted avidin 6 --> 5) and their fusion, pseudotetrameric dual chain avidin, were biologically active, i.e. showed biotin binding, and also displayed structural characteristics similar to those of wild-type avidin. Dual chain avidin facilitates the development of dual affinity avidins by allowing adjustment of the ligand-binding properties in half of the binding sites independent of the other half. In addition, the subunit fusion strategy described in this study can be used, where applicable, to modify oligomeric proteins in general.     

Amyloid fibril formation by circularly permuted and C-terminally deleted mutants

The natural N- and C-termini, i.e., the given order of secondary structure segments, are critical for protein folding and stability, as shown by several studies using circularly permuted proteins, mutants that have their N- and C-termini linked and are then digested at another site to create new termini. A previous work showed that circularly permuted mutants of sperm whale myoglobin (Mb) are functional, have native-like folding and bind heme, but are less stable than the wild-type protein and aggregate. The ability of wild-type myoglobin to form amyloid fibrils has been established recently, and because circularly permuted mutations are destabilizing, we asked whether these permutations would also affect the rate of amyloid fibril formation. Our investigations revealed that, indeed, the circularly permuted mutants formed cytotoxic fibrils at a rate higher than that of the wild-type. To further investigate the role of the C-terminus in the overall stability of the protein, we investigated two C-terminally deleted mutant, Mb(1-123) and Mb(1-99), and found that Mb(1-123) formed cytotoxic fibrils at a higher rate than that of the wild-type while Mb(1-99) formed cytotoxic fibrils at a similar rate than that of the wild-type. Collectively, our findings show that the native position of both the N-and C-termini is important for the precise structural architecture of myoglobin.     

Circularly permuted variants of the green fluorescent protein.

Folding of the green fluorescent protein (GFP) from Aequorea victoria is characterized by autocatalytic formation of its p-hydroxybenzylideneimidazolidone chromophore, which is located in the center of an 11-stranded beta-barrel. We have analyzed the in vivo folding of 20 circularly permuted variants of GFP and find a relatively low tolerance towards disruption of the polypeptide chain by introduction of new termini. All permuted variants with termini in strands of the beta-barrel and about half of the variants with termini in loops lost the ability to form the chromophore. The thermal stability of the permuted GFPs with intact chromophore is very similar to that of the wild-type, indicating that chromophore-side chain interactions strongly contribute to the extraordinary stability of GFP.     

In vivo assembly of aspartate transcarbamoylase from fragmented and circularly permuted catalytic polypeptide chains

Previous studies on Escherichia coli aspartate transcarbamoylase (ATCase) demonstrated that active, stable enzyme was formed in vivo from complementing polypeptides of the catalytic (c) chain encoded by gene fragments derived from the pyrBI operon. However, the enzyme lacked the allosteric properties characteristic of wild-type ATCase. In order to determine whether the loss of homotropic and heterotropic properties was attributable to the location of the interruption in the polypeptide chain rather than to the lack of continuity, we constructed a series of fragmented genes so that the breaks in the polypeptide chains would be dispersed in different domains and diverse regions of the structure. Also, analogous molecules containing circularly permuted c chains with altered termini were constructed for comparison with the ATCase molecules containing fragmented c chains. Studies were performed on four sets of ATCase molecules containing cleaved c chains at positions between residues 98 and 99, 121 and 122, 180 and 181, and 221 and 222; the corresponding circularly permuted chains had N termini at positions 99, 122, 181, and 222. All of the ATCase molecules containing fragmented or circularly permuted c chains exhibited the homotropic and heterotropic properties characteristic of the wild-type enzyme. Hill coefficients (n(H:)) and changes in them upon the addition of ATP and CTP were similar to those observed with wild-type ATCase. In addition, the conformational changes revealed by the decrease in sedimentation coefficient upon the addition of a bisubstrate analog were virtually identical to that for the wild-type enzyme. Differential scanning calorimetry showed that neither the breakage of the polypeptide chains nor the newly formed covalent bond between the termini in the wild-type enzyme had a significant impact on the thermal stability of the assembled dodecamers. The studies demonstrate that continuity of the polypeptide chain within structural domains is not essential for the assembly, activity, and allosteric properties of ATCase.     

