Protein engineering using circular permutation - structure, function, stability, and applications.

Protein engineering is important for creating novel variants from natural proteins, enabling a wide range of applications. Approaches such as rational design and directed evolution are routinely used to make new protein variants. Computational tools like de novo design can introduce new protein folds. Expanding the amino acid repertoire to include unnatural amino acids with non-canonical side chains in vitro by native chemical ligation and in vivo via codon expansion methods broadens sequence and structural possibilities. Circular permutation (CP) is an invaluable approach to redesigning a protein by rearranging the amino acid sequence, where the connectivity of the secondary structural elements is altered without changing the overall structure of the protein. Artificial CP proteins (CPs) are employed in various applications such as biocatalysis, sensing of small molecules by fluorescence, genome editing, ligand-binding protein switches, and optogenetic engineering. Many studies have shown that CP can lead to either reduced or enhanced stability or catalytic efficiency. The effects of CP on a protein's energy landscape cannot be predicted a priori. Thus, it is important to understand how CP can affect the thermodynamic and kinetic stability of a protein. In this review, we discuss the discovery and advancement of techniques to create protein CP, and existing reviews on CP. We delve into the plethora of biological applications for designed CP proteins. We subsequently discuss the experimental and computational reports on the effects of CP on the thermodynamic and kinetic stabilities of proteins of various topologies. An understanding of the various aspects of CP will allow the reader to design robust CP proteins for their specific purposes.

Image recognition-based petal arrangement estimation.

Flowers exhibit morphological diversity in the number and positional arrangement of their floral organs, such as petals. The petal arrangements of blooming flowers are represented by the overlap position relation between neighboring petals, an indicator of the floral developmental process; however, only specialists are capable of the petal arrangement identification. Therefore, we propose a method to support the estimation of the arrangement of the perianth organs, including petals and tepals, using image recognition techniques. The problem for realizing the method is that it is not possible to prepare a large number of image datasets: we cannot apply the latest machine learning based image processing methods, which require a large number of images. Therefore, we describe the tepal arrangement as a sequence of interior-exterior patterns of tepal overlap in the image, and estimate the tepal arrangement by matching the pattern with the known patterns. We also use methods that require less or no training data to implement the method: the fine-tuned YOLO v5 model for flower detection, GrubCut for flower segmentation, the Harris corner detector for tepal overlap detection, MAML-based interior-exterior estimation, and circular permutation matching for tepal arrangement estimation. Experimental results showed good accuracy when flower detection, segmentation, overlap location estimation, interior-exterior estimation, and circle permutation matching-based tepal arrangement estimation were evaluated independently. However, the accuracy decreased when they were integrated. Therefore, we developed a user interface for manual correction of the position of overlap estimation and interior-exterior pattern estimation, which ensures the quality of tepal arrangement estimation.

Computational insights into the circular permutation roles on ConA binding and structural stability.

The mechanisms behind Concanavalin A (ConA) circular permutation have been under investigation since 1985. Although a vast amount of information is available about this lectin and its applications, the exact purpose of its processing remains unclear. To shed light on this, this study employed computer simulations to compare the unprocessed ProConA with the mature ConA. This approach aimed to reveal the importance of the post-translational modifications, especially how they affect the lectin stability and carbohydrate-binding properties. To achieve these goals, we conducted 200 ns molecular dynamics simulations and trajectory analyses on the monomeric forms of ProConA and ConA (both unbound and in complex with D-mannose and the GlcNAc2Man9 N-glycan), as well as on their oligomeric forms. Our findings reveal significant stability differences between ProConA and ConA at both the monomeric and tetrameric levels, with ProConA exhibiting consistently lower stability parameters compared to ConA. In terms of carbohydrate binding properties, however, both lectins showed remarkable similarities in their interaction profiles, contact numbers, and binding free energies with D-mannose and the high-mannose N-glycan. Overall, our results suggest that the processing of ProConA significantly enhances the stability of the mature lectin, especially in maintaining the tetrameric oligomer, without substantially affecting its carbohydrate-binding properties.

Construction and characterization of a functional variant hFGF7 with enhanced properties by circular permutation.

