Changes In Solvent Conditions Research Articles

Exploration of various solid forms of novel (1H-Benzimidazole, 2-[4-(1,1-dimethylethyl) phenyl

Polymorphism/solid forms are acknowledged to have important implications in the pharmaceutical industry, hence regulating polymorph/solid forms formation is essential. 1H-Benzimidazole, 2-[4-(1,1-dimethylethyl)phenyl] (BDP) drug has been discovered as a transcriptional activator of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in certain metabolic tissues. Up to date, there is no solid forms of BDP have been reported. The purpose of this study was to see how changing solvent conditions and co-formers influenced the formation of distinct solid forms of BDP. The solvents were chosen based on their polarity, as different polarity solvents are anticipated to interact differently with APIs, impacting the creation of diverse solid forms. Similarly, two GRAS (Generally Recognized As Safe) co-formers, Oxalic Acid (OA) and Malonic Acid (MA), were employed to test the potential of various forms of BDP to form diverse supramolecular networks that might improve BDP's physicochemical qualities. The experimental finding reveals that one polymorph, two solvates and three molecular salts have been obtained by solvent evaporation (SE) and liquid assisted grinding (LAG) methods. Interestingly, BDP creates a similar channel structure in all the two solvates (acetone and isopropanol). Furthermore, different forms of BDP using the same co-former show different molecular assemblages, confirming the occurrence of multiple forms. PXRD, SCXRD, and thermal analyses were used to characterize all of the synthesized forms. The findings of this study indicate that solvent choice and synthesis process are important factors in polymorph screening and can influence the creation of diverse molecular forms.

Frenkel excitons in heat-stressed supramolecular nanocomposites enabled by tunable cage-like scaffolding.

Delocalized Frenkel excitons-coherently shared excitations among chromophores-are responsible for the remarkable efficiency of supramolecular light-harvesting assemblies within photosynthetic organisms. The translation of nature's design principles to applications in optoelectronic devices has been limited by the fragility of the supramolecular structures used and the delicate nature of Frenkel excitons, particularly under mildly changing solvent conditions and elevated temperatures and upon deposition onto solid substrates. Here, we overcome those functionalization barriers through composition of stable supramolecular light-harvesting nanotubes enabled by tunable (~4.3-4.9 nm), uniform (±0.3 nm) cage-like scaffolds. High-resolution cryogenic electron microscopy, combined with scanning electron microscopy, broadband femtosecond transient absorption spectroscopy and near-field scanning optical microscopy revealed that excitons within the cage-like scaffolds are robust, even under extreme heat stress, and control over nanocomposite dimensions is maintained on solid substrates. Our bio-inspired nanocomposites provide a general framework for the development of next-generation organic devices made from stable supramolecular materials.

Thermodynamic framework for switching the lower critical solution temperature of thermo-sensitive particle gels in aqueous solvent

Switching the lower critical solution temperatures (LCSTs) of thermo-sensitive particle gels including poly(N-isopropylacrylamide) (PNIPAM), poly(N-diethylacrylamide) (PNDEAM), and poly(N-vinylcaprolactam) (PNVCL) was investigated in various aqueous solvents. Three prepared particle gels represented a single LCST in a pure water solution near human body temperature. By adding a chaotropic co-solvent, such as dimethylformamide (DMF) or dimethyl sulfoxide (DMSO), to given gels in aqueous solution, the degree of influence on change of LCST was significantly different due to the difference in chemical structures. LCSTs of polymer solutions were affected by changing solvent conditions from pure water to mixed solvents. The LCST behaviors of linear PNIPAM, PNDEAM, and PNVCL in water/DMF or DMSO were determined by thermo-optical analysis (TOA), which supported the switchability of LCSTs in hydrogel systems. We used the photon correlation spectroscopy (PCS) technique to measure the switched LCSTs of hydrogel particles in mixed solvent systems. The switched LCSTs of the investigated systems exhibited a non-linear trend by varying co-solvent contents. A molecular thermodynamic framework, a combination of the modified double lattice with chain length dependence (MDL-CL) model and the Flory-Rehner (F-R) model, was utilized to describe and correlate the liquid-liquid equilibrium (LLE) and thermosensitive swelling behaviors between linear and cross-linked polymer solutions. With the addition of chaotropic co-solvents, a non-linear trend for phase behaviors of the three polymers was described after taking into account temperature and composition dependence on the association fraction (ηij) between the species. We assumed the donor-acceptor concept and competitive hydrogen bonding between polymer/water and water/co-solvent. The association fraction for the secondary lattice model was modified from an arbitrarily determined value to a physically meaningful value. Accordingly, the phase equilibrium of the amphiphilic polymers influenced by the structure-breaker characteristics of the co-solute was properly implemented in both experimental and modeling aspects.

