Green Fluorescent Protein Mutants Research Articles

Na+/H+ exchanger isoform 1 (NHE1) confers resistance to apoptosis in pulmonary arterial smooth muscle cells (PASMCs)

Pulmonary hypertension (PH) is a condition caused by remodeling of the lung vasculature due to abnormal cell turnover involving increased cell proliferation and resistance to apoptosis. The mechanisms controlling abnormal cell growth and survival are incompletely understood. We previously found that Na+/H+ exchanger (NHE) activity is increased in PASMCs from PH patients and the Sugen Hypoxia (SuHx) animal model of PH. NHE isoform 1 (NHE1) is a membrane protein that regulates intracellular pH (pHi) and cell volume via export of H+ and import of Na+. This protein also has a cytosolic tail region shown to be involved in non-canonical functions such as cytoskeletal interactions and cell signaling via protein-protein interactions. NHE1 has been associated with increased PASMC proliferation; however, it is unclear if increased NHE activity, dysregulation of pHi or cytoskeletal interactions are involved in resistance to apoptosis. In this study, we tested the hypothesis that increased expression of NHE1 leads to apoptosis resistance in PASMCs via dysregulation of pHi. We first used the rat SuHx model of PH in which male Wistar rats receive a single subcutaneous injection of the vascular endothelial growth factor receptor inhibitor, SU5416, followed by 3 wks of exposure to hypoxia (10% O2) and 2 wks of normoxia. PASMCs were isolated from distal pulmonary arteries of control and SuHx Wistar rats and treated with the NHE inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 1 uM), or vehicle for 24 h before analyzing cell apoptosis via Hoechst staining under baseline and stimulated conditions (250 uM H2O2). Endoplasmic reticulum stress was measured using the fluorescent stain, Thioflavin T (ThT). In some experiments, cells from control rats were infected with adenovirus containing green fluorescent protein (AdGFP; control for infection), wild-type NHE1 (AdNHE1-WT) or NHE1 mutants: AdNHE1-E262I lacking ion translocation activity and AdNHE1-EM lacking the ezrin binding site which prevents cytoskeletal interactions. NHE1 protein expression and localization were assessed via immunoblotting (total and surface proteins). Effcacy of Na+/H+ translocation was assessed from Na+-dependent recovery following an ammonium pulse. At baseline, despite increased ThT levels in SuHx cells, apoptosis was not different compared to control cells, indicating SuHx cells have suppressed apoptotic response to increased ER stress. Following treatment with EIPA, apoptosis was increased in SuHx cells, possibly indicating they became sensitized to the high levels of ER stress and were able to proceed into the apoptotic pathway. Similarly, following treatment with H2O2, SuHx PASMCs showed resistance to apoptosis compared to control cells, which was alleviated with pre-treatment with EIPA. In control cells, wild-type NHE1 and NHE1 mutants were expressed in similar amounts which led to an expected increase in NHE activity for WT and NHE1-EM, but not for NHE1-E262I when compared to GFP. We found that enhanced expression of NHE1-WT was suffcient to inhibit apoptosis following treatment with H2O2. Interestingly, both NHE1 mutants also significantly decreased PASMC apoptosis following H2O2 stimulation. Our results indicate NHE1 is necessary for apoptosis resistance in PASMCs from animals with PH and that increased expression is suffcient to confer resistance to apoptosis in control PASMCs. Additionally, these results suggest neither pHi regulation nor interaction between NHE1 and ezrin are essential for NHE1-induced apoptosis resistance, indicating another mechanism through which NHE1 confers resistance to apoptosis in PH. Based on these findings, we speculate that development of NHE1-related therapeutic targets could potentially restore cell apoptosis and reverse disease. AHA 23PRE1022720, HL159906, HL073859. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Efficient disruption of CmHk1 using CRISPR/Cas9 ribonucleoprotein delivery in Cordyceps militaris.