Relocation or duplication of the helix A sequence of T4 lysozyme causes only modest changes in structure but can increase or decrease the rate of folding

In T4 lysozyme, helix A is located at the amino terminus of the sequence but is associated with the C-terminal domain in the folded structure. To investigate the implications of this arrangement for the folding of the protein, we first created a circularly permuted variant with a new amino terminus at residue 12. In effect, this moves the sequence corresponding to helix A from the N- to the C-terminus of the molecule. The protein crystallized nonisomorphously with the wild type but has a very similar structure, showing that the unit consisting of helix A and the C-terminal domain can be reconstituted from a contiguous polypeptide chain. The protein is less stable than the wild type but folds slightly faster. We then produced a second variant in which the helix A sequence was appended at the C-terminus (as in the first variant), but was also restored at the N-terminus (as in the wild type). This variant has two helix A sequences, one at the N-terminus and the other at the C-terminus, each of which can compete for the same site in the folded protein. The crystal structure shows that it is the N-terminal sequence that folds in a manner similar to that of the wild type, whereas the copy at the C-terminus is forced to loop out. The stability of this protein is much closer to that of the wild type, but its rate of folding is significantly slower. The reduction in rate is attributed to the presence of the two identical sequence segments which compete for a single, mutually exclusive, site.     

Circular permutation within the coenzyme binding domain of the tetrameric glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus.

A circularly permuted (cp) variant of the phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been constructed with N- and C-termini created within the coenzyme binding domain. The cp variant has a kcat value equal to 40% of the wild-type value, whereas Km and KD values for NAD show a threefold decrease compared to wild type. These results indicate that the folding process and the conformational changes that accompany NAD binding during the catalytic event occur efficiently in the permuted variant and that NAD binding is tighter. Reversible denaturation experiments show that the stability of the variant is only reduced by 0.7 kcal/mol compared to the wild-type enzyme. These experiments confirm and extend results obtained recently on other permuted proteins. For multimeric proteins, such as GAPDH, which harbor subunits with two structural domains, the natural location of the N- and C-termini is not a prerequisite for optimal folding and biological activity.     

Towards Tricking a Pathogen’s Protease into Fighting Infection: The 3D Structure of a Stable Circularly Permuted Onconase Variant Cleavedby HIV-1 Protease

Onconase® is a highly cytotoxic amphibian homolog of Ribonuclease A. Here, we describe the construction of circularly permuted Onconase® variants by connecting the N- and C-termini of this enzyme with amino acid residues that are recognized and cleaved by the human immunodeficiency virus protease. Uncleaved circularly permuted Onconase® variants are unusually stable, non-cytotoxic and can internalize in human T-lymphocyte Jurkat cells. The structure, stability and dynamics of an intact and a cleaved circularly permuted Onconase® variant were determined by Nuclear Magnetic Resonance spectroscopy and provide valuable insight into the changes in catalytic efficiency caused by the cleavage. The understanding of the structural environment and the dynamics of the activation process represents a first step toward the development of more effective drugs for the treatment of diseases related to pathogens expressing a specific protease. By taking advantage of the protease’s activity to initiate a cytotoxic cascade, this approach is thought to be less susceptible to known resistance mechanisms.     

Circular permutation of granulocyte colony-stimulating factor

The sequence of granulocyte colony-stimulating factor (G-CSF) has been circularly permuted by introducing new chain termini into interhelical loops and by constraining the N- and C-terminal helices, either by direct linkage of the termini (L0) or by substitution of the amino-terminal 10-residue segment with a seven-residue linker composed of glycines and serines (L1). All the circularly permuted G-CSFs (cpG-CSFs) were able to fold into biologically active structures that could recognize the G-CSF receptor. CD and NMR spectroscopy demonstrated that all of the cpG-CSFs adopted a fold similar to that of the native molecule, except for one [cpG-CSF(L1)[142/141]] which has the new termini at the end of loop 34 with the shorter L1 linker. All of the cpG-CSFs underwent cooperative unfolding by urea, and a systematically lower free energy change (DeltaGurea) was observed for molecules with the shorter L1 linker than for those molecules in which the original termini were directly linked (the L0 linker). The thermodynamic stability of the cpG-CSFs toward urea was found to correlate with their relative ability to stimulate proliferation of G-CSF responsive cells. Taken together, these results indicate that the G-CSF sequence is robust in its ability to undergo linear rearrangement and adopt a biologically active conformation. The choice of linker, with its effect on stability, seems to be important for realizing the full biological activity of the three-dimensional structure. The breakpoint and linker together are the ultimate determinants of the structural and biological profiles of these circularly permuted cytokines. In the following paper [McWherter, C. A., et al. (1999) Biochemistry 38, 4564-4571], McWherter and co-workers have used circularly permuted G-CSF sequences to engineer chimeric dual IL-3 and G-CSF receptor agonists in which the relative spatial orientation of the receptor agonist domains is varied. Interpreting the differences in activity for the chimeric molecules in terms of the connectivity between domains depends critically on the results reported here for the isolated cpG-CSF domains.     

