Alternative Folding Research Articles

Engineering A Modular Protein Color Switch using An Entropy‐driven Beta Strand Exchange

Protein conformational switches are widely used in functional regulation and biosensing to visualize biological substances in vitro and in vivo. A major challenge in protein switch design strategies is coupling the target recognition domain to an output domain to produce a change in fluorescence wavelength or intensity. To overcome this challenge, we tested a rational design strategy that exploits two key protein engineering principles (1) loop entropy, by which long insertions into a loop of a host protein destabilizes the host due to an entropic cost associated with loop closure unless the inserted sequence adopts a folded structure; (2) alternate frame folding (AFF), which allows a protein (GFP variants, in this case) to switch between two mutually exclusive folds. For proof-of-concept, we chose an unfolded, circularly permuted FK506-binding protein (cpFKBP) as the input recognition domain, and inserted it in one of the two mutually exclusive folds of the GFP-AFF fusion protein. Upon addition of ligand, cpFKBP folding effects a conformational change in which the 10th beta strand of GFP exchanges, replacing Thr203 (green state) with Tyr203 (yellow state). Single point mutations and insertions were introduced to thermodynamically balance the protein switch for response within a reasonable timescale. We observe a shift in emission wavelength within an hour in response to ligand in vitro and in cellulo with a yellow:green color change of ~2.5-fold. This protein switch design has the advantages of genetic encodability, potential modularity and a ratiometric response. The design strategy further provides insight into protein engineering principles for application in molecular diagnostics, cellular biosensors and activatable proteins.

Evolutionary insights into shape-shifting proteins

Over millions of years a protein that now folds into two stable structures likely favored first one configuration, then the other, before settling on both.

Specificity of mRNA Folding and Its Association with Evolutionarily Adaptive mRNA Secondary Structures

The secondary structure is a fundamental feature of both non-coding RNAs (ncRNAs) and messenger RNAs (mRNAs). However, our understanding of the secondary structures of mRNAs, especially those of the coding regions, remains elusive, likely due to translation and the lack of RNA-binding proteins that sustain the consensus structure like those binding to ncRNAs. Indeed, mRNAs have recently been found to adopt diverse alternative structures, but the overall functional significance remains untested. We hereby approach this problem by estimating the folding specificity, i.e., the probability that a fragment of an mRNA folds back to the same partner once refolded. We show that the folding specificity of mRNAs is lower than that of ncRNAs and exhibits moderate evolutionary conservation. Notably, we find that specific rather than alternative folding is likely evolutionarily adaptive since specific folding is frequently associated with functionally important genes or sites within a gene. Additional analysis in combination with ribosome density suggests the ability to modulate ribosome movement as one potential functional advantage provided by specific folding. Our findings reveal a novel facet of the RNA structurome with important functional and evolutionary implications and indicate a potential method for distinguishing the mRNA secondary structures maintained by natural selection from molecular noise.

Order and stochasticity in the folding of individual Drosophila genomes

Mammalian and Drosophila genomes are partitioned into topologically associating domains (TADs). Although this partitioning has been reported to be functionally relevant, it is unclear whether TADs represent true physical units located at the same genomic positions in each cell nucleus or emerge as an average of numerous alternative chromatin folding patterns in a cell population. Here, we use a single-nucleus Hi-C technique to construct high-resolution Hi-C maps in individual Drosophila genomes. These maps demonstrate chromatin compartmentalization at the megabase scale and partitioning of the genome into non-hierarchical TADs at the scale of 100 kb, which closely resembles the TAD profile in the bulk in situ Hi-C data. Over 40% of TAD boundaries are conserved between individual nuclei and possess a high level of active epigenetic marks. Polymer simulations demonstrate that chromatin folding is best described by the random walk model within TADs and is most suitably approximated by a crumpled globule build of Gaussian blobs at longer distances. We observe prominent cell-to-cell variability in the long-range contacts between either active genome loci or between Polycomb-bound regions, suggesting an important contribution of stochastic processes to the formation of the Drosophila 3D genome.