A Decoy Folding Nucleus can Modulate Protein Folding Kinetics

The ability to modulate the folding kinetics of a specific protein in a milieu can be an important tool for chemical biology and pharmacology. Kinetics and thermodynamics of folding are routinely modulated by changing solvent conditions, but such changes are not protein specific. Here we demonstrate a general principle by which specific kinetic modulation can be achieved for any protein whose folding route is known. Our strategy is to design a pre-stabilized folding nucleus of a protein. We show that the β1- β2 part (residues 1 to 17) of ubiquitin, which is the segment known to fold first (into a β-hairpin shape)[1], can make folding faster when introduced as a separate conformationally constrained peptide at excess concentrations. A weaker but still significant effect is observed when this ‘decoy nucleus’ peptide is not conformationally constrained, or is in an unfolding buffer to start with. Interestingly, the thermodynamic stability of the protein remains unchanged, as the unfolding rate also becomes faster. We simulate the system for both a weakly constrained and a strongly constrained decoy nucleus, and observe that the decoy interacts strongly with the protein only when it is conformationally constrained. Together, our results suggest that the folding of almost any protein which possesses a well-defined folding nucleus can be modulated, by introducing a nucleus-mimicking molecule with a stabilized structure. It also implies that in vivo, fragments of proteins which are naturally present as degradation by-products can in principle affect the folding of the full length proteins. [1] Atomic-level description of ubiquitin folding. Piana et al., PNAS, 2013, 110 (15), 5915-5920

Protein Interactions that Enable Safe and Efficient Copper Ion Transport in the Human Cytoplasm

Although copper (Cu) is an essential metal for most living organisms, high levels and free such ions are toxic. In humans, after cellular uptake via the membrane-bound importer Ctr1, Cu is transported to targets by cytoplasmic Cu chaperones: Atox1 delivers Cu to membrane-bound P1B-type ATPases (i.e., ATP7B or Wilson disease protein) in the Golgi (secretory path; here, most Cu-dependent enzymes are loaded with Cu) whereas CCS delivers Cu specifically to cytoplasmic superoxide dismutase. In contrast to bacterial and yeast homologs, ATP7B has six similar metal-binding domains protruding into the cytoplasm: possibly, conformational changes among these regulate overall ATP7B activity. To reveal underlying molecular mechanisms as well as thermodynamic and kinetic driving forces for human Cu transport - from the cell membrane to the Golgi - our strategy involves a range of complementary biophysical experiments on purified proteins, domain constructs and engineered variants. From our studies, we have discovered that (a) the cytoplasmic C-terminus of Ctr1 binds Cu through its HCH motif with a moderate affinity that allows for Cu delivery to Atox1, (b) Atox1 can interact with CCS and exchange Cu implying cross-reactivity between cytoplasmic chaperones, (c) transfer of Cu from Atox1 to metal-binding domains in ATP7B proceeds through Cu-bridged hetero-protein dimers displaying enthalpy-entropy compensation, (d) conformational changes and domain-domain interactions within ATP7B depend on Cu loading status and minute changes in solvent conditions, and (e), in addition to its cytoplasmic chaperone activity, Atox1 may have functionality in the nucleus as it interacted with several DNA-binding proteins in a yeast two-hybrid screen.

Ultrafast Hydrogen Exchange Reveals Specific Structural Events during the Initial Stages of Folding of Cytochrome c

Many proteins undergo a sharp decrease in chain dimensions during early stages of folding, prior to the rate-limiting step in folding. However, it remains unclear whether compact states are the result of specific folding events or a general hydrophobic collapse of the poly peptide chain driven by the change in solvent conditions. To address this fundamental question, we extended the temporal resolution of NMR-detected H/D exchange labeling experiments into the microsecond regime by adopting a microfluidics approach. By observing the competition between H/D exchange and folding as a function of labeling pH, coupled with direct measurement of exchange rates in the unfolded state, we were able to monitor hydrogen-bond formation for over 50 individual backbone NH groups within the initial 140 microseconds of folding of horse cytochrome c. Clusters of solvent-shielded amide protons were observed in two α-helical segments in the C-terminal half of the protein, while the N-terminal helix remained largely unstructured, suggesting that proximity in the primary structure is a major factor in promoting helix formation and association at early stages of folding, while the entropically more costly long-range contacts between the N- and C-terminal helices are established only during later stages. Our findings clearly indicate that the initial chain condensation in cytochrome c is driven by specific interactions among a subset of α-helical segments rather than a general hydrophobic collapse.