Cordyceps militaris, an entomopathogenic ascomycete, produces edible medicinal mushrooms known to have medicinal and therapeutic functions. To develop the genetic transformation system in C. militaris, green fluorescent protein (GFP) mutants of C. militaris were generated by PEG-mediated protoplast transformation. The CRISPR/Cas9 ribonucleoprotein (RNP) targeting the class III histidine kinase of C. militaris (CmHk1) was then delivered into protoplasts of C. militaris through the transformation system. Mutations induced by the RNP in selected mutants were detected: 1nt deletion (6 mutants), 3 nt deletion with substitution of 1nt (1 mutant), insertion of 85 nts (1 mutant), 41 nts (2 mutants), and 35 nts (5 mutants). An in vitro sensitivity assay of the mutants indicated that knockout of CmHk1 reduced sensitivity to two fungicides, iprodione and fludioxonil, but increased sensitivity to osmotic stresses compared to the wild type. Summing up, the CRISPR/Cas9 RNP delivery system was successfully developed, and our results revealed that CmHk1 was involved in the fungicide resistance and osmotic stress in C. militaris.

Recombinant Adeno-Associated Virus-Mediated Editing of the G551D Cystic Fibrosis Transmembrane Conductance Regulator Mutation in Ferret Airway Basal Cells.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), a chronic disease that affects multiple organs, including the lung. We developed a CF ferret model of a scarless G551→D substitution in CFTR (CFTRG551D-KI), enabling approaches to correct this gating mutation in CF airways via gene editing. Homology-directed repair (HDR) was tested in Cas9-expressing CF airway basal cells (Cas9-GKI) from this model, as well as reporter basal cells (Y66S-Cas9-GKI) that express an integrated nonfluorescent Y66S-EGFP (enhanced green fluorescent protein) mutant gene to facilitate rapid assessment of HDR by the restoration of fluorescence. Recombinant adeno-associated virus (rAAV) vectors were used to deliver two DNA templates and sgRNAs for dual-gene editing at the EGFP and CFTR genes, followed by fluorescence-activated cell sorting of EGFPY66S-corrected cells. When gene-edited airway basal cells were polarized at an air-liquid interface, unsorted and EGFPY66S-corrected sorted populations gave rise to 26.0% and 70.4% CFTR-mediated Cl- transport of that observed in non-CF cultures, respectively. The consequences of gene editing at the CFTRG551D locus by HDR and nonhomologous end joining (NHEJ) were assessed by targeted gene next-generation sequencing (NGS) against a specific amplicon. NGS revealed HDR corrections of 3.1% of G551 sequences in the unsorted population of rAAV-infected cells, and 18.4% in the EGFPY66S-corrected cells. However, the largest proportion of sequences had indels surrounding the CRISPR (clustered regularly interspaced short palindromic repeats) cut site, demonstrating that NHEJ was the dominant repair pathway. This approach to simultaneously coedit at two genomic loci using rAAV may have utility as a model system for optimizing gene-editing efficiencies in proliferating airway basal cells through the modulation of DNA repair pathways in favor of HDR.

Fluorescence Lifetime Imaging of pH along the Secretory Pathway.

Many cellular processes are dependent on correct pH levels, and this is especially important for the secretory pathway. Defects in pH homeostasis in distinct organelles cause a wide range of diseases, including disorders of glycosylation and lysosomal storage diseases. Ratiometric imaging of the pH-sensitive mutant of green fluorescent protein, pHLuorin, has allowed for targeted pH measurements in various organelles, but the required sequential image acquisition is intrinsically slow and therefore the temporal resolution is unsuitable to follow the rapid transit of cargo between organelles. Therefore, we applied fluorescence lifetime imaging microscopy (FLIM) to measure intraorganellar pH with just a single excitation wavelength. We first validated this method by confirming the pH in multiple compartments along the secretory pathway and compared the pH values obtained by the FLIM-based measurements with those obtained by conventional ratiometric imaging. Then, we analyzed the dynamic pH changes within cells treated with Bafilomycin A1, to block the vesicular ATPase, and Brefeldin A, to block endoplasmic reticulum (ER)–Golgi trafficking. Finally, we followed the pH changes of newly synthesized molecules of the inflammatory cytokine tumor necrosis factor-α while they were in transit from the ER via the Golgi to the plasma membrane. The toolbox we present here can be applied to measure intracellular pH with high spatial and temporal resolution and can be used to assess organellar pH in disease models.