Cézanne's Gravity by Carol Armstrong

Reviewed by: Cézanne's Gravity by Carol Armstrong Lucy Whelan Cézanne's Gravity. Carol Armstrong. New Haven, CT: Yale University Press, 2018. Pp. xii + 280. $65.00 (cloth). If there is a single artist who defines the tendency of histories of Western modern art to cling to a teleological timeline—in which art evolves steadily from realism towards the freedom of abstraction—that artist is surely Paul Cézanne. In the decades after his death in 1906, artists, art historians, and critics such as Clement Greenberg placed him at the origin of that lineage, dubbing him the "most copious source of modern art."1 For Greenberg especially, this status was primarily due to the apparent emphasis Cézanne's tactile brushstrokes gave to the flatness of the [End Page 204] canvas support, that Greenberg retrospectively cast as the primary origin of modern painting's self-reflexive way of pointing to itself and the specific conditions of its medium. According to this narrative, Cézanne's flatness would eventually find its teleological endpoint in the rectangles of color produced by (overwhelmingly male) abstract expressionist artists after the Second World War. This account of modernism has been critiqued and disrupted over the last four decades, yet still it seems indelible: modernism in its paradigmatic form, with Cézanne at its head, remains as temporally linear as it is spatially flat. Carol Armstrong's stimulating and beautifully written study offers a bold reappraisal of this rectilinear view of modernism, insisting on alternative possible folds in its history while simultaneously recovering the warped strangeness of Cézanne's work. It does so primarily through a series of pairings, sometimes groupings, of Cézanne with various twentieth-century writers, painters, and thinkers that followed him: Roger Fry, Virginia Woolf, Vanessa Bell, Albert Einstein, Maurice Merleau-Ponty, Rainer Maria Rilke, R. D. Laing, Greenberg, and Helen Frankenthaler. The reason for these pairings is not simply in order to trace Cézanne's reception, but rather in order to see him refracted by a plurality of perspectives, all of which reveal a different painter from the one shaped retroactively by art history's desire for linearity. In this way, Armstrong advances a "heterochronic" model for approaching art through multiple and overlapping timelines that builds upon recent work in the field (20). Art historians countering traditional linear chronologies with more complex temporalities include Mieke Bal, Michael Ann Holly, Alexander Nagel, Amy Powell, and, most pertinently here, Keith Moxey. Yet Armstrong's book is to my knowledge the first to bring this approach to modernism. By focusing on such a complex painter as Cézanne, she offers a powerful demonstration of how taking a heterochronic approach to time enables us to treat artworks as agents, rather than products, of history. For as she shows, Cézanne's works have more than enough material and intellectual presence to be capable of eluding linear, pre-ordained art historical narratives. Armstrong's quest to retrieve Cézanne's strangeness likewise expands and builds on a wealth of writing on the artist produced in recent decades. Art historians have explored his work's curious form of sensitivity to the visual world, its sensuous physicality, its peculiar erotic charge, uncanny non-humanity, and apparent lack of "finish," and these and other topics weave lightly in and out of this study. That said, it would be possible for readers approaching this book from outside of nineteenth-century art history not to realise how much authors such as Yve-Alain Bois, T. J. Clark, Tamar Garb, and Aruna D'Souza have already successfully refocused discussions around Cézanne away from the Greenbergian model that Cézanne's Gravity positions itself so assertively against. There is nothing inherently wrong in this, however—indeed Armstrong's own beautifully observed study of Cézanne's watercolors from 2004 is among the best of this literature—and as the first study to counter Greenberg's emphasis on the artist's flatness so directly, Cézanne's Gravity merits positioning itself as transformative in this sense. In any case, writing on Cézanne already forms a large part of this study, in that its first, third, and fourth chapters...

Elastic Properties of Alloy ZE10 Sheets Evaluation by Kerns Texture Parameters

The ZE10 magnesium alloy with the rare-earth metal additives, which contribute to a better forming of the alloy, was used as studied material. The ZE10 magnesium alloy with the rare-earth metal additives, which contribute to a better forming of the alloy, was used as studied material. Sheet material is usually straightened on roller levelers to relieve residual stresses and improve flatness. The metal is subjected to alternating deformation by bending when straightening. The changes in the structure, crystallographic texture and, as a result, physical and mechanical properties occur in the metal are often not taken into account in the future. The elastic modulus is an important parameter, for example, in the production of products using bending. In this work, the elastic modulus of sheets of magnesium alloy ZE10 was estimated in three main directions. A starting sheet was obtained by extruding an ingot, then rolling in the longitudinal direction and then rolling with a change in direction by 90° after each pass in combination with heating to 350°C. The original sheets were subsequently subjected to alternate folding. Evaluations were made of the elastic modulus of the original sheet, as well as the sheets after 0.5, 1.0, 3.0 and 5.0 alternating bending cycles. To estimate the elastic modulus, we used the Kearns texture parameters , which we calculated from the inverse pole figures, as well as the elastic constants of the single crystal of the ZE10 alloy found by us. The maximum deviation of the calculated and experimental values of the elastic modulus did not exceed 5.2%. Strong correlations and quadratic regression equations have been established between the values of the elastic modulus, mechanical characteristics (tensile strength, yield stress, elongation), on the one hand, and the above-mentioned parameters of the Kerns texture, on the other hand. The approximation reliability coefficients are 0.76 - 0.99.