Solvent Microenvironments and Copper Binding Alters the Conformation and Toxicity of a Prion Fragment

The secondary structures of amyloidogenic proteins are largely influenced by various intra and extra cellular microenvironments and metal ions that govern cytotoxicity. The secondary structure of a prion fragment, PrP(111-126), was determined using circular dichroism (CD) spectroscopy in various microenvironments. The conformational preferences of the prion peptide fragment were examined by changing solvent conditions and pH, and by introducing external stress (sonication). These physical and chemical environments simulate various cellular components at the water-membrane interface, namely differing aqueous environments and metal chelating ions. The results show that PrP(111-126) adopts different conformations in assembled and non-assembled forms. Aging studies on the PrP(111-126) peptide fragment in aqueous buffer demonstrated a structural transition from random coil to a stable β-sheet structure. A similar, but significantly accelerated structural transition was observed upon sonication in aqueous environment. With increasing TFE concentrations, the helical content of PrP(111-126) increased persistently during the structural transition process from random coil. In aqueous SDS solution, PrP(111-126) exhibited β-sheet conformation with greater α-helical content. No significant conformational changes were observed under various pH conditions. Addition of Cu2+ ions inhibited the structural transition and fibril formation of the peptide in a cell free in vitro system. The fact that Cu2+ supplementation attenuates the fibrillar assemblies and cytotoxicity of PrP(111-126) was witnessed through structural morphology studies using AFM as well as cytotoxicity using MTT measurements. We observed negligible effects during both physical and chemical stimulation on conformation of the prion fragment in the presence of Cu2+ ions. The toxicity of PrP(111-126) to cultured astrocytes was reduced following the addition of Cu2+ ions, owing to binding affinity of copper towards histidine moiety present in the peptide.

Nonuniform chain collapse during early stages of staphylococcal nuclease folding detected by fluorescence resonance energy transfer and ultrarapid mixing methods

The development of tertiary structure during folding of staphylococcal nuclease (SNase) was studied by time-resolved fluorescence resonance energy transfer measured using continuous- and stopped-flow techniques. Variants of this two-domain protein containing intradomain and interdomain fluorescence donor/acceptor pairs (Trp and Cys-linked fluorophore or quencher) were prepared to probe the intradomain and interdomain structural evolution accompanying SNase folding. The intra-domain donor/acceptor pairs are within the β-barrel domain (Trp27/Cys64 and Trp27/Cys97) and the interdomain pair is between the α-helical domain and the β-barrel domain (Trp140/Cys64). Time-resolved energy transfer efficiency accompanying folding and unfolding at different urea concentrations was measured over a time range from 30 μs to ≈ 10 s. Information on average donor/acceptor distances at different stages of the folding process was obtained by using a quantitative kinetic modeling approach. The average distance for the donor/acceptor pairs in the β-barrel domain decreases to nearly native values whereas that of the interdomain donor/acceptor pairs remains unchanged in the earliest intermediate (<500 μs of refolding). This indicates a rapid nonuniform collapse resulting in an ensemble of heterogeneous conformations in which the central region of the β-barrel domain is well developed while the C-terminal α-helical domain remains disordered. The distance between Trp140 and Cys64 decreases to native values on the 100-ms time scale, indicating that the α-helical domain docks onto the preformed β-barrel at a late stage of the folding. In addition, the unfolded state is found to be more compact under native conditions, suggesting that changes in solvent conditions may induce a nonspecific hydrophobic collapse.