Nuclear transition protein 2 (TP2) of mammalian spermatids has a very basic carboxyl terminal domain

Nuclear transition protein 2 (TP2) along with TP1 are major basic chromosomal proteins of rat spermatids during the period of transition from histone-associated to protamine-associated DNA. TP2 isolated by reversed phase high pressure liquid chromatography was cleaved with S. aureus V8 protease to yield two fragments. The complete amino acid sequence of the 27 residue peptide assigned to the carboxyl terminus was established. It contains most of the basic residues of the protein and is likely to be a major site of DNA binding. Thus, TP2 is differentiated from core histones in having its basic domain at the carboxyl rather than amino terminal end.     

Interaction between the noncatalytic region of Sid1p kinase and Cdc14p is required for full catalytic activity and localization of Sid1p

Sid1p is a group II p21-activated kinase/germinal center kinase family member that is part of a signaling network required for cytokinesis in fission yeast. Germinal center kinases are characterized by well conserved amino-terminal catalytic domains followed by less conserved carboxyl termini. The carboxyl termini among group I germinal center kinases are moderately conserved and thought to be regulatory regions. Little is known about the carboxyl termini of group II family members. Sid1p has been shown to bind the novel protein Cdc14p; however, the functional significance of this interaction is unknown. Here we report that the carboxyl terminus of Sid1p is an essential regulatory region. Our results indicate that this region contains the binding domain for Cdc14p, and this association is required for full Sid1p catalytic activity as well as intracellular localization. Furthermore, overexpression of the carboxyl terminus of Sid1p alone compromises the signaling of cytokinesis. We conclude that Cdc14p positively regulates the Sid1p kinase by binding the noncatalytic carboxyl-terminal region of the protein.     

Carbohydrate transporters of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS)

The glucose transporter of Escherichia coli couples translocation with phosphorylation of glucose. The IICB(Glc) subunit spans the membrane eight times. Split, circularly permuted and cyclized forms of IICB(Glc) are described. The split variant was 30 times more active when the two proteins were encoded by a dicistronic mRNA than by two genes. The stability and activity of circularly permuted forms was improved when they were expressed as fusion proteins with alkaline phosphatase. Cyclized IICB(Glc) and IIA(Glc) were produced in vivo by RecA intein-mediated trans-splicing. Purified, cyclized IIA(Glc) and IICB(Glc) had 100% and 30% of wild-type glucose phosphotransferase activity, respectively. Cyclized IIA(Glc) displayed increased stability against temperature and GuHCl-induced unfolding.     

Truncated variants of gp120 bind CD4 with high affinity and suggest a minimum CD4 binding region.

The envelope glycoprotein, gp120, of human immunodeficiency virus type 1 (HIV-1) binds the cellular protein CD4 with high affinity. By deletion we show that 62 N- and 20 C-terminal residues along with the V1, V2 and V3 variable regions of gp 120 are unnecessary for CD4 binding. A 287 residue variant (ENV59), missing those 197 amino acids, binds to CD4 with high affinity. A polyclonal antibody failed to efficiently precipitate ENV59 which is consistent with the loss of immunodominant antigenic structures in the regions deleted. This suggests that ENV59 may have potential as an immunogen, able to elicit antibodies against more conserved regions of gp120. Additionally, complementing co-expressed gp120 fragments as well as a circularly permuted molecule bind CD4, and suggest either that the molecular termini are adjacent in the folded structure, or that an N-terminal region folds into the structure unconstrained by its method of attachment to the rest of the molecule.     

Engineering a circularly permuted GFP scaffold for peptide presentation

The use of peptides as in vivo and in vitro ligand binding agents is hampered by the high flexibility, low stability and lack of intrinsic detection signal of peptide aptamers. Recent attempts to overcome these limitations included the integration of the binding peptide into a stable protein scaffold. In this paper, we present the optimization and testing of a circularly permuted variant of the green fluorescent protein (GFP). We examined the ability of the optimized scaffold to accept peptide insertions at three different regions. The three regions chosen are localized in close spatial proximity to each other and support different conformations of the inserted peptides. In all the three regions peptides with a biased, but still comprehensive, amino acid repertoire could be presented without disturbing the function of the optimized GFP-scaffold.     

Characterization of NAD:arginine ADP-ribosyltransferases

NAD:arginine mono-ADP-ribosyltransferases catalyze the transfer of ADP-ribose from NAD to the guanidino group of arginine on a target protein. Deduced amino acid sequences of one family (ART1) of mammalian ADP-ribosyltransferases, cloned from muscle and lymphocytes, show hydrophobic amino and carboxyl termini consistent with glycosylphosphatidylinositol (GPI)-anchored proteins. The proteins, overexpressed in mammalian cells transfected with the transferase cDNAs, are released from the cell surface with phosphatidylinositol-specific phospholipase C (PI-PLC), and display immunological and biochemical characteristics consistent with a cell surface, GPI-anchored protein. In contrast, the deduced amino acid sequence of a second family (ART5) of transferases, cloned from murine lymphoma cells and expressed in high abundance in testis, displays a hydrophobic amino terminus, consistent with a signal sequence, but lacks a hydrophobic signal sequence at its carboxyl terminus, suggesting that the protein is destined for export. Consistent with the surface localization of the GPI-linked transferases, multiple surface substrates have been identified in myotubes and activated lymphocytes, and, notably, include integrin subunits. Similar to the bacterial toxin ADP-ribosyltransferases, the mammalian transferases contain the characteristic domains involved in NAD binding and ADP-ribose transfer, including a highly acidic region near the carboxy terminus, which, when disrupted by in vitro mutagenesis, results in a loss of enzymatic activity. The carboxyl half of the protein, synthesized as a fusion protein in E. coli, possessed NADase, but not ADP-ribosyltransferase activity. These findings are consistent with the existence at the carboxyl terminus of ART1 of a catalytically active domain, capable of hydrolyzing NAD, but not of transferring ADP-ribose to a guanidino acceptor.     

Redesigning the single-chain variant of the restriction endonuclease PvuII by circular permutation

The restriction endonuclease PvuII has been introduced as a sequence-specific cleavage module in highly-specific nucleases for gene targeting. Here, a structural reorganization of the single-chain variant of PvuII (scPvuII) was performed by circular permutation as a proof-of-concept in order to find out whether the relocated, new termini next to structural elements important for DNA recognition and catalysis could be used for the fusion with other regulatory protein domains. Three circularly permuted variants of scPvuII were obtained that all maintain the specific endonucleolytic activity of scPvuII.     

The 43-kilodalton protein of Torpedo nicotinic postsynaptic membranes: purification and determination of primary structure

The primary structure of the 43-kilodalton peripheral membrane protein (43-kDa protein) of Torpedo nicotinic postsynaptic membrane has been determined. The 43-kDa protein, which was isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, has an amino terminus resistant to Edman degradation, while the sequence at the carboxyl terminus is Tyr-Val. An amino acid sequence of 405 residues was obtained by NH2-terminal sequence analysis of complementary peptides generated by digestion with trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and endoproteinase Lys-C, as well as by chemical cleavage at methionine. This sequence of molecular mass 45,618 daltons lacks the amino terminus but extends to the carboxyl terminus of the 43-kDa protein. Unusual structural features of the 43-kDa protein include two regions of approximately 80 residues, each containing 10% cysteine, as well as stretches predicted to exist as amphipathic alpha-helices. Other than the group blocking the amino terminus, no evidence was found for posttranslational modification of amino acids. The 43-kDa protein may represent a novel protein family because a computer search of this sequence with the National Biomedical Research Foundation data base (Release 12.0) did not reveal any significant homology to known protein sequences.