Fabrication of rigidity and space variable protein oligomers with two peptide linkers.
ABSTRACT: Supramolecular protein assemblies have garnered considerable interest due to their potential in diverse fields with unrivaled attainable functionalities and structural accuracy. Despite significant advances in protein assembly strategies, inserting long linkers with varied lengths and rigidity between assembling protein building blocks remains extremely difficult. Here we report a series of green fluorescent protein (GFP) oligomers, where protein building blocks were linked via two independent peptide strands. Assembling protein units for this two-peptide assembly were designed by flopped fusion of three self-assembling GFP fragments with two peptide linkers. Diverse flexible and rigid peptide linkers were successfully inserted into high-valent GFP oligomers. In addition, oligomers with one flexible linker and one rigid linker could also be fabricated, allowing more versatile linker rigidity control. Linker length could be varied from 10 amino acids (aa) even up to 76 aa, which is the longest among reported protein assembling peptide linkers. Discrete GFP oligomers containing diverse linkers with valencies between monomers to decamers were monodispersely purified by gel elution. Furthermore, various functional proteins could be multivalently fused to the present GFP oligomers. Binding assays, size exclusion chromatography, dynamic light scattering, circular dichroism, differential scanning calorimetry, and transmission electron microscopy suggested circular geometries of the GFP oligomers and showed distinct characteristics of GFP oligomers with length/rigidity varied linkers. Lastly, a surface binding study indicated that more spaced oligomeric binding modules offered more effective multivalent interactions than less spaced modules.
Project description:Inspired by natural multienzyme complexes, many types of artificial multienzyme complexes have recently been constructed. We previously constructed a self-assembled complex of a bacterial cytochrome P450 and its ferredoxin and ferredoxin reductase partners using heterotrimerization of proliferating cell nuclear antigen (PCNA) from Sulfolobus solfataricus. In this study, we inserted different peptide linkers between ferredoxin and the PCNA subunit, and examined the effect on activity of the self-assembled multienzyme complex. Although the activity was affected by the lengths of both the rigid poly-L-proline-rich linkers and the flexible Gly4-Ser repeating linkers, the poly-L-proline-rich linkers provided the greatest activity enhancement. The optimized poly-L-proline-rich linker enhanced the activity 1.9-fold compared with the GGGGSLVPRGSGGGGS linker used in the previously reported complex, while the Gly4-Ser repeating linkers, (G4S)n (n = 1-6), did not yield higher activity than the maximum activity by the optimized poly-L-proline linker. Both the rigidity/flexibility and length of the peptide linker were found to be important for enhancing the overall activity of the multienzyme complex.
Project description:Engineered fusion proteins containing two or more functional polypeptides joined by a peptide or protein linker are important for many fields of biological research. The separation distance between functional units can impact epitope access and the ability to bind with avidity; thus the availability of a variety of linkers with different lengths and degrees of rigidity would be valuable for protein design efforts. Here, we report a series of designed structured protein linkers incorporating naturally occurring protein domains and compare their properties to commonly used Gly4Ser repeat linkers. When incorporated into the hinge region of an immunoglobulin G (IgG) molecule, flexible Gly4Ser repeats did not result in detectable extensions of the IgG antigen-binding domains, in contrast to linkers including more rigid domains such as ?2-microglobulin, Zn-?2-glycoprotein and tetratricopeptide repeats. This study adds an additional set of linkers with varying lengths and rigidities to the available linker repertoire, which may be useful for the construction of antibodies with enhanced binding properties or other fusion proteins.
Project description:In this work we addressed the problem how to fabricate self-assembling tubular nanostructures displaying target recognition functionalities. Bacterial flagellar filaments, composed of thousands of flagellin subunits, were used as scaffolds to display single-domain antibodies (nanobodies) on their surface. As a representative example, an anti-GFP nanobody was successfully inserted into the middle part of flagellin replacing the hypervariable surface-exposed D3 domain. A novel procedure was developed to select appropriate linkers required for functional internal insertion. Linkers of various lengths and conformational properties were chosen from a linker database and they were randomly attached to both ends of an anti-GFP nanobody to facilitate insertion. Functional fusion constructs capable of forming filaments on the surface of flagellin-deficient host cells were selected by magnetic microparticles covered by target GFP molecules and appropriate linkers were identified. TEM studies revealed that short filaments of 2-900?nm were formed on the cell surface. ITC and fluorescent measurements demonstrated that the fusion protein exhibited high binding affinity towards GFP. Our approach allows the development of functionalized flagellar nanotubes against a variety of important target molecules offering potential applications in biosensorics and bio-nanotechnology.