Human fibroblast growth factor 7 (hFGF7) is a member of the paracrine-acting FGF family and mediates various reactions such as wound healing, tissue homeostasis, and liver regeneration. These activities make it a plausible candidate for pharmaceutical applications as a drug. However, the low expression level and stability of the recombinant hFGF7 were known to be major hurdles for further applications. Here, the expression level and stability of hFGF7 were attempted to improve by changing the order of amino acids through circular permutation (CP), thereby expecting an alternative fate according to the N-end rule. CP-hFGF7 variants were constructed systematically by using putative amino acid residues in the loop region that avoided the disruption of the structural integrity especially in the functional motif. Among them, cp-hFGF7115-114 revealed a relatively higher expression level in the soluble fraction than the wild-type hFGF7 and was efficiently purified (7 mg L-1) to apparent homogeneity. The activity and stability of the purified variant cp-hFGF7115-114 were comparable or superior to that of the wild-type hFGF7, thereby strongly suggesting that CP could be an alternative tool for the functional expression of hFGF7 in Escherichia coli.

Rational Design of a Circularly Permuted Flavin-Based Fluorescent Protein.

Flavin-based fluorescent proteins are oxygen-independent reporters that hold great promise for imaging anaerobic and hypoxic biological systems. In this study, we explored the feasibility of applying circular permutation, a valuable method for the creation of fluorescent sensors, to flavin-based fluorescent proteins. We used rational design and structural data to identify a suitable location for circular permutation in iLOV, a flavin-based reporter derived from A. thaliana. However, relocating the N- and C-termini to this position resulted in a significant reduction in fluorescence. This loss of fluorescence was reversible, however, by fusing dimerizing coiled coils at the new N- and C-termini to compensate for the increase in local chain entropy. Additionally, by inserting protease cleavage sites in circularly permuted iLOV, we developed two protease sensors and demonstrated their application in mammalian cells. In summary, our work establishes the first approach to engineer circularly permuted FbFPs optimized for high fluorescence and further showcases the utility of circularly permuted FbFPs to serve as a scaffold for sensor engineering.

Elucidation of the folding pathway of a circular permutant of topologically knotted YbeA by tryptophan substitutions.

CP74 is an engineered circular permutant of a deep trefoil knotted SpoU-TrmD (SPOUT) RNA methyl transferase protein YbeA from E. coli. We have previously established that the circular permutation unties the knotted topology of YbeA and CP74 forms a domain-swapped dimer with a large dimeric interface of ca. 4600 Å2. To understand the impact of domain-swapping and the newly formed hinge region joining the two folded domains on the folding and stability of CP74, the five equally spaced tryptophan residues were individually substituted into phenylalanine to monitor their conformational and stability changes by a battery of biophysical tools. Far-UV circular dichroism, intrinsic fluorescence, and small-angle X-ray scattering dictated minimal global conformational perturbations to the native structures in the tryptophan variants. The structures of the tryptophan variants also showed the conservation of the domain-swapped ternary structure with the exception that the W72F exhibited significant asymmetry in the α-helix 5. Comparative global thermal and chemical stability analyses indicated the pivotal role of W100 in the folding of CP74 followed by W19 and W72. Solution-state NMR spectroscopy and hydrogen-deuterium exchange mass spectrometry further revealed the accumulation of a native-like intermediate state in which the hinge region made important contributions to maintain the domain-swapped ternary structure of CP74.

Structures of permuted halves of a modern ribose-binding protein.

Periplasmic binding proteins (PBPs) are a class of proteins that participate in the cellular transport of various ligands. They have been used as model systems to study mechanisms in protein evolution, such as duplication, recombination and domain swapping. It has been suggested that PBPs evolved from precursors half their size. Here, the crystal structures of two permuted halves of a modern ribose-binding protein (RBP) from Thermotoga maritima are reported. The overexpressed proteins are well folded and show a monomer-dimer equilibrium in solution. Their crystal structures show partially noncanonical PBP-like fold type I conformations with structural deviations from modern RBPs. One of the half variants forms a dimer via segment swapping, suggesting a high degree of malleability. The structural findings on these permuted halves support the evolutionary hypothesis that PBPs arose via a duplication event of a flavodoxin-like protein and further support a domain-swapping step that might have occurred during the evolution of the PBP-like fold, a process that is necessary to generate the characteristic motion of PBPs essential to perform their functions.

