Application of N-Terminal Site-Specific Biotin and Digoxigenin Conjugates to Clinical Anti-drug Antibody Assay Development.

Biotin- and digoxigenin (DIG)-conjugated therapeutic drugs are critical reagents used for the development of anti-drug antibody (ADA) assays for the assessment of immunogenicity. The current practice of generating biotin and DIG conjugates is to label a therapeutic antibody with biotin or DIG via primary amine groups on lysine or N-terminal residues. This approach modifies lysine residues nonselectively, which can impact the ability of an ADA assay to detect those ADAs that recognize epitopes located at or near the modified lysine residue(s). The impact of the lysine modification is considered greater for therapeutic antibodies that have a limited number of lysine residues, such as the variable heavy domain of heavy chain (VHH) antibodies. In this paper, for the first time, we report the application of site-specifically conjugated biotin- and DIG-VHH reagents to clinical ADA assay development using a model molecule, VHHA. The site-specific conjugation of biotin or DIG to VHHA was achieved by using an optimized reductive alkylation approach, which enabled the majority of VHHA molecules labeled with biotin or DIG at the desirable N-terminus, thereby minimizing modification of the protein after labeling and reducing the possibility of missing detection of ADAs. Head-to-head comparison of biophysical characterization data revealed that the site-specific biotin and DIG conjugates demonstrated overall superior quality to biotin- and DIG-VHHA prepared using the conventional amine coupling method, and the performance of the ADA assay developed using site-specific biotin and DIG conjugates met all acceptance criteria. The approach described here can be applied to the production of other therapeutic-protein- or antibody-based critical reagents that are used to support ligand binding assays.

Sustainability Challenges and Opportunities in Oligonucleotide Manufacturing.

With a renewed and growing interest in therapeutic oligonucleotides across the pharmaceutical industry, pressure is increasing on drug developers to take more seriously the sustainability ramifications of this modality. With 12 oligonucleotide drugs reaching the market to date and hundreds more in clinical trials and preclinical development, the current state of the art in oligonucleotide production poses a waste and cost burden to manufacturers. Legacy technologies make use of large volumes of hazardous reagents and solvents, as well as energy-intensive processes in synthesis, purification, and isolation. In 2016, the American Chemical Society (ACS) Green Chemistry Institute Pharmaceutical Roundtable (GCIPR) identified the development of greener processes for oligonucleotide Active Pharmaceutical Ingredients (APIs) as a critical unmet need. As a result, the Roundtable formed a focus team with the remit of identifying green chemistry and engineering improvements that would make oligonucleotide production more sustainable. In this Perspective, we summarize the present challenges in oligonucleotide synthesis, purification, and isolation; highlight potential solutions; and encourage synergies between academia; contract research, development and manufacturing organizations; and the pharmaceutical industry. A critical part of our assessment includes Process Mass Intensity (PMI) data from multiple companies to provide preliminary baseline metrics for current oligonucleotide manufacturing processes.

Thermal vesiculation during volcanic eruptions.

Terrestrial volcanic eruptions are the consequence of magmas ascending to the surface of the Earth. This ascent is driven by buoyancy forces, which are enhanced by bubble nucleation and growth (vesiculation) that reduce the density of magma. The development of vesicularity also greatly reduces the 'strength' of magma, a material parameter controlling fragmentation and thus the explosive potential of the liquid rock. The development of vesicularity in magmas has until now been viewed (both thermodynamically and kinetically) in terms of the pressure dependence of the solubility of water in the magma, and its role in driving gas saturation, exsolution and expansion during decompression. In contrast, the possible effects of the well documented negative temperature dependence of solubility of water in magma has largely been ignored. Recently, petrological constraints have demonstrated that considerable heating of magma may indeed be a common result of the latent heat of crystallization as well as viscous and frictional heating in areas of strain localization. Here we present field and experimental observations of magma vesiculation and fragmentation resulting from heating (rather than decompression). Textural analysis of volcanic ash from Santiaguito volcano in Guatemala reveals the presence of chemically heterogeneous filaments hosting micrometre-scale vesicles. The textures mirror those developed by disequilibrium melting induced via rapid heating during fault friction experiments, demonstrating that friction can generate sufficient heat to induce melting and vesiculation of hydrated silicic magma. Consideration of the experimentally determined temperature and pressure dependence of water solubility in magma reveals that, for many ascent paths, exsolution may be more efficiently achieved by heating than by decompression. We conclude that the thermal path experienced by magma during ascent strongly controls degassing, vesiculation, magma strength and the effusive-explosive transition in volcanic eruptions.