Adsorption, structural alteration and elution of peptides at pendant PEO layers

An experimentally based, quantitative understanding of the entrapment and function of small peptides within PEO brush layers does not currently exist. Earlier work provided a rationale for expecting that an ordered, compact peptide will enter the PEO phase more readily than a peptide of similar size that adopts a less ordered, less compact form, and that amphiphilicity will promote peptide retention within the hydrophobic region of the PEO brush. Here we more deliberately describe criteria for peptide integration and structural change within the PEO brush, and discuss the reversibility of peptide entrapment with changing solvent conditions. For this purpose, circular dichroism (CD) was used to record the adsorption and conformational changes of (amphiphilic) WLBU2 and (non-amphiphilic) polyarginine peptides at uncoated (hydrophobic) and PEO-coated silica nanoparticles. Peptide conformation was controlled between disordered and α-helical forms by varying the concentration of perchlorate ion. We show an initially more ordered (α-helical) structure promotes peptide adsorption into the PEO layer. Further, a partially helical peptide undergoes an increase in helicity after entry, likely due to concomitant loss of capacity for peptide-solvent hydrogen bonding. Peptide interaction with the PEO chains resulted in entrapment and conformational change that was irreversible to elution with changing solution conditions in the case of the amphiphilic peptide. In contrast, the adsorption and conformational change of the non-amphiphilic peptide was reversible. These results indicate that responsive drug delivery systems based on peptide-loaded PEO layers can be controlled by modulation of solution conditions and peptide amphiphilicity.

Photoinduced fibrils formation of chicken egg white lysozyme under native conditions

AbstractRecent findings showed that transiently accessing structurally native‐like yet energetically higher conformational states is sufficient to trigger the formation of protein fibrils. Typically, these conformational states are made available through changing solvent conditions or introducing mutations. Here we show a novel way to initialize fibril formation for Chicken egg white lysozyme (CEWL) under native conditions via controlled UV illumination. Through a cassette of tryptophan‐based photochemistry, the two terminal disulfide bonds in CEWL can be selectively reduced. The reduced CEWL is then converted to conformational states with the C‐terminal fragment floppy upon thermal fluctuation. These states serve as precursors for the fibrillar aggregation. Intriguingly, the CEWL fibrils are stabilized by intermolecular disulfide bonds instead of noncovalent β‐sheet structures, distinct from the amyloid‐like lysozyme fibrils reported before. Based on the experimental evidences and all‐atom molecular dynamics simulation, we proposed a “runaway domain‐swapping” model for the structure of the CEWL fibrils, in which each CEWL molecule swaps the C‐terminal fragment into the complementary position of the adjacent molecule along the fibrils. We anticipate that this fibrillation mechanism can be extended to many other disulfide‐containing proteins. Our study stands for the first example of formation of protein fibrils under native conditions upon UV illumination and poses the potential danger of low UV dose to organisms at the protein level. Proteins 2012. © 2012 Wiley Periodicals, Inc.

The Mobility of Water Molecules through Hydrated Pores

To achieve a more exact understanding of the water transport in natural channels, a series of non-natural structures have been developed. They have been studied by far-infrared spectroscopy, solid-state nuclear magnetic resonance, differential scanning calorimetry, thermogravimetric analysis, and variable-temperature powder X-ray diffraction to examine the behavior of water at the molecular level. Water in these predominantly nonpolar pores can be metastable, with filling and emptying occurring upon changes in solvent conditions. The water contained in these pores exhibits a dynamics that might be controlled, since it depends on the structural features of the monomers that form the pore “skeleton”. We have observed changes in the pore diameter depending on the selected isomer. This provokes at a given temperature differences in the arrangement and dynamics of the contained water. The water dynamics increases with both temperature and pore diameter in a process that is reversible over a temperature range specific for each structure. Beyond this particular temperature threshold, the pore water can be irreversibly evacuated, and at this point a decrease of the dynamics is observed. The slower dynamics of the remaining water in partially evacuated pores is probably due to the increased interaction with the inner-pore surface owing to a concomitant narrowing of the pore. These findings not only highlight the need for the presence of freely moving water inside the pore to sustain its permeability by water, but also point to the decrease in the dynamics of the remaining water in partially evacuated pores.

Antibody Recognition of a Human Chorionic Gonadotropin Epitope (hCGβ66–80) Depends on Local Structure Retained in the Free Peptide

Human chorionic gonadotropin (hCG) is an important biomarker in pregnancy and oncology, where it is routinely detected and quantified by specific immunoassays. Intelligent epitope selection is essential to achieving the required assay performance. We present binding affinity measurements demonstrating that a typical β3-loop-specific monoclonal antibody (8G5) is highly selective in competitive immunoassays and distinguishes between hCGβ(66-80) and the closely related luteinizing hormone (LH) fragment LHβ(86-100), which differ only by a single amino acid residue. A combination of optical spectroscopic measurements and atomistic computer simulations on these free peptides reveals differences in turn type stabilized by specific hydrogen bonding motifs. We propose that these structural differences are the basis for the observed selectivity in the full protein.