A disulphide bond-mediated hetero-dimer of a hemoprotein and a fluorescent protein exhibiting efficient energy transfer.

Artificial protein hetero-dimerization is one of the promising strategies to construct protein-based chemical tools. In this work, cytochrome b 562, an electron transfer hemoprotein, and green fluorescent protein (GFP) mutants with cysteine residues added to their surfaces were conjugated via a pyridyl disulphide-based thiol-disulfide exchange reaction. The eight hetero-dimers, which have cysteine residues at different positions to form the disulphide bonds, were obtained and characterized by gel-electrophoresis, mass spectrometry and size exclusion chromatography. The fluorescence properties of the hetero-dimers were evaluated by fluorescence spectroscopy and fluorescence lifetime measurements. Efficient photoinduced energy transfer from the GFP chromophore to the heme cofactor was observed in each of the hetero-dimers. The energy transfer efficiency is strongly dependent on the cross-linking residues, reaching 96%. Furthermore, the estimated Förster distance and the structure-based maximum possible distances of the donor and acceptor suggest that one of the hetero-dimers has a rigid protein-protein structure with favourable properties for energy transfer. The disulphide bond-mediated protein hetero-dimerization is useful for screening functional protein systems towards further developments.

Targeted mutagenesis in plants using Beet curly top virus for efficient delivery of CRISPR/Cas12a components

Genome editing using CRISPR/Cas is rapidly being developed for gene targeting in eukaryotes including plants. However, gene targeting by homology-directed DNA recombination (HDR) is an infrequent event compared to the dominant DNA repair by non-homologous end-joining. Another bottleneck is the ineffective delivery of CRISPR/Cas components into plant cells. To overcome these constraints, here a geminiviral replicon from Beet curly top virus (BCTV) has been produced with a wide host range and high DNA accumulation capacity for efficient delivery of CRISPR/Cas12a components into plant cells. Initially, a BCTV replicon was prepared after removing the virion sense genes from an infectious full-length clone for agrobacterium mediated infection. This replicon expressed a green fluorescent protein (GFP) marker gene at a high level compared to T-DNA binary vector. In transient assay, the BCTV replicon produced a higher rate of mutagenesis and HDR in the GFP transgene in Nicotiana benthamiana through efficient delivery of CRISPR/Cas12a components compared to the cognate T-DNA control. This was through a range of complete or partial HDR for conversion of GFP into YFP after exchange of a single amino acid (Thr224Tyr) in the target gene. In addition, induced mutagenesis and HDR in the target gene were heritable. Thus, the BCTV replicon provides a new tool for efficient delivery of CRISPR/Cas12a components that could be used in a wide range of dicotyledonous plants. The established GFP to YFP system and the GFP mutant line produced also enable further optimization and understanding of HDR in plants via CRISPR/Cas12a system using geminiviral replicons.

Accurate and efficient structure-based computational mutagenesis for modeling fluorescence levels of Aequorea victoria green fluorescent protein mutants.