Haemolysin coregulated protein is an exported receptor and chaperone of type VI secretion substrates.

Secretion systems require high-fidelity mechanisms to discriminate substrates among the vast cytoplasmic pool of proteins. Factors mediating substrate recognition by the type VI secretion system (T6SS) of Gram-negative bacteria, a widespread pathway that translocates effector proteins into target bacterial cells, have not been defined. We report that haemolysin coregulated protein (Hcp), a ring-shaped hexamer secreted by all characterized T6SSs, binds specifically to cognate effector molecules. Electron microscopy analysis of an Hcp-effector complex from Pseudomonas aeruginosa revealed the effector bound to the inner surface of Hcp. Further studies demonstrated that interaction with the Hcp pore is a general requirement for secretion of diverse effectors encompassing several enzymatic classes. Though previous models depict Hcp as a static conduit, our data indicate it is a chaperone and receptor of substrates. These unique functions of a secreted protein highlight fundamental differences between the export mechanism of T6 and other characterized secretory pathways.

Trace Explosives Vapor Generation and Quantitation at Parts per Quadrillion Concentrations.

The generation of trace 2,4,6-trinitrotoluene (TNT), cyclotrimethylenetrinitramine (RDX), and pentaerythritol tetranitrate (PETN) vapors using a pneumatically modulated liquid delivery system (PMLDS) coupled to a polytetrafluoroethylene (PTFE) total-consumption micronebulizer is presented. The vapor generator operates in a continuous manner with final vapor concentrations proportional to the explosive concentration in aqueous solution delivered through the nebulizer and the diluent air flow rate. For quantitation of concentrations in the parts per billionvolume (ppbv) to parts per trillionvolume (pptrv) range, Tenax-TA thermal desorption tubes were used for vapor collection with subsequent analysis on a thermal-desorption system programmable-temperature vaporization gas chromatograph (TDS-PTV-GC) with a µ-ECD detector. With 30 min sample times and an average sampling rate of 100 mL min(-1), vapor concentrations of 38 pptrv for TNT, 25 pptrv for RDX, and 26 pptrv for PETN were determined. For parts per quadrillionvolume (ppqv) vapor quantitation of TNT and RDX, an online PTV-GC system with a negative-ion chemical ionization mass spectrometer (methane reagent gas) was used for direct sampling and capture of the vapor on the PTV inlet. Vapor concentrations as low as 160 ppqv and 710 ppqv for TNT and RDX were quantified, respectively, with an instrument duty cycle as low as 4 min.

Phosphorothioate anti-sense oligonucleotides: the kinetics and mechanism of the generation of the sulfurising agent from phenylacetyl disulfide (PADS).

The synthesis of phosphorothioate oligonucleotides is often accomplished in the pharmaceutical industry by the sulfurisation of the nucleotide-phosphite using phenylacetyl disulfide (PADS) which has an optimal combination of properties. This is best achieved by an initial 'ageing' of PADS for 48 h in acetonitrile with 3-picoline to generate polysulfides. The initial base-catalysed degradation of PADS occurs by an E1cB-type elimination to generate a ketene and acyldisulfide anion. Proton abstraction to reversibly generate a carbanion is demonstrated by H/D exchange, the rate of which is greatly increased by electron-withdrawing substituents in the aromatic ring of PADS. The ketene can be trapped intramolecularly by an o-allyl group. The disulfide anion generated subsequently attacks unreacted PADS on sulfur to give polysulfides, the active sulfurising agent. The rate of degradation of PADS is decreased by less basic substituted pyridines and is only first order in PADS indicating that the rate-limiting step is formation of the disulfide anion from the carbanion.

Phosphorothioate anti-sense oligonucleotides: the kinetics and mechanism of the sulfurisation of phosphites by phenylacetyl disulfide (PADS).

In the pharmaceutical industry the sulfurisation of nucleotide-phosphites to produce more biologically stable thiophosphates is often achieved using 'aged' solutions of phenylacetyl disulfide (PADS) which consist of a mixture of polysulfides that are more efficient sulfur transfer reagents. However, both 'fresh' and 'aged' solutions of PADS are capable of the sulfurisation of phosphites. The rates of both processes in acetonitrile are first order in sulfurising agent, phosphite and a pyridine base, although with 'aged' PADS the rate becomes independent of base at high concentrations. The Brönsted ß values for sulfurisation using 'fresh' and 'aged' PADS with substituted pyridines are 0.43 and 0.26, respectively. With 'fresh' PADS the Brönsted ßnuc = 0.51 for substituted trialkyl phosphites is consistent with a mechanism involving nucleophilic attack of the phosphite on the PADS disulfide bond to reversibly generate a phosphonium intermediate, the rate-limiting breakdown of which occurs by a base catalysed elimination process, confirmed by replacing the ionisable hydrogens in PADS with methyl groups. The comparable polysulfide phosphonium ion intermediate seen with 'aged' PADS presents a more facile pathway for product formation involving S-S bond fission as opposed to C-S bond fission.

Strong subunit coordination drives a powerful viral DNA packaging motor.

Terminase enzymes are viral motors that package DNA into a preformed capsid and are of interest both therapeutically and as potential nano-machines. The enzymes excise a single genome from a concatemeric precursor (genome maturation) and then package the duplex to near-crystalline density (genome packaging). The functional motors are oligomers of protomeric subunits and are the most powerful motors currently known. Here, we present mechanistic studies on the terminase motor from bacteriophage λ. We identify a mutant (K76R) that is specifically deficient in packaging activity. Biochemical analysis of this enzyme provides insight into the linkage between ATP hydrolysis and motor translocation. We further use this mutant to assemble chimeric motors with WT enzyme and characterize the catalytic activity of the complexes. The data demonstrate that strong coordination between the motor protomers is required for DNA packaging and that incorporation of even a single mutant protomer poisons motor activity. Significant coordination is similarly observed in the genome maturation reaction; however, although the motor is composed of a symmetric tetramer of protomers, the maturation complex is better described as a "dimer-of-dimers" with half-site reactivity. We describe a model for how the motor alternates between a stable genome maturation complex and a dynamic genome packaging complex. The fundamental features of coordinated ATP hydrolysis, DNA movement, and tight association between the motor and the duplex during translocation are recapitulated in all of the viral motors. This work is thus of relevance to all terminase enzymes, both prokaryotic and eukaryotic.

Pathophysiology and Prevention of Intraoperative Atelectasis: A Review of the Literature.

Atelectasis is a common problem in the perioperative setting, affecting a significant number of surgical patients receiving general anesthesia. Absorption, compression, and reduced surfactant are the three mechanisms implicated in the etiology of atelectasis. Interventions designed to minimize the risk of intraoperative atelectasis such as positioning, positive end-expiratory pressure, and administration of the least amount of fraction of inspired oxygen can be used to maintain patency of small airways and ultimately improve gas exchange in the surgical patient.

Demonstration of Physicochemical and Functional Similarity of Biosimilar Pegfilgrastim-cbqv to Pegfilgrastim.