Refolding of Cold‐Denatured Barstar Induced by Radio‐Frequency Heating: A New Method to Study Protein Folding by Real‐Time NMR Spectroscopy

AbstractThe C40A/C82A double mutant of barstar has been shown to undergo cold denaturation above the water freezing point. By rapidly applying radio‐frequency power to lossy aqueous samples, refolding of barstar from its cold‐denatured state can be followed by real‐time NMR spectroscopy. Since temperature‐induced unfolding and refolding is reversible for this double mutant, multiple cycling can be utilized to obtain 2D real‐time NMR data. Barstar contains two proline residues that adopt a mix of cis and trans conformations in the low‐temperature‐unfolded state, which can potentially induce multiple folding pathways. The high time resolution real‐time 2D‐NMR measurements reported here show evidence for multiple folding pathways related to proline isomerization, and stable intermediates are populated. By application of advanced heating cycles and state‐correlated spectroscopy, an alternative folding pathway circumventing the rate‐limiting cis‐trans isomerization could be observed. The kinetic data revealed intermediates on both, the slow and the fast folding pathway.

Refolding of Cold-Denatured Barstar Induced by Radio-Frequency Heating: A New Method to Study Protein Folding by Real-Time NMR Spectroscopy.

The C40A/C82A double mutant of barstar has been shown to undergo cold denaturation above the water freezing point. By rapidly applying radio‐frequency power to lossy aqueous samples, refolding of barstar from its cold‐denatured state can be followed by real‐time NMR spectroscopy. Since temperature‐induced unfolding and refolding is reversible for this double mutant, multiple cycling can be utilized to obtain 2D real‐time NMR data. Barstar contains two proline residues that adopt a mix of cis and trans conformations in the low‐temperature‐unfolded state, which can potentially induce multiple folding pathways. The high time resolution real‐time 2D‐NMR measurements reported here show evidence for multiple folding pathways related to proline isomerization, and stable intermediates are populated. By application of advanced heating cycles and state‐correlated spectroscopy, an alternative folding pathway circumventing the rate‐limiting cis‐trans isomerization could be observed. The kinetic data revealed intermediates on both, the slow and the fast folding pathway.

Oligomerization of a symmetric β-trefoil protein in response to folding nucleus perturbation.

Gene duplication and fusion events in protein evolution are postulated to be responsible for the common protein folds exhibiting internal rotational symmetry. Such evolutionary processes can also potentially yield regions of repetitive primary structure. Repetitive primary structure offers the potential for alternative definitions of critical regions, such as the folding nucleus (FN). In principle, more than one instance of the FN potentially enables an alternative folding pathway in the face of a subsequent deleterious mutation. We describe the targeted mutation of the carboxyl-terminal region of the (internally located) FN of the de novo designed purely-symmetric β-trefoil protein Symfoil-4P. This mutation involves wholesale replacement of a repeating trefoil-fold motif with a "blade" motif from a β-propeller protein, and postulated to trap that region of the Symfoil-4P FN in a nonproductive folding intermediate. The resulting protein (termed "Bladefoil") is shown to be cooperatively folding, but as a trimeric oligomer. The results illustrate how symmetric protein architectures have potentially diverse folding alternatives available to them, including oligomerization, when preferred pathways are perturbed.