Project description:In the transformation of multiple genes, gene fusion is an attractive alternative to other methods, including sexual crossing, re-transformation, and co-transformation, among others. The 2A peptide from the foot-and-mouth disease virus (FMDV) causes the co-translational "cleavage" of polyprotein and operates in a wide variety of eukaryotic cells. LP4, a linker peptide that originates from a natural polyprotein occurring in the seed of Impatiens balsamina, can be split between the first and second amino acids in post-translational processing. LP4/2A is a hybrid linker peptide that contains the first nine amino acids of LP4 and 20 amino acids of 2A. The three linkers have been used as a suitable technique to link the expression of genes in some transgenic plants, but to date the cleavage efficiency of three linkers have not been comprehensively demonstrated in the same transformation system, especially in the staple crop. To verify the functions of 2A, LP4, and LP4/2A linker peptides in transgenic maize, six fusion protein vectors that each encoded a single open reading frame (ORF) incorporating two report genes, Green Fluorescent Protein (GFP) and ?-glucuronidase (GUS), separated by 2A (or modified 2A), LP4 or LP4/2A were assembled to compare the cleavage efficiency of the three linkers in a maize transient expression system. The results demonstrated the more protein production and higher cleavage splicing efficiency with the polyprotein construct linked by the LP4/2A peptide than those of the polyprotein constructs linked by 2A or LP4 alone. Seven other fusion proteins that each encoded a single ORF incorporating two different genes GFP and Red Fluorecent Protein (RFP) with different signal peptides were assembled to study the subcellular localization of genes linked by LP4/2A. The subcellular localization experiments suggested that both types of signal peptide, co-translational and post-translational, could lead their proteins to the target localization in maize protoplast transformed by LP4/2A polyprotein construct and it implied the LP4/2A linker peptide could alleviate the inhibition of 2A processing by the carboxy-terminal region of upstream protein of 2A when translocated into the ER.
Project description:Protein fusions are of fundamental importance in the study of cellular biology and the elucidation of cell signaling pathways, and the importance of linkers for the proper function of protein fusions is well documented in the literature. However, there are few convenient methods available to experimentalists for the systematic implementation of linkers in protein fusions. In this work, we describe a universal approach to the creation and insertion of focused linker libraries into protein fusions. This process, deemed protaTETHER, utilizes reiterative oligomer design, PCR-mediated linker library generation, and restriction enzyme-free cloning methods in a straightforward, three-step cloning process. We utilize a fusion between the catalytic subunit of cAMP-dependent protein kinase A (PKAc) and green fluorescent protein (GFP) for the development of the protaTETHER method, implementing small linker libraries that vary by length, sequence, and predicted secondary structural elements. We analyze the impact of linker length and sequence on the expression, activity, and subcellular localization of the PKAc-GFP fusions, and use these results to select a PKAc-GFP fusion construct with robust expression and enzymatic activity. Based upon the results of both biochemical experiments and molecular modeling, we determine that linker flexibility is more important than linker length for optimal kinase activity and expression.
Project description:In this investigation, we report evidence for energy transfer in new protein-based megamolecules with tunable distances between donor and acceptor fluorescent proteins. The megamolecules used in this work are monodisperse oligomers, with molecular weights of ?100-300 kDa and lengths of ?5-20 nm, and are precisely defined structures of fusion protein building blocks and covalent cross-linkers. Such structures are promising because the study of energy transfer in protein complexes is usually difficult in this long length regime due to synthetic limitations. We incorporated fluorescent proteins into the megamolecule structure and varied the separation distance between donor and acceptor by changing the length of the cross-linker in dimer conjugates and inserting nonfluorescent spacer proteins to create oligomers. Two-photon absorption measurements demonstrated strong coupling between donor and acceptor dipoles in the megamolecules. For the dimer systems, no effect of the cross-linker length on energy transfer efficiency was observed with the steady-state fluorescence investigation. However, for the same dimer conjugates, energy transfer rates decreased upon increasing cross-linker length, as evaluated by fluorescence up-conversion. Molecular dynamics simulations were used to rationalize the results, providing quantitative agreement between measured and calculated energy transfer lengths for steady-state results, and showing that the differences between the time-resolved and steady-state measurements arise from the long time scale for large-scale fluctuations in the megamolecule structure. Our results show an increase in energy transfer length with increasing megamolecule size. This is evidence for long-range energy transfer in large protein megamolecules.
Project description:Peptide inter-domain linkers are peptide segments covalently linking two adjacent domains within a protein. Linkers play a variety of structural and functional roles in naturally occurring proteins. In this work we analyze the sequence properties of the predicted linker regions of the bacterial transcriptional regulators belonging to the recently discovered MocR subfamily of the GntR regulators. Analyses were carried out on the MocR sequences taken from the phyla Actinobacteria, Firmicutes, Alpha-, Beta- and Gammaproteobacteria. The results suggest that MocR linkers display phylum-specific characteristics and unique features different from those already described for other classes of inter-domain linkers. They show an average length significantly higher: 31.8 ± 14.3 residues reaching a maximum of about 150 residues. Compositional propensities displayed general and phylum-specific trends. Pro is dominating in all linkers. Dyad propensity analysis indicate Pro-Pro as the most frequent amino acid pair in all linkers. Physicochemical properties of the linker regions were assessed using amino acid indices relative to different features: in general, MocR linkers are flexible, hydrophilic and display propensity for β-turn or coil conformations. Linker sequences are hypervariable: only similarities between MocR linkers from organisms related at the level of species or genus could be found with sequence searches. The results shed light on the properties of the linker regions of the new MocR subfamily of bacterial regulators and may provide knowledge-based rules for designing artificial linkers with desired properties.