BACKGROUND: Pegfilgrastim-cbqv/CHS-1701 (UDENYCA®) (hereafter referred to as pegfilgrastim-cbqv) was approved in 2018 by the US Food and Drug Administration as a biosimilar for pegfilgrastim (Neulasta®) (hereafter referred to as pegfilgrastim). Both pegfilgrastim-cbqv and pegfilgrastim are conjugates of recombinant human granulocyte colony stimulating factor (r-metHuG-CSF) with a 20 kDa polyethylene glycol (PEG) indicated to decrease the incidence of infection, as manifested by febrile neutropenia, in patients receiving myelosuppressive anticancer drugs. The demonstration of analytical similarity for PEG-protein conjugates presents unique challenges since both the protein and PEG attributes must be characterized. OBJECTIVE: The current study demonstrates the analytical similarity of pegfilgrastim-cbqv and the reference product, pegfilgrastim. In addition to the physicochemical and functional characterization of the protein, the study assessed attributes specific to PEGylation including PEG size and polydispersity, site of attachment, linker composition, and PEGylation process-related variants. METHODS: The structural, functional, and stability attributes of pegfilgrastim-cbqv and pegfilgrastim were compared using state-of-the-art analytical methods. For the protein, the primary structure, disulfide structure, and secondary and tertiary structures were assessed using traditional protein characterization techniques such as mass spectrometry (MS), circular dichroism (CD), intrinsic fluorescence, and differential scanning calorimetry (DSC), as well as more advanced techniques such as two-dimensional (2D) nuclear magnetic resonance (NMR) and hydrogen deuterium exchange (HDX). For the PEG moiety, the site of attachment, occupancy, linker composition, size and polydispersity were compared using mass spectrometry (both intact and after endoprotease digestion), multiangle light scattering detection (MALS), and Edman degradation. Purity assessments included the assessment of both protein variants and PEGylation variants using chromatographic and electrophoretic analytical separation techniques. The functional similarity between pegfilgrastim-cbqv and pegfilgrastim was compared using both a cell-based bioassay and surface plasmon resonance (SPR). The degradation rates and stability profiles were compared under accelerated and stressed conditions. RESULTS: Biosimilarity was demonstrated by a thorough assessment of physiochemical and functional attributes, as well as comparative stability, of pegfilgrastim-cbqv relative to pegfilgrastim. These studies demonstrated identical primary structure and disulfide structure, highly similar secondary and tertiary structure, as well as functional similarity. The impurity profile of pegfilgrastim-cbqv was comparable to that of pegfilgrastim with only minor differences in PEGylation variants and a slight offset in the PEG molar mass. These differences were not clinically relevant. The degradation profiles were qualitatively and quantitatively similar under accelerated and stress conditions. CONCLUSION: The structural, functional, and stability data demonstrate that pegfilgrastim-cbqv is highly similar to the reference product, pegfilgrastim.

Bat cellular immunity varies by year and dietary habit amidst land conversion.

Monitoring the health of wildlife populations is essential in the face of increased agricultural expansion and forest fragmentation. Loss of habitat and habitat degradation can negatively affect an animal's physiological state, possibly resulting in immunosuppression and increased morbidity or mortality. We sought to determine how land conversion may differentially impact cellular immunity and infection risk in Neotropical bats species regularly infected with bloodborne pathogens, and to evaluate how effects may vary over time and by dietary habit. We studied common vampire bats (Desmodus rotundus), northern yellow-shouldered bats (Sturnira parvidens) and Mesoamerican mustached bats (Pteronotus mesoamericanus), representing the dietary habits of sanguivory, frugivory and insectivory respectively, in northern Belize. We compared estimated total white blood cell count, leukocyte differentials, neutrophil to lymphocyte ratio and infection status with two bloodborne bacterial pathogens (Bartonella spp. and hemoplasmas) of 118 bats captured in a broadleaf, secondary forest over three years (2017-2019). During this period, tree cover decreased by 14.5% while rangeland expanded by 14.3%, indicating increasing habitat loss and fragmentation. We found evidence for bat species-specific responses of cellular immunity between years, with neutrophil counts significantly decreasing in S. parvidens from 2017 to 2018, but marginally increasing in D. rotundus. However, the odds of infection with Bartonella spp. and hemoplasmas between 2017 and 2019 did not differ between bat species, contrary to our prediction that pathogen prevalence may increase with land conversion. We conclude that each bat species invested differently in cellular immunity in ways that changed over years of increasing habitat loss and fragmentation. We recommend further research on the interactions between land conversion, immunity and infection across dietary habits of Neotropical bats for informed management and conservation.

Sortase-Modified Cholera Toxoids Show Specific Golgi Localization.

Cholera toxoid is an established tool for use in cellular tracing in neuroscience and cell biology. We use a sortase labeling approach to generate site-specific N-terminally modified variants of both the A2-B5 heterohexamer and B5 pentamer forms of the toxoid. Both forms of the toxoid are endocytosed by GM1-positive mammalian cells, and while the heterohexameric toxoid was principally localized in the ER, the B5 pentamer showed an unexpectedly specific localization in the medial/trans-Golgi. This study suggests a future role for specifically labeled cholera toxoids in live-cell imaging beyond their current applications in neuronal tracing and labeling of lipid rafts in fixed cells.

Artifact Adaptive Ideal Filtering of EMG Signals Contaminated by Spinal Cord Transcutaneous Stimulation.