Photo-Induced Fibrillar Formation of Chicken Egg White Lysozyme Under Native Conditions

Recent findings showed that transiently accessing structurally native-like yet energetically higher conformational states is sufficient to trigger the formation of protein fibrils. Typically, these conformational states are made available through changing solvent conditions or introducing mutations. Here we show a novel way to initialize fibril formation for Chicken egg white lysozyme (CEWL) under native conditions via controlled UV illumination. Through a cassette of tryptophan-based photochemistry, the two terminal disulfide bonds in CEWL can be selectively reduced. The reduced CEWL is then converted to conformational states with the C-terminal fragment floppy upon thermal fluctuation. These states serve as precursors for the fibrillar aggregation. Intriguingly, the CEWL fibrils are stabilized by intermolecular disulfide bonds instead of noncovalent β-sheet structures, distinct from the amyloid-like lysozyme fibrils reported before. Based on the experimental evidences and all-atom molecular dynamics simulation, we proposed a runaway domain-swapping model for the structure of the CEWL fibrils, in which each CEWL molecule swaps the C-terminal fragment into the complementary position of the adjacent molecule along the fibrils. We anticipate that this fibrillation mechanism can be extended to many other disulfide-containing proteins. Our study stands for the first example of formation of protein fibrils under native conditions upon UV illumination and poses the potential danger of low UV dose to organisms at the protein level.

Determination of the relative pKa's of mixtures of organic acids using NMR titration experiments based on aliased 1H–13C HSQC spectra

NMR titrations can be used to simultaneously determine the pKa's of mixtures of organic acids and bases provided chemical shifts are sensitive to their change of protonation states. Carbons are in principle better probes than protons because their chemical shifts are typically more sensitive to the changes in the electronic densities of the functional groups they are bound to. They are also less prone to overlap, do not suffer from second-order effects and are less sensitive to changes in solvent conditions. Recording HSQC spectra satisfactorily reduces the lack of sensitivity of carbon-detected experiments and the signal dispersion in two dimensions allows to automatically and finely track the drifts of a large number of signals simultaneously. The application of spectral aliasing to the carbon dimensions allows to quickly reach the high resolution required to precisely measure variations in carbon chemical shifts. The paper extends the methodology to the treatment of the data stemming from the simultaneous analysis of mixtures of compounds. It also introduces methods to improve the normalization process and deal with missing points. The application is not limited to the determination of pKa and can be readily extended to the determination of other chemical constants in experiments where the components of the mixture dynamically compete for substrates other than protons.

What Drives Amyloid Molecules To Assemble into Oligomers and Fibrils?

We develop a theory for three states of equilibrium of amyloid peptides: the monomer, oligomer, and fibril. We assume that the oligomeric state is a disordered micellelike collection of a few peptide chains held together loosely by hydrophobic interactions into a spherical hydrophobic core. We assume that fibrillar amyloid chains are aligned and further stabilized by steric zipper interactions—hydrogen bonding, steric packing, and specific hydrophobic side-chain contacts. The model makes a broad set of predictions that are consistent with experimental results: 1), Similar to surfactant micellization, amyloid oligomerization should increase with peptide concentration in solution. 2), The onset of fibrillization limits the concentration of oligomers in the solution. 3), The extent of A β fibrillization increases with peptide concentration. 4), The predicted average fibril length versus monomer concentration agrees with data on α-synuclein. 5), Full fibril length distributions agree with data on α-synuclein. 6), Denaturants should melt out fibrils. And finally, 7), added salt should stabilize fibrils by reducing repulsions between amyloid peptide chains. It is of interest that small changes in solvent conditions can tip the equilibrium balance between oligomer and fibril and cause large changes in rates through effects on the transition-state barrier. This model may provide useful insights into the physical processes underlying amyloid diseases.