Project description:Paramyxoviruses include several insidious and ubiquitous pathogens of humans and animals, with measles virus (MeV) being a prominent one. The MeV membrane fusion apparatus consists of a receptor binding protein (hemagglutinin [H]) tetramer and a fusion (F) protein trimer. Four globular MeV H heads are connected to a tetrameric stalk through flexible linkers. We sought here to characterize the function of a 17-residue H-head segment proximal to the stalk that was unresolved in all five MeV H-head crystal or cocrystal structures. In particular, we assessed whether its primary sequence and length are critical for proper protein oligomerization and intracellular transport or for membrane fusion triggering. Extensive alanine substitutions had no effect on fusion triggering, suggesting that sequence identity is not critical for this function. Excessive shortening of this segment reduced or completely abrogated fusion trigger function, while length compensation restored it. We then characterized the mechanism of function loss. Mutated H proteins were efficiently transported to the cell surface, but certain alterations enhancing linker flexibility resulted in accumulation of high-molecular-weight H oligomers. Some oligomers had reduced fusion trigger capacity, while others retained this function. Thus, length and rigidity of the unresolved head segment favor proper H tetramerization and counteract interactions between subunits from different tetramers. The structurally unresolved H-head segment, together with the top of the stalk, may act as a leash to provide the right degree of freedom for the heads of individual tetramers to adopt a triggering-permissive conformation while avoiding improper contacts with heads of neighboring tetramers.Understanding the molecular mechanism of membrane fusion triggering may allow development of new antiviral strategies. The fusion apparatus of paramyxoviruses consists of a receptor binding tetramer and a fusion protein trimer. Structural analyses of the receptor binding hemagglutinin-neuraminidases of certain paramyxoviruses suggest that fusion triggering is preceded by relocation of its head domains, facilitated by flexible linkers. Having noted a structurally unresolved 17-residue segment linking the globular heads to the tetrameric stalk of the measles virus hemagglutinin (H), we asked whether and how it may facilitate membrane fusion triggering. We conclude that, together with the top of the stalk, the flexible linker keeps H heads on a leash long enough to adopt a triggering-permissive conformation but short enough to limit roaming and improper contacts with heads of neighboring tetramers. All morbillivirus H-protein heads appear to be connected to their stalks through a "leash," suggesting a conserved triggering mechanism.
Project description:MRI contrast agents providing very high relaxivity values can be obtained through the attachment of multiple gadolinium(III) complexes to the interior surfaces of genome-free viral capsids. In previous studies, the contrast enhancement was predicted to depend on the rigidity of the linker attaching the MRI agents to the protein surface. To test this hypothesis, a new set of Gd-hydroxypyridonate based MRI agents was prepared and attached to genetically introduced cysteine residues through flexible and rigid linkers. Greater contrast enhancements were seen for MRI agents that were attached via rigid linkers, validating the design concept and outlining a path for future improvements of nanoscale MRI contrast agents.
Project description:Polyvalent antigen display is an effective strategy to enhance the immunogenicity of subunit vaccines by clustering them in an array-like manner on a scaffold system. This strategy results in a higher local density of antigens, increased high avidity interactions with B cells and other antigen presenting cells, and therefore a more effective presentation of vaccine antigens. In this study, we used lumazine synthase (LS), an icosahedral symmetry capsid derived from Bacillus anthracis, as a scaffold to present 60 copies of a linear B cell epitope (PB10) from the ricin toxin fused to the C terminus of LS via four different linkers. We then investigated the effects of linker length, linker rigidity and formaldehyde crosslinking on the protein assembly, conformational integrity, thermal stability, in vitro antibody binding, and immunogenicity in mice. Fusion of the PB10 peptide onto LS, with varying linker lengths, did not affect protein assembly, thermal stability or exposure of the epitope, but had a minor impact on protein conformation. Formaldehyde crosslinking considerably improved protein thermal stability with only minor impact on protein conformation. All LS_PB10 constructs, when administered to mice by injection without adjuvant, elicited measurable anti-ricin serum IgG titers, although the titers were not sufficient to confer protection against a 10× lethal dose ricin challenge. This work sheds light on the biophysical properties, immunogenicity and potential feasibility of LS from B. anthracis as a scaffold system for polyvalent antigen display.