X-ray Crystallographic and Molecular Dynamic Analyses of Drosophila melanogaster Embryonic Muscle Myosin Define Domains Responsible for Isoform-Specific Properties

Drosophila melanogaster is a powerful system for characterizing alternative myosin isoforms and modeling muscle diseases, but high-resolution structures of fruit fly contractile proteins have not been determined. Here we report the first x-ray crystal structure of an insect myosin: the D melanogaster skeletal muscle myosin II embryonic isoform (EMB). Using our system for recombinant expression of myosin heavy chain (MHC) proteins in whole transgenic flies, we prepared and crystallized stable proteolytic S1-like fragments containing the entire EMB motor domain bound to an essential light chain. We solved the x-ray crystal structure by molecular replacement and refined the resulting model against diffraction data to 2.2 Å resolution. The protein is captured in two slightly different renditions of the rigor-like conformation with a citrate of crystallization at the nucleotide binding site and exhibits structural features common to myosins of diverse classes from all kingdoms of life. All atom molecular dynamics simulations on EMB in its nucleotide-free state and a derivative homology model containing 61 amino acid substitutions unique to the indirect flight muscle isoform (IFI) suggest that differences in the identity of residues within the relay and the converter that are encoded for by MHC alternative exons 9 and 11, respectively, directly contribute to increased mobility of these regions in IFI relative to EMB. This suggests the possibility that alternative folding or conformational stability within these regions contribute to the observed functional differences in Drosophila EMB and IFI myosins.

Alternative folding to a monomer or homopolymer is a common feature of the type 1 pilus subunit FimA from enteroinvasive bacteria

Adhesive type 1 pili from enteroinvasive, Gram-negative bacteria mediate attachment to host cells. Up to 3000 copies of the main pilus subunit, FimA, assemble into the filamentous, helical quaternary structure of the pilus rod via a mechanism termed donor-strand complementation, in which the N-terminal extension of each subunit, the donor strand, is inserted into the incomplete immunoglobulin-like fold of the preceding FimA subunit. For FimA from Escherichia coli, it has been previously shown that the protein can also adopt a monomeric, self-complemented conformation in which the donor strand is inserted intramolecularly in the opposite orientation relative to that observed for FimA polymers. Notably, soluble FimA monomers can act as apoptosis inhibitors in epithelial cells after uptake of type 1-piliated pathogens. Here, we show that the FimA orthologues from Escherichia coli, Shigella flexneri, and Salmonella enterica can all fold to form self-complemented monomers. We solved X-ray structures of all three FimA monomers at 0.89–1.69 Å resolutions, revealing identical, intramolecular donor-strand complementation mechanisms. Our results also showed that the pseudo-palindromic sequences of the donor strands in all FimA proteins permit their alternative folding possibilities. All FimA monomers proved to be 50–60 kJ/mol less stable against unfolding than their pilus rod-like counterparts (which exhibited very high energy barriers of unfolding and refolding). We conclude that the ability of FimA to adopt an alternative, monomeric state with anti-apoptotic activity is a general feature of FimA proteins of type 1-piliated bacteria.

Topological descriptions of protein folding

How knotted proteins fold has remained controversial since the identification of deeply knotted proteins nearly two decades ago. Both computational and experimental approaches have been used to investigate protein knot formation. Motivated by the computer simulations of Bölinger et al. [Bölinger D, et al. (2010) PLoS Comput Biol 6:e1000731] for the folding of the [Formula: see text]-knotted α-haloacid dehalogenase (DehI) protein, we introduce a topological description of knot folding that could describe pathways for the formation of all currently known protein knot types and predicts knot types that might be identified in the future. We analyze fingerprint data from crystal structures of protein knots as evidence that particular protein knots may fold according to specific pathways from our theory. Our results confirm Taylor's twisted hairpin theory of knot folding for the [Formula: see text]-knotted proteins and the [Formula: see text]-knotted ketol-acid reductoisomerases and present alternative folding mechanisms for the [Formula: see text]-knotted phytochromes and the [Formula: see text]- and [Formula: see text]-knotted proteins.

An alternative N-terminal fold of the intestine-specific annexin A13a induces dimerization and regulates membrane-binding

Annexin proteins function as Ca2+-dependent regulators of membrane trafficking and repair that may also modulate membrane curvature. Here, using high-resolution confocal imaging, we report that the intestine-specific annexin A13 (ANX A13) localizes to the tips of intestinal microvilli and determined the crystal structure of the ANX A13a isoform to 2.6 Å resolution. The structure revealed that the N terminus exhibits an alternative fold that converts the first two helices and the associated helix-loop-helix motif into a continuous α-helix, as stabilized by a domain-swapped dimer. We also found that the dimer is present in solution and partially occludes the membrane-binding surfaces of annexin, suggesting that dimerization may function as a means for regulating membrane binding. Accordingly, as revealed by in vitro binding and cellular localization assays, ANX A13a variants that favor a monomeric state exhibited increased membrane association relative to variants that favor the dimeric form. Together, our findings support a mechanism for how the association of the ANX A13a isoform with the membrane is regulated.