Microfluidic dialysis cell for characterization of macromolecule interactions

Microfluidic dialysis is a promising tool for characterisation of macromolecule interactions and responsive materials, but until now it has been limited to the characterisation of high molecular weight molecules. Here, we introduce a low molecular weight cut-off dialysis membrane fabricated by coating a rigid membrane filter with a layer of crosslinked hydrogel. The modified hybrid silicone/glass microfluidic cells retain low molecular weight samples at a well-defined concentration for long periods of time, while allowing timely changes to be made in the fluid's supporting solvent (i.e. pH, ionic strength, ionic species). Straightforward access to the sample using high-resolution optical microscopy enables in situ characterisation of the sample's physical properties in response to changes in solvent conditions. A proof of principle measurement investigating the pH-sensitive binding between the protein/polysaccharide pair, neurocan and hyaluronan, is presented to demonstrate the integration of protein retention, solvent switching and fluorescence recovery after photobleaching (FRAP).

Role of partial protein unfolding in alcohol‐induced protein aggregation

Proteins aggregate in response to various stresses including changes in solvent conditions. Addition of alcohols has been recently shown to induce aggregation of disease-related as well as nondisease-related proteins. Here we probed the biophysical mechanisms underlying alcohol-induced protein aggregation, in particular the role of partial protein unfolding in aggregation. We have studied aggregation mechanisms due to benzyl alcohol which is used in numerous biochemical and biotechnological applications. We chose cytochrome c as a model protein, for the reason that various optical and structural probes are available to monitor its global and partial unfolding reactions. Benzyl alcohol induced the aggregation of cytochrome c in isothermal conditions and decreased the temperature at which the protein aggregates. However, benzyl alcohol did not perturb the overall native conformation of cytochrome c. Instead, it caused partial unfolding of a local protein region around the methionine residue at position 80. Site-specific optical probes, two-dimensional NMR titrations, and hydrogen exchange all support this conclusion. The protein aggregation temperature varied linearly with the melting temperature of the Met80 region. Stabilizing the Met80 region by heme iron reduction drastically decreased protein aggregation, which confirmed that the local unfolding of this region causes protein aggregation. These results indicate that a possible mechanism by which alcohols induce protein aggregation is through partial rather than complete unfolding of native proteins.

Protein Scaffold Engineering Towards Tunable Surface Attachment

Ringing the changes: A selective, controlled, and tunable attachment of proteins to a metal or to insulating surfaces is presented. A ring-shaped protein encapsulating SiO2-binding peptides (see picture: yellow) was engineered to expose multiple binding sites simply by changing solvent conditions, thereby creating a single monolayer without the need for surface modification.

Stabilization of conformationally dynamic helices by covalently attached acyl chains

Acylation of proteins is known to mediate membrane attachment and to influence subcellular sorting. Here, we report that acylation can stabilize secondary structure. Circular dichroism spectroscopy showed that N-terminal attachment of acyl chains decreases the ability of an intrinsically flexible hydrophobic model peptide to refold from an alpha-helical state to beta-sheet in response to changing solvent conditions. Acylation also stabilized the membrane-embedded alpha-helix. This increase of global helix stability did not result from decreased local conformational dynamics of the helix backbone as assessed by deuterium/hydrogen-exchange experiments. We concluded that acylation can stabilize the structure of intrinsically dynamic helices and may thus prevent misfolding.

The Effect of Increasing the Stability of Non-native Interactions on the Folding Landscape of the Bacterial Immunity Protein Im9

How stabilising non-native interactions influence protein folding energy landscapes is currently not well understood: such interactions could speed folding by reducing the conformational search to the native state, or could slow folding by increasing ruggedness. Here, we examine the influence of non-native interactions in the folding process of the bacterial immunity protein Im9, by exploiting our ability to manipulate the stability of the intermediate and rate-limiting transition state (TS) in the folding of this protein by minor alteration of its sequence or changes in solvent conditions. By analysing the properties of these species using Φ-value analysis, and exploration of the structural properties of the TS ensemble using molecular dynamics simulations, we demonstrate the importance of non-native interactions in immunity protein folding and demonstrate that the rate-limiting step involves partial reorganisation of these interactions as the TS ensemble is traversed. Moreover, we show that increasing the contribution to stability made by non-native interactions results in an increase in Φ-values of the TS ensemble without altering its structural properties or solvent-accessible surface area. The data suggest that the immunity proteins fold on multiple, but closely related, micropathways, resulting in a heterogeneous TS ensemble that responds subtly to mutation or changes in the solvent conditions. Thus, altering the relative strength of native and non-native interactions influences the search to the native state by restricting the pathways through the folding energy landscape.