A computational mutagenesis technique was used to characterize the structural effects associated with over 46000 single and multiple amino acid variants of Aequorea victoria green fluorescent protein (GFP), whose functional effects (fluorescence levels) were recently measured by experimental researchers. For each GFP mutant, the approach generated a single score reflecting the overall change in sequence-structure compatibility relative to native GFP, as well as a vector of environmental perturbation (EP) scores characterizing the impact at all GFP residue positions. A significant GFP structure-function relationship (P<0.0001) was elucidated by comparing the sequence-structure compatibility scores with the functional data. Next, the computed vectors for GFP mutants were used to train predictive models of fluorescence by implementing random forest (RF) classification and tree regression machine learning algorithms. Classification performance reached 0.93 for sensitivity, 0.91 for precision and 0.90 for balanced accuracy, and regression models led to Pearson's correlation as high as r=0.83 between experimental and predicted GFP mutant fluorescence. An RF model trained on a subset of over 1000 experimental single residue GFP mutants with measured fluorescence was used for predicting the 3300 remaining unstudied single residue mutants, with results complementing known GFP biochemical and biophysical properties. In addition, models trained on the subset of experimental GFP mutants harboring multiple residue replacements successfully predicted fluorescence of the single residue GFP mutants. The models developed for this study were accurate and efficient, and their predictions outperformed those of several related state-of-the-art methods.

Effect of shaking speed on immobilization of cephalosporin C acylase: Correlation between protein distribution and properties of the immobilized enzymes.

During enzyme immobilization, enzyme activity and protein distribution are affected by various factors such as enzyme load, temperature, and pH. In general, two types of protein distribution patterns (heterogeneous or homogeneous) are observed inside a porous carrier, owing to differences in preparation parameters. During the immobilization of a fusion protein (CCApH) of cephalosporin C acylase (CCA) and pHluorin (a pH-sensitive mutant of green fluorescent protein), different shaking speeds induced obvious differences in protein distribution on an epoxy carrier, LX-1000EPC. Enzyme immobilization with a homogeneous distribution pattern was observed at a low shaking speed (120 rpm) with an operational stability of 10 batches at 37°C. The operational stability of an immobilisate with heterogeneous protein distribution prepared at a high shaking speed (200 rpm) was six batches. Given the pH-sensitive characteristics of pHluorin in the fusion protein, the intraparticle pH of CCApH immobilisates during catalysis was monitored using confocal laser scanning microscopy. The microenvironmental pH of the immobilisate with heterogeneous protein distribution sharply decreased by about 2 units; this decrease in the pH may be detrimental to the life-span of immobilized CCA. Thus, this work demonstrates the good operational stability of pH-sensitive proton-forming immobilized enzymes with homogeneous protein distribution.

Effect of Protein Surface Charge Distribution on Protein–Polyelectrolyte Complexation

Charge anisotropy or the presence of charge patches at protein surfaces has long been thought to shift the coacervation curves of proteins and has been used to explain the ability of some proteins to coacervate on the "wrong side" of their isoelectric point. This work makes use of a panel of engineered superfolder green fluorescent protein mutants with varying surface charge distributions but equivalent net charge and a suite of strong and weak polyelectrolytes to explore this concept. A patchiness parameter, which assessed the charge correlation between points on the surface of the protein, was used to quantify the patchiness of the designed mutants. Complexation between the polyelectrolytes and proteins showed that the mutant with the largest patchiness parameter was the most likely to form complexes, while the smallest was the least likely to do so. The patchiness parameter was found to correlate well with the phase behavior of the protein-polymer mixtures, where both macrophase separation and the formation of soluble aggregates were promoted by increasing the patchiness depending on the polyelectrolyte with which the protein was mixed. Increasing total charge and increasing strength of the polyelectrolyte promote interactions for oppositely charged polyelectrolytes, while charge regulation is also key to interactions for similarly charged polyelectrolytes, which must interact selectively with oppositely charged patches.

Mechanism of Color and Photoacidity Tuning for the Protonated Green Fluorescent Protein Chromophore.