Metal-binding and circular permutation-dependent thermodynamic and kinetic stability of azurin.

Native topology is known to determine the folding kinetics and the energy landscape of proteins. Furthermore, the circular permutation (CP) of proteins alters the order of the secondary structure connectivity while retaining the three-dimensional structure, making it an elegant and powerful approach to altering native topology. Previous studies elucidated the influence of CP in proteins with different folds such as Greek key ß-barrel, ß-sandwich, ß-α-ß, and all α-Greek key. CP mainly affects the protein stability and unfolding kinetics, while folding kinetics remains mostly unaltered. However, the effect of CP on metalloproteins is yet to be elaborately studied. The active site of metalloproteins poses an additional complexity in studying protein folding. Here, we investigate a CP variant (cpN42) of azurin-in both metal-free and metal-bound (holo) forms. As observed earlier in other proteins, apo-forms of wild-type (WT) and cpN42 fold with similar rates. In contrast, zinc-binding accelerates the folding of WT but decelerates the folding of cpN42. On zinc-binding, the spontaneous folding rate of WT increases by >250 times that of cpN42, which is unprecedented and the highest for any CP to date. On the other hand, zinc-binding reduces the spontaneous unfolding rate of cpN42 by ~100 times, making the WT and CP azurins unfold at similar rates. Our study demonstrates metal binding as a novel way to modulate the unfolding and folding rates of CPs compared to their WT counterparts. We hope our study increases the understanding of the effect of CP on the folding mechanism and energy landscape of metalloproteins.

Ribonuclease T2 represents a distinct circularly permutated version of the BECR RNases.

Detection of homologous relationships among proteins and understanding their mechanisms of diversification are major topics in the fields of protein science, bioinformatics, and phylogenetics. Recent developments in sequence/profile-based and structural similarity-based methods have greatly facilitated the unification and classification of many protein families into superfamilies or folds, yet many proteins remain unclassified in current protein databases. As one of the three earliest identified RNases in biology, ribonuclease T2, also known as RNase I in Escherichia coli, RNase Rh in fungi, or S-RNase in plant, is thought to be an ancient RNase family due to its widespread distribution and distinct structure. In this study, we present evidence that RNase T2 represents a circularly permutated version of the BECR (Barnase-EndoU-Colicin E5/D-RelE) fold RNases. This subtle relationship cannot be detected by traditional methods such as sequence/profile-based comparisons, structure-similarity searches, and circular permutation detections. However, we were able to identify the structural similarity using rational reconstruction of a theoretical RNase T2 ancestor via a reverse circular permutation process, followed by structural modeling using AlphaFold2, and structural comparisons. This relationship is further supported by the fact that RNase T2 and other typical BECR RNases, namely Colicin D, RNase A, and BrnT, share similar catalytic site configurations, all involving an analogous set of conserved residues on the α0 helix and the ß4 strand of the BECR fold. This study revealed a hidden root of RNase T2 in bacterial toxin systems and demonstrated that reconstruction and modeling of ancestral topology is an effective strategy to identify remote relationship between proteins.

SeqCP: A sequence-based algorithm for searching circularly permuted proteins.

Circular permutation (CP) is a protein sequence rearrangement in which the amino- and carboxyl-termini of a protein can be created in different positions along the imaginary circularized sequence. Circularly permutated proteins usually exhibit conserved three-dimensional structures and functions. By comparing the structures of circular permutants (CPMs), protein research and bioengineering applications can be approached in ways that are difficult to achieve by traditional mutagenesis. Most current CP detection algorithms depend on structural information. Because there is a vast number of proteins with unknown structures, many CP pairs may remain unidentified. An efficient sequence-based CP detector will help identify more CP pairs and advance many protein studies. For instance, some hypothetical proteins may have CPMs with known functions and structures that are informative for functional annotation, but existing structure-based CP search methods cannot be applied when those hypothetical proteins lack structural information. Despite the considerable potential for applications, sequence-based CP search methods have not been well developed. We present a sequence-based method, SeqCP, which analyzes normal and duplicated sequence alignments to identify CPMs and determine candidate CP sites for proteins. SeqCP was trained by data obtained from the Circular Permutation Database and tested with nonredundant datasets from the Protein Data Bank. It shows high reliability in CP identification and achieves an AUC of 0.9. SeqCP has been implemented into a web server available at: http://pcnas.life.nthu.edu.tw/SeqCP/.