The aims of this study are to characterize the contamination of EMG signals by artifacts generated by the delivery of spinal cord transcutaneous stimulation (scTS) and to evaluate the performance of an Artifact Adaptive Ideal Filtering (AA-IF) technique to remove scTS artifacts from EMG signals. METHODS: In five participants with spinal cord injury (SCI), scTS was delivered at different combinations of intensity (from 20 to 55 mA) and frequencies (from 30 to 60 Hz) while Biceps Brachii (BB) and Triceps Brachii (TB) muscles were at rest or voluntarily activated. Using a Fast Fourier Transform (FFT), we characterized peak amplitude of scTS artifacts and boundaries of contaminated frequency bands in the EMG signals recorded from BB and TB muscles. Then, we applied the AA-IF technique and the empirical mode decomposition Butterworth filtering method (EMD-BF) to identify and remove scTS artifacts. Finally, we compared the content of the FFT that was preserved and the root mean square of the EMG signals (EMGrms) following application of the AA-IF and EMD-BF techniques. RESULTS: Frequency bands of ~2Hz width were contaminated by scTS artifact at frequencies nearby the main frequency set for the stimulator and its harmonics. The width of the frequency bands contaminated by scTS artifacts increased with current intensity delivered using scTS ( [Formula: see text]), was lower when EMG signals were recorded during voluntary contractions compared to rest ( [Formula: see text]), and was larger in BB muscle compared to TB muscle ( [Formula: see text]). A larger portion of the FFT was preserved using the AA-IF technique compared to the EMD-BF technique (96±5% vs. 75±6%, [Formula: see text]). CONCLUSION: The AA-IF technique allows for a precise identification of the frequency bands contaminated by scTS artifacts and ultimately preserves a larger amount of uncontaminated content from the EMG signals.

Expanded diversity of novel hemoplasmas in rare and undersampled Neotropical bats.

Hemotropic mycoplasmas are emerging as a model system for studying bacterial pathogens in bats, but taxonomic coverage of sampled host species remains biased. We leveraged a long-term field study in Belize to uncover novel hemoplasma diversity in bats by analyzing 80 samples from 19 species, most of which are infrequently encountered. PCR targeting the partial 16S rRNA gene found 41% of bats positive for hemoplasmas. Phylogenetic analyses found two novel host shifts of hemoplasmas, four entirely new hemoplasma genotypes, and the first hemoplasma detections in four bat species. One of these novel hemoplasmas (from Neoeptesicus furinalis) shared 97.6% identity in the partial 16S rRNA gene to a human hemoplasma (Candidatus Mycoplasma haemohominis). Additional analysis of the partial 23S rRNA gene allowed us to also designate two novel hemoplasma species, in Myotis elegans and Phyllostomus discolor, with the proposed names Candidatus Mycoplasma haematomyotis sp. nov. and Candidatus Mycoplasma haematophyllostomi sp. nov., respectively. Our analyses show that additional hemoplasma diversity in bats can be uncovered by targeting rare or undersampled host species.

Energy-independent helicase activity of a viral genome packaging motor.

The assembly of complex double-stranded DNA viruses includes a genome packaging step where viral DNA is translocated into the confines of a preformed procapsid shell. In most cases, the preferred packaging substrate is a linear concatemer of viral genomes linked head-to-tail. Viral terminase enzymes are responsible for both excision of an individual genome from the concatemer (DNA maturation) and translocation of the duplex into the capsid (DNA packaging). Bacteriophage λ terminase site-specifically nicks viral DNA at the cos site in a concatemer and then physically separates the nicked, annealed strands to mature the genome in preparation for packaging. Here we present biochemical studies on the so-called helicase activity of λ terminase. Previous studies reported that ATP is required for strand separation, and it has been presumed that ATP hydrolysis is required to drive the reaction. We show that ADP and nonhydrolyzable ATP analogues also support strand separation at low (micromolar) concentrations. In addition, the Escherichia coli integration host factor protein (IHF) strongly stimulates the reaction in a nucleotide-independent manner. Finally, we show that elevated concentrations of nucleotide inhibit both ATP- and IHF-stimulated strand separation by λ terminase. We present a model where nucleotide and IHF interact with the large terminase subunit and viral DNA, respectively, to engender a site-specifically bound, catalytically competent genome maturation complex. In contrast, binding of nucleotide to the low-affinity ATP binding site in the small terminase subunit mediates a conformational switch that down-regulates maturation activities and activates the DNA packaging activity of the enzyme. This affords a motor complex that binds tightly, but nonspecifically, to DNA as it translocates the duplex into the capsid shell. These studies have yielded mechanistic insight into the assembly of the maturation complex on viral DNA and its transition to a mobile packaging motor that may be common to all of the complex double-stranded DNA viruses.