The neutral or A state of the green fluorescent protein (GFP) chromophore is a remarkable example of a photoacid naturally embedded in the protein environment and accounts for the large Stokes shift of GFP in response to near UV excitation. Its color tuning mechanism has been largely overlooked, as it is less preferred for imaging applications than the redder anionic or B state. Past studies, based on site-directed mutagenesis or solvatochromism of the isolated chromophore, have concluded that its color tuning range is much narrower than its anionic counterpart. However, as we performed extensive investigation on more GFP mutants, we found that the color of the neutral chromophore can be more sensitive to protein electrostatics than can the anionic counterpart. Electronic Stark spectroscopy reveals a fundamentally different electrostatic color tuning mechanism for the neutral state of the chromophore that demands a three-form model as compared to that of the anionic state, which requires only two forms ( J. Am. Chem. Soc. 2019, 141, 15250-15265). Specifically, an underlying zwitterionic charge-transfer state is required to explain its sensitivity to electrostatics. As the Stokes shift is tightly linked to excited-state proton transfer (ESPT) of the protonated chromophore, we infer design principles of the GFP chromophore as a photoacid through the color tuning mechanisms of both protonation states. The three-form model could also be applied to similar biological and nonbiological dyes and complements the failure of the two-form model for donor-acceptor systems with localized ground-state electronic distributions.

Controlling the DNA Hybridization Chain Reaction

A novel method for controlling the oligomerization of metastable DNA hairpins using the hybridization chain reaction (HCR) is reported. Control was achieved through the introduction of a base-pair mismatch in the duplex of the hairpins. The mismatch modification allows one to kinetically differentiate initiation versus propagation events, leading to DNA oligomers up to 10 monomers long and improving dispersities from 2.5 to 1.3-1.6. Importantly, even after two consecutive chain extensions, dispersity remained unaffected, showing that well-defined block co-oligomers can be achieved. As a proof-of-concept, this technique was then applied to hairpin monomers functionalized with a mutant green fluorescent protein to prepare protein oligomers. Taken together, this work introduces an effective method for controlling living macromolecular HCR oligomerization in a manner analogous to the controlled polymerization of small molecules.

How to Become a Protein Crystallographer in a Nanoscience Lab

Download : Download high-res image (240KB) Download : Download full-size image Peter H. Winegar obtained his BS in chemistry at Michigan Technological University in 2017 under the mentorship of Prof. Loredana Valenzano. Currently, he is a PhD candidate in chemistry at Northwestern University and is advised by Prof. Chad A. Mirkin. Peter’s current research includes the directed crystallization and oligomerization of proteins using DNA interactions. He serves on the board of Northwestern’s Phi Lambda Upsilon Chemistry Honor Society and participates in science outreach to elementary students through Northwestern’s Science in the Classroom program.

DNA-Directed Protein Packing within Single Crystals

Designed DNA-DNA interactions are investigated for their ability to modulate protein packing within single crystals of mutant green fluorescent proteins (mGFPs) functionalized with a single DNA strand (mGFP-DNA). We probe the effects of DNA sequence, length, and protein-attachment position on the formation and protein packing of mGFP-DNA crystals. Notably, when complementary mGFP-DNA conjugates are introduced to one another, crystals form with nearly identical packing parameters, regardless of sequence if the number of bases is equivalent. DNA complementarity is essential, because experiments with non-complementary sequences produce crystals with different protein arrangements. Importantly, the DNA length and its position of attachment on the protein markedly influence the formation of and protein packing within single crystals. This work shows how designed DNA interactions can be used to influence the growth and packing in X-ray diffraction quality protein single crystals and is thus an important step forward in protein crystal engineering.

An Antiviral Drug Screening Platform with a FRET Biosensor for Measurement of Arenavirus Z Assembly.