Increased sequence hydrophobicity reduces conformational specificity: A mutational case study of the Arc repressor protein.

The amino-acid sequences of soluble, globular proteins must have hydrophobic residues to form a stable core, but excess sequence hydrophobicity can lead to loss of native state conformational specificity and aggregation. Previous studies of polar-to-hydrophobic mutations in the β-sheet of the Arc repressor dimer showed that a single substitution at position 11 (N11L) leads to population of an alternate dimeric fold in which the β-sheet is replaced by helix. Two additional hydrophobic mutations at positions 9 and 13 (Q9V and R13V) lead to population of a differently folded octamer along with both dimeric folds. Here we conduct a comprehensive study of the sequence determinants of this progressive loss of fold specificity. We find that the alternate dimer-fold specifically results from the N11L substitution and is not promoted by other hydrophobic substitutions in the β-sheet. We also find that three highly hydrophobic substitutions at positions 9, 11, and 13 are necessary and sufficient for oligomer formation, but the oligomer size depends on the identity of the hydrophobic residue in question. The hydrophobic substitutions increase thermal stability, illustrating how increased hydrophobicity can increase folding stability even as it degrades conformational specificity. The oligomeric variants are predicted to be aggregation-prone but may be hindered from doing so by proline residues that flank the β-sheet region. Loss of conformational specificity due to increased hydrophobicity can manifest itself at any level of structure, depending upon the specific mutations and the context in which they occur.

Folding Mechanism of the SH3 Domain from Grb2.

SH3 domains are small protein modules involved in the regulation of important cellular pathways. These domains mediate protein-protein interactions recognizing motifs rich in proline on the target protein. The SH3 domain from Grb2 (Grb2-SH3) presents the typical structure of an SH3 domain composed of two three-stranded antiparallel β-sheets orthogonally packed onto each other, to form a single hydrophobic core. Grb2 interacts, via SH3 domain, with Gab2, a scaffolding disordered protein, triggering some key metabolic pathways involved in cell death and differentiation. In this work we report a mutational analysis (Φ value analysis) of the folding pathway of Grb2-SH3 that, coupled with molecular dynamic simulations, allows us to assess the structure of the transition state and the mechanism of folding of this domain. Data suggest that Grb2-SH3 folds via a native-like, diffused transition state with a concurrent formation of native-like secondary and tertiary structure (nucleation-condensation mechanism) and without the accumulation of folding intermediates. The comparison between our data and previous folding studies on SH3 domains belonging to other proteins highlights that proteins of this class may fold via alternative pathways, stabilized by different nuclei leading or not to accumulation of folding intermediates. This comparative analysis suggests that the alternative folding pathways for this class of SH3 domains can be selectively regulated by the specific amino acid sequences.

Effects of Non-native Interactions on Frustrated Proteins Folding under Confinement.

In vitro, kinetically significant non-native interactions have been identified experimentally during the folding of proteins Im7, Im9, and A39V/N53P/V55L Fyn SH3 domain. To understand the role of non-native interactions on the folding of some frustrated proteins in chaperone, we employed native-centric models with and without additional transferable, sequence-dependent non-native hydrophobic interactions to comparatively study the folding behaviors of the three proteins confined in spherical cages. Under purely repulsive confinement, as a decrease of cavity size, the non-native interactions increase, especially in the unfolded state, enhancing the roughness of the folding energy landscape. As a result, the increase in native stability for the three proteins by the model incorporated non-native interactions (db + MJ hϕ model) is much smaller than that by the purely native-centric model (desolvation-barrier (db) model); the acceleration of folding simulated by the db + MJ hϕ model is much slower than that via the db model; in particular, the folding rate of Im7 decreases when reducing the cavity size under zero-denaturant condition. The repulsive confinement can also promote formation of specific non-native contacts in the transition state and favor more folding pathways passing through the misfolded state, leading to a higher population of the misfolded intermediate. In an attractive cage, the attractive interactions could inhibit the formation of intrachain non-native contacts and provide alternate folding pathways to the native state so that the population of the misfolded intermediate decreases when increasing the strength of attractive interaction between the substrate protein and cavity wall. This study should be helpful in general to understand how the chaperonins reshape the folding energy landscape of some frustrated proteins.