Genetically encoded fluorescent biosensors for GPCR research.

G protein-coupled receptors (GPCRs) regulate a wide range of physiological and pathophysiological cellular processes, thus it is important to understand how GPCRs are activated and function in various cellular contexts. In particular, the activation process of GPCRs is dynamically regulated upon various extracellular stimuli, and emerging evidence suggests the subcellular functions of GPCRs at endosomes and other organelles. Therefore, precise monitoring of the GPCR activation process with high spatiotemporal resolution is required to investigate the underlying molecular mechanisms of GPCR functions. In this review, we will introduce genetically encoded fluorescent biosensors that can precisely monitor the real-time GPCR activation process in live cells. The process includes the binding of extracellular GPCR ligands, conformational change of GPCR, recruitment of G proteins or ß-arrestin, GPCR internalization and trafficking, and the GPCR-related downstream signaling events. We will introduce fluorescent GPCR biosensors based on a variety of strategies such as fluorescent resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), circular permuted fluorescent protein (cpFP), and nanobody. We will discuss the pros and cons of these GPCR biosensors as well as their applications in GPCR research.

An Improved Genetically Encoded Fluorescent cAMP Indicator for Sensitive cAMP Imaging and Fast Drug Screening.

Cyclic adenosine 3',5'-monophosphate (cAMP) is an important intracellular second messenger molecule downstream of many G protein-coupled receptors (GPCRs). Fluorescence imaging with bright and sensitive cAMP indicators allows not only dissecting the spatiotemporal dynamics of intracellular cAMP, but also high-content screening of compounds against GPCRs. We previously reported the high-performance circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1. Here, we developed improved G-Flamp1 variants G-Flamp2 and G-Flamp2b. Compared to G-Flamp1, G-Flamp2 exhibited increased baseline fluorescence (1.6-fold) and larger fluorescence change (ΔF/F0) (1,300% vs. 1,100%) in HEK293T cells, while G-Flamp2b showed increased baseline fluorescence (3.1-fold) and smaller ΔF/F0 (400% vs. 1,100%). Furthermore, live cell imaging of mitochondrial matrix-targeted G-Flamp2 confirmed cytosolic cAMP was able to enter the mitochondrial matrix. G-Flamp2 imaging also showed that adipose tissue extract activated the Gi protein-coupled orphan GPCR GPR50 in HEK293T cells. Taken together, our results showed that the high-performance of G-Flamp2 would facilitate sensitive intracellular cAMP imaging and activity measurement of compounds targeting GPCR-cAMP signaling pathway during early drug development.

Expression and Purification of BsaXI Restriction Endonuclease and Engineering New Specificity From BsaXI Specificity Subunit.

It is stated that BsaXI is a Type IIB restriction endonuclease (REase) that cleaves both sides of its recognition sequence 5'↓N9 AC N5 CTCC N10↓ 3' (complement strand 5' ↓N7 GGAG N5 GT N12↓ 3'), creating 3-base 3' overhangs. Here we report the cloning and expression of bsaXIS and bsaXIRM genes in Escherichia coli. The BsaXI activity was successfully reconstituted by mixing the BsaXI RM fusion subunit with the BsaXI S subunit and the enzyme complex further purified by chromatography over 6 columns. As expected, the S subunit consisted of two subdomains encoding TRD1-CR1 [target recognition domain (TRD), conserved region (CR)] for 5' AC 3', and TRD2-CR2 presumably specifying 5' CTCC 3'. TRD1-CR1 (TRD2-CR2 deletion) or duplication of TRD1 (TRD1-CR1-TRD1-CR2) both generated a new specificity 5' AC N5 GT 3' when the S variants were complexed with the RM subunits. The circular permutation of TRD1 and TRD2, i.e., the relocation of TRD2-CR2 to the N-terminus and TRD1-CR1 to the C-terminus generated the same specificity with the RM subunits, although some wobble cleavage was detected. The TRD2 domain in the BsaXI S subunit can be substituted by a close homolog (∼59% sequence identity) and generated the same specificity. However, TRD2-CR2 domain alone failed to express in E. coli, but CR1-TRD2-CR2 protein could be expressed and purified which showed partial nicking activity with the RM subunits. This work demonstrated that like Type I restriction systems, the S subunit of a Type IIB system could also be manipulated to create new specificities. The genome mining of BsaXI TRD2 homologs in GenBank found more than 36 orphan TRD2 homologs, implying that quite a few orphan TRD2s are present in microbial genomes that may be potentially paired with other TRDs to create new restriction specificities.