The smallest arenavirus gene product, Z protein, plays critical roles in the virus life cycle. Z is the major driving force of budding and particle production because of a unique property that defines self-assembly. In addition to the roles in budding, Z also participates in the suppression of type I interferon production to evade host antiviral immunity. Therefore, Z and its assembled form are an attractive drug target for arenaviral hemorrhagic fever, such as Lassa fever. Here, we developed a biosensor that enabled the evaluation of the prototype arenavirus, lymphocytic choriomeningitis virus (LCMV), Z assembly using the principle of Förster resonance energy transfer (FRET). This FRET biosensor consisted of three tandem Z that were sandwiched between super-enhanced cyan-emitting fluorescent protein and variant of a yellow-emitting mutant of green fluorescent protein so that Z-Z intermolecular binding via the really interesting new gene finger domain increased the emission ratio. To identify novel anti-arenavirus compounds, the FRET biosensor was employed to screen the PathogenBox400 for inhibitors of Z assembly in a 96-well plate format. The assay performed well, with a Z'-factor of 0.89, and identified two compounds that decreased the emission ratio of the FRET biosensor in a dose-dependent manner. Of them, the compound, 5,6,7,8-tetrahydro-7-(benzyl)-pyrido[4',3':4,5]thieno[2,3-d]pyrimidin-2,4-diamine, was found to significantly inhibit LCMV propagation in infected cells. Thereby, the present study demonstrated that a novel FRET biosensor incorporating Z assembly built on FRET and named Zabton, was a valuable screening tool to identify anti-arenavirus compounds in the context of inhibition of Z assembly.Key words: Arenavirus, Förster resonance energy transfer, anti-viral drugs, Z protein.

Direct Observation of the Protonation States in the Mutant Green Fluorescent Protein.

Neutron crystallography has been used to elucidate the protonation states for the enhanced green fluorescent protein, which has revolutionized imaging technologies. The structure has a deprotonated hydroxyl group in the fluorescent chromophore. Also, the protonation states of His148 and Thr203, as well as the orientation of a critical water molecule in direct contact with the chromophore, could be determined. The results demonstrate that the deprotonated hydroxyl group in the chromophore and the nitrogen atom ND1 in His148 are charged negatively and positively, respectively, forming an ion pair. The position of the two deuterium atoms in the critical water molecule appears to be displaced slightly toward the acceptor oxygen atoms according to their omit maps. This displacement implies the formation of an intriguing electrostatic potential realized inside of the protein. Our findings provide new insights into future protein design strategies along with developments in quantum chemical calculations.

Anti-viral Effects of Superpositively Charged Mutant of Green Fluorescent Protein.

Supercharged GFP proteins were known as effective carriers for delivery of macromolecules into eukaryotic cells as well as fluorescent fusion tags for in vitro and in vivo detection. Herein, anti-viral effects of +36 GFP and its anti-tumor effects were studied in vitro and in vivo, respectively. We evaluated anti-HIV, anti-HSV, and anti-HCV effects of +36 GFP in vitro using ELISA, and real time PCR as common techniques for their detection, respectively. Moreover, we assessed the role of +36 GFP for eliciting HPV-related anti-tumor effects in mice due to the lack of HPV replication in vitro. Our data showed that +36 GFP efficiently enter the cells and augment the transfection rate of HPV16E7 antigen, as well. Furthermore, +36 GFP significantly reduced HCV, HIV and HSV replication up to 75%, 49% and 43% in HCV-infected Huh7.5 cells, HIV-infected Hela cells and HSV-infected Vero cells, respectively. On the other hand, mice immunization with +36 GFP complexed with HPV16 E7 antigen (+36GFP + E7) or fused to HPV16 E7 antigen (+36GFP-E7) elicited a higher Th1 cellular immune response with the predominant IgG2a, IgG2b, IFN-γ and Granzyme B levels than those induced by other groups. These regimens protected mice against TC- 1 tumor challenge (~ 67%) compared to E7 protein alone (~ 33%). These data suggested that +36 GFP can act as an anti-viral agent at certain dose due to its high efficiency in cell penetration in vitro and in vivo. Generally, +36 GFP targets viral replication in vitro as well as helps to suppress the growth of HPV-related tumors in vivo.