The enzymology of a viral genome packaging motor is influenced by the assembly state of the motor subunits.

Terminase enzymes are responsible for the excision of a single genome from a concatemeric precursor (genome maturation) and concomitant packaging of DNA into the capsid shell. Here, we demonstrate that lambda terminase can be purified as a homogeneous "protomer" species, and we present a kinetic analysis of the genome maturation and packaging activities of the protomeric enzyme. The protomer assembles into a distinct maturation complex at the cos sequence of a concatemer. This complex rapidly nicks the duplex to form the mature left end of the viral genome, which is followed by procapsid binding, activation of the packaging ATPase, and translocation of the duplex into the capsid interior by the terminase motor complex. Genome packaging by the protomer shows high fidelity with only the mature left end of the duplex inserted into the capsid shell. In sum, the data show that the terminase protomer exhibits catalytic activity commensurate with that expected of a bone fide genome maturation and packaging complex in vivo and that both catalytically competent complexes are composed of four terminase protomers assembled into a ringlike structure that encircles duplex DNA. This work provides mechanistic insight into the coordinated catalytic activities of terminase enzymes in virus assembly that can be generalized to all of the double-stranded DNA viruses.

Mutations altering a structurally conserved loop-helix-loop region of a viral packaging motor change DNA translocation velocity and processivity.

Many double-stranded DNA viruses employ ATP-driven motors to translocate their genomes into small, preformed viral capsids against large forces resisting confinement. Here, we show via direct single-molecule measurements that a mutation T194M downstream of the Walker B motif in the phage lambda gpA packaging motor causes an 8-fold reduction in translocation velocity without substantially changing processivity or force dependence, whereas the mutation G212S in the putative C (coupling) motif causes a 3-fold reduction in velocity and a 6-fold reduction in processivity. Meanwhile a T194M pseudorevertant (T194V) showed a near restoration of the wild-type dynamics. Structural comparisons and modeling show that these mutations are in a loop-helix-loop region that positions the key residues of the catalytic motifs, Walker B and C, in the ATPase center and is structurally homologous with analogous regions in chromosome transporters and SF2 RNA helicases. Together with recently published studies of SpoIIIE chromosome transporter and Ded1 RNA helicase mutants, these findings suggest the presence of a structurally conserved region that may be a part of the mechanism that determines motor velocity and processivity in several different types of nucleic acid translocases.

Spore-terminated cantilevers for chemical patterning on complex architectures.

Atomic force microscope tips terminated with spore cells are used to directly pattern onto glass and tissue surfaces. The spore cells act as sponges and eliminate the need to use microfabricated ink reservoirs during lithography.

Developmental continuity in theory of mind: speed and accuracy of belief-desire reasoning in children and adults.

On belief-desire reasoning tasks, children first pass tasks involving true belief before those involving false belief, and tasks involving positive desire before those involving negative desire. The current study examined belief-desire reasoning in participants old enough to pass all such tasks. Eighty-three 6- to 11-year-olds and 20 adult participants completed simple, computer-based tests of belief-desire reasoning, which recorded response times as well as error rates. Both measures suggested that, like young children, older children and adults find it more difficult to reason about false belief and negative desires than true beliefs and positive desires. It is argued that this developmental continuity is most consistent with either executive competence or executive performance accounts of the development of belief-desire reasoning.

The dual-basin landscape in GFP folding.

Recent experimental studies suggest that the mature GFP has an unconventional landscape composed of an early folding event with a typical funneled landscape, followed by a very slow search and rearrangement step into the locked, active chromophore-containing structure. As we have shown previously, the substantial difference in time scales is what generates the observed hysteresis in thermodynamic folding. The interconversion between locked and the soft folding structures at intermediate denaturant concentrations is so slow that it is not observed under the typical experimental observation time. Simulations of a coarse-grained model were used to describe the fast folding event as well as identify native-like intermediates on energy landscapes enroute to the fluorescent native fold. Interestingly, these simulations reveal structural features of the slow dynamic transition to chromophore activation. Experimental evidence presented here shows that the trapped, native-like intermediate has structural heterogeneity in residues previously linked to chromophore formation. We propose that the final step of GFP folding is a "locking" mechanism leading to chromophore formation and high stability. The combination of previous experimental work and current simulation work is explained in the context of a dual-basin folding mechanism described above.