Application of Restriction Free (RF) Cloning in Circular Permutation.

The restriction free (RF) cloning has emerged as one of the highly efficient techniques in the area of genetic engineering. RF cloning has wide range of applications in plasmid DNA manipulation including cloning of a single gene, simultaneous assembly of multiple DNA fragments, and mutagenesis from single to multiple simultaneous alterations of a target DNA. Recently, we have developed a new technique of circular permutation using RF cloning. Circular permutation is widely used to investigate the mechanisms of protein folding and function. Previously, restriction enzyme based cloning was used to introduce circular permutation. Our RF cloning method made the protocol faster and more cost-effective. In this chapter, we describe a step-by-step protocol for generating circular permutants using RF methodology.

Circularly Permuted Far-Red Fluorescent Proteins.

The color palette of genetically encoded fluorescent protein indicators (GEFPIs) has expanded rapidly in recent years. GEFPIs with excitation and emission within the "optical window" above 600 nm are expected to be superior in many aspects, such as enhanced tissue penetration, reduced autofluorescence and scattering, and lower phototoxicity. Circular permutation of fluorescent proteins (FPs) is often the first step in the process of developing single-FP-based GEFPIs. This study explored the tolerance of two far-red FPs, mMaroon1 and mCarmine, towards circular permutation. Several initial constructs were built according to previously reported circularly permuted topologies for other FP analogs. Mutagenesis was then performed on these constructs and screened for fluorescent variants. As a result, five circularly permuted far-red FPs (cpFrFPs) with excitation and emission maxima longer than 600 nm were identified. Some displayed appreciable brightness and efficient chromophore maturation. These cpFrFPs variants could be intriguing starting points to further engineer far-red GEFPIs for in vivo tissue imaging.

CirPred, the first structure modeling and linker design system for circularly permuted proteins.

BACKGROUND: This work aims to help develop new protein engineering techniques based on a structural rearrangement phenomenon called circular permutation (CP), equivalent to connecting the native termini of a protein followed by creating new termini at another site. Although CP has been applied in many fields, its implementation is still costly because of inevitable trials and errors. RESULTS: Here we present CirPred, a structure modeling and termini linker design method for circularly permuted proteins. Compared with state-of-the-art protein structure modeling methods, CirPred is the only one fully capable of both circularly-permuted modeling and traditional co-linear modeling. CirPred performs well when the permutant shares low sequence identity with the native protein and even when the permutant adopts a different conformation from the native protein because of three-dimensional (3D) domain swapping. Linker redesign experiments demonstrated that the linker design algorithm of CirPred achieved subangstrom accuracy. CONCLUSIONS: The CirPred system is capable of (1) predicting the structure of circular permutants, (2) designing termini linkers, (3) performing traditional co-linear protein structure modeling, and (4) identifying the CP-induced occurrence of 3D domain swapping. This method is supposed helpful for broadening the application of CP, and its web server is available at http://10.life.nctu.edu.tw/CirPred/ and http://lo.life.nctu.edu.tw/CirPred/ .

PROFICS: A bacterial selection system for directed evolution of proteases.

Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial selection system, which enables the optimization of proteases for biotechnology, therapeutics or diagnosis in a simple overnight process. During the PROFICS process, proteases are selected for their ability to specifically cut a tag from a reporter enzyme and leave a native N-terminus. Precise and efficient cleavage after the recognition sequence reverses the phenotype of an Escherichia coli knockout strain deficient in an essential enzyme of pyrimidine synthesis. A toolbox was generated to select for proteases with different preferences for P1' residues (the residue immediately following the cleavage site). The functionality of PROFICS is demonstrated with viral proteases and human caspase-2. PROFICS improved caspase-2 activity up to 25-fold after only one round of mutation and selection. Additionally, we found a significantly improved tolerance for all P1' residues caused by a mutation in a substrate interaction site. We showed that this improved activity enables cells containing the new variant to outgrow cells containing all other mutants, facilitating its straightforward selection. Apart from optimizing enzymatic activity and P1' tolerance, PROFICS can be used to reprogram specificities, erase off-target activity, optimize expression via tags/codon usage, or even to screen for potential drug-resistance-conferring mutations in therapeutic targets such as viral proteases in an unbiased manner.

Circularly-Permuted Pistol Ribozyme: A Synthetic Ribozyme Scaffold for Mammalian Riboswitches.

A small molecule-responsive self-cleaving ribozyme (aptazyme) embedded in the untranslated region of an mRNA functions as a riboswitch that allows chemical regulation of gene expression in mammalian cells. Aptazymes are engineered by fusing a self-cleaving ribozyme with an RNA aptamer that recognizes a small molecule so that the ribozyme is either activated or inhibited in the presence of the small molecule. However, the variety of aptamers, ribozymes, and aptazyme design strategies suitable for mammalian riboswitch applications is still limited. This work focuses on a new ribozyme scaffold for engineering aptazymes and riboswitches that function in mammalian cells. We investigated circularly permuted variants of the pistol ribozyme class (CPP) as a synthetic ribozyme scaffold for mammalian riboswitch applications. Through semirational design and high-throughput screening, we designed guanine and tetracycline activated riboswitches based on three distinct aptazyme architectures, resulting in riboswitches with ON/OFF ratios as high as 8.6. Our work adds CPP to the limited ribozyme scaffold toolbox for mammalian synthetic biology applications and highlights the opportunities in exploring ribozymes beyond natural motifs.

cAMP Biosensors Based on Genetically Encoded Fluorescent/Luminescent Proteins.

Cyclic adenosine monophosphate (cAMP) plays a key role in signal transduction pathways as a second messenger. Studies on the cAMP dynamics provided useful scientific insights for drug development and treatment of cAMP-related diseases such as some cancers and prefrontal cortex disorders. For example, modulation of cAMP-mediated intracellular signaling pathways by anti-tumor drugs could reduce tumor growth. However, most early stage tools used for measuring the cAMP level in living organisms require cell disruption, which is not appropriate for live cell imaging or animal imaging. Thus, in the last decades, tools were developed for real-time monitoring of cAMP distribution or signaling dynamics in a non-invasive manner. Genetically-encoded sensors based on fluorescent proteins and luciferases could be powerful tools to overcome these drawbacks. In this review, we discuss the recent genetically-encoded cAMP sensors advances, based on single fluorescent protein (FP), Föster resonance energy transfer (FRET), single luciferase, and bioluminescence resonance energy transfer (BRET) for real-time non-invasive imaging.

Genetically Encoded Biosensors Based on Fluorescent Proteins.

Genetically encoded biosensors based on fluorescent proteins (FPs) allow for the real-time monitoring of molecular dynamics in space and time, which are crucial for the proper functioning and regulation of complex cellular processes. Depending on the types of molecular events to be monitored, different sensing strategies need to be applied for the best design of FP-based biosensors. Here, we review genetically encoded biosensors based on FPs with various sensing strategies, for example, translocation, fluorescence resonance energy transfer (FRET), reconstitution of split FP, pH sensitivity, maturation speed, and so on. We introduce general principles of each sensing strategy and discuss critical factors to be considered if available, then provide representative examples of these FP-based biosensors. These will help in designing the best sensing strategy for the successful development of new genetically encoded biosensors based on FPs.