Specific modification at the C-terminal lysine residue of the green fluorescent protein variant, GFPuv, expressed in Escherichia coli

Green fluorescent protein (GFP) is amenable to recombinant expression in various kinds of cells and is widely used in life science research. We found that the recombinant expression of GFPuv, a commonly-used mutant of GFP, in E. coli produced two distinct molecular species as judged by in-gel fluorescence SDS-PAGE. These molecular species, namely form I and II, could be separately purified by anion-exchange chromatography without any remarkable differences in the fluorescence spectra. Mass spectrometric analyses revealed that the molecular mass of form I is almost the same as the calculated value, while that of form II is approximately 1 Da larger than that of form I. Further mass spectrometric top-down sequencing pinpointed the modification in GFPuv form II, where the ε-amino group of the C-terminal Lys238 residue is converted into the hydroxyl group. No equivalent modification was observed in the native GFP in jellyfish Aequorea victoria, suggesting that this modification is not physiologically relevant. Crystal structure analysis of the two species verified the structural identity of the backbone and the vicinity of the chromophore. The modification found in this study may also be generated in other GFP variants as well as in other recombinant expression systems.

Modeling of the glycine tripeptide cyclization in the Ser65Gly/Tyr66Gly mutant of green fluorescent protein

The quantum mechanics/molecular mechanics approach is used to model the chain of elementary reactions involved in the backbone cyclization of the initially non-cyclized Gly65–Gly66–Gly67 tripeptide inside the protein matrix of the green fluorescent protein (GFP) Ser65Gly/Tyr66Gly mutant. The computationally characterized reaction mechanism provides support for understanding chromophore maturation in GFP-like fluorescent proteins.

Excited State Electronic Interconversion and Structural Transformation of Engineered Red-Emitting Green Fluorescent Protein Mutant.

Red fluorescent proteins with a large Stokes shift offer a limited autofluorescence background and are used in deep tissue imaging. Here, by introducing the free amino group in Aequorea victoria, the electrostatic charges of the p-hydroxybenzylidene imidazolinone chromophore of green fluorescent protein (GFP) have been altered resulting in an unusual, 85 nm red-shifted fluorescence. The structural and biophysical analysis suggested that the red shift is due to positional shift occupancy of Glu222 and Arg96, resulting in extended conjugation and a relaxed chromophore. Femtosecond transient absorption spectra exhibited that the excited state relaxation dynamics of red-shifted GFP (rGFP) (τ4 = 234 ps) are faster compared to the A. victoria green fluorescent protein (τ4 = 3.0 ns). The nanosecond time-resolved emission spectra of rGFP reveal the continuous spectral shift during emission by a solvent reorientation in the chromophore. Finally, the molecular dynamics simulations revealed the rearrangement of the hydrogen bond interactions in the chromophore vicinity, reshaping the symmetric distribution of van der Waals space to fine tune the GFP structure resulting from highly red-shifted rGFP.

Programming Protein Polymerization with DNA

A strategy that utilizes DNA for controlling the association pathway of proteins is described. This strategy uses sequence-specific DNA interactions to program energy barriers for polymerization, allowing for either step-growth or chain-growth pathways to be accessed. Two sets of mutant green fluorescent protein (mGFP)-DNA monomers with single DNA modifications have been synthesized and characterized. Depending on the deliberately controlled sequence and conformation of the appended DNA, these monomers can be polymerized through either a step-growth or chain-growth pathway. Cryo-electron microscopy with Volta phase plate technology enables the visualization of the distribution of the oligomer and polymer products, and even the small mGFP-DNA monomers. Whereas cyclic and linear polymer distributions were observed for the step-growth DNA design, in the case of the chain-growth system linear chains exclusively were observed, and a dependence of the chain length on the concentration of the initiator strand was noted. Importantly, the chain-growth system possesses a living character whereby chains can be extended with the addition of fresh monomer. This work represents an important and early example of mechanistic control over protein assembly, thereby establishing a robust methodology for synthesizing oligomeric and polymeric protein-based materials with exceptional control over architecture.