MicroRNA-122 amplifies hepatitis C virus translation by shaping the structure of the internal ribosomal entry site

The liver-specific microRNA-122 (miR-122) recognizes two conserved sites at the 5′ end of the hepatitis C virus (HCV) genome and contributes to stability, translation, and replication of the viral RNA. We show that stimulation of the HCV internal ribosome entry site (IRES) by miR-122 is essential for efficient viral replication. The mechanism relies on a dual function of the 5′ terminal sequence in the complementary positive (translation) and negative strand (replication), requiring different secondary structures. Predictions and experimental evidence argue for several alternative folds involving the miR-binding region (MBR) adjacent to the IRES and interfering with its function. Mutations in the MBR, designed to suppress these dysfunctional structures indeed stimulate translation independently of miR-122. Conversely, MBR mutants favoring alternative folds show impaired IRES activity. Our results therefore suggest that miR-122 binding assists the folding of a functional IRES in an RNA chaperone-like manner by suppressing energetically favorable alternative secondary structures.

Aminoacyl-tRNA synthetase evolution and sectoring of the genetic code

ABSTRACTThe genetic code sectored via tRNA charging errors, and the code progressed toward closure and universality because of evolution of aminoacyl-tRNA synthetase (aaRS) fidelity and translational fidelity mechanisms. Class I and class II aaRS folds are identified as homologs. From sequence alignments, a structurally conserved Zn-binding domain common to class I and class II aaRS was identified. A model for the class I and class II aaRS alternate folding pathways is posited. Five mechanisms toward code closure are highlighted: 1) aaRS proofreading to remove mischarged amino acids from tRNA; 2) accurate aaRS active site specification of amino acid substrates; 3) aaRS-tRNA anticodon recognition; 4) conformational coupling proofreading of the anticodon-codon interaction; and 5) deamination of tRNA wobble adenine to inosine. In tRNA anticodons there is strong wobble sequence preference that results in a broader spectrum of contacts to synonymous mRNA codon wobble bases. Adenine is excluded from the anticodon wobble position of tRNA unless it is modified to inosine. Uracil is generally preferred to cytosine in the tRNA anticodon wobble position. Because of wobble ambiguity when tRNA reads mRNA, the maximal coding capacity of the three nucleotide code read by tRNA is 31 amino acids + stops.

Fn3 proteins engineered to recognize tumor biomarker mesothelin internalize upon binding.

Mesothelin is a cell surface protein that is overexpressed in numerous cancers, including breast, ovarian, lung, liver, and pancreatic tumors. Aberrant expression of mesothelin has been shown to promote tumor progression and metastasis through interaction with established tumor biomarker CA125. Therefore, molecules that specifically bind to mesothelin have potential therapeutic and diagnostic applications. However, no mesothelin-targeting molecules are currently approved for routine clinical use. While antibodies that target mesothelin are in development, some clinical applications may require a targeting molecule with an alternative protein fold. For example, non-antibody proteins are more suitable for molecular imaging and may facilitate diverse chemical conjugation strategies to create drug delivery complexes. In this work, we engineered variants of the fibronectin type III domain (Fn3) non-antibody protein scaffold to bind to mesothelin with high affinity, using directed evolution and yeast surface display. Lead engineered Fn3 variants were solubly produced and purified from bacterial culture at high yield. Upon specific binding to mesothelin on human cancer cell lines, the engineered Fn3 proteins internalized and co-localized to early endosomes. To our knowledge, this is the first report of non-antibody proteins engineered to bind mesothelin. The results validate that non-antibody proteins can be engineered to bind to tumor biomarker mesothelin, and encourage the continued development of engineered variants for applications such as targeted diagnostics and therapeutics.

Fluorine-Mediated Editing of a G-Quadruplex Folding Pathway.

A (3+1)-hybrid-type G-quadruplex was substituted within its central tetrad by a single 2'-fluoro-modified guanosine. Driven by the anti-favoring nucleoside analogue, a novel quadruplex fold with inversion of a single G-tract and conversion of a propeller loop into a lateral loop emerges. In addition, scalar couplings across hydrogen bonds demonstrate the formation of intra- and inter-residual F⋅⋅⋅H8-C8 pseudo-hydrogen bonds within the modified quadruplexes. Alternative folding can be rationalized by the impact of fluorine on intermediate species on the basis of a kinetic partitioning mechanism. Apparently, chemical or other environmental perturbations are able to redirect folding of a quadruplex, possibly modulating its regulatory role in physiological processes.