Concentrations Of Guanidinium Chloride Research Articles

Investigating functional and folding stability of an engineered E. coli L-asparaginase harboring Y176F/S241C mutations

Purpose: L-asparaginase has been widely recognized as a critical component in the treatment of various types of lymphoproliferative disorders, since its introduction in 1960s. However, its use in some cases leads to allergic reactions rendering the continuation of treatment unfeasible. Thus, the development of L-asparaginase from alternative sources or the production of engineered enzymes have always been considered. This study aimed to produce and evaluate a novel enzyme designed based on the sequence of L-asparaginase from E. coli bacteria with Y176F/S241C mutations. Methods: The Y176F/S241C mutant L-asparaginase was successfully expressed as the GST-fusion protein in E. coli, and then was subjected to affinity and size exclusion chromatography. The activity of the purified enzyme was determined based on the released ammonia as the result of substrate hydrolysis using Nessler’s reagent. Chemical denaturation experiment in the presence of increasing concentration of guanidinium chloride was applied to determine the folding stability of the purified enzyme. Results: The mutant enzyme was purified with an efficiency of 77-fold but at a low recovery of 0.7%. The determined kinetic parameters Km, Vmax, kcat, specific activity and catalytic efficiency were 13.96 (mM), 2.218(mM/min), 273.9 (min-1), 237.8 (IU/mg) and 19.62 (mM-1 min-1), respectively. Moreover, unfolding free energy determined by guanidinium chloride induced denaturation for mutated and commercial L-asparaginase enzymes were 8421 J/mol and 5274 J/mol, respectively. Conclusion: The mutant enzyme showed improved stability over the wild-type. Although the expression level and recovery were low, the mutant L-asparaginase demonstrated promising activity and stability, with potential clinical and industrial applications.

Electro-osmotic Flow Generation via a Sticky Ion Action.

Selective transport of ions through nanometer-sized pores is fundamental to cell biology and central to many technological processes such as water desalination and electrical energy storage. Conventional methods for generating ion selectivity include placement of fixed electrical charges at the inner surface of a nanopore through either point mutations in a protein pore or chemical treatment of a solid-state nanopore surface, with each nanopore type requiring a custom approach. Here, we describe a general method for transforming a nanoscale pore into a highly selective, anion-conducting channel capable of generating a giant electro-osmotic effect. Our molecular dynamics simulations and reverse potential measurements show that exposure of a biological nanopore to high concentrations of guanidinium chloride renders the nanopore surface positively charged due to transient binding of guanidinium cations to the protein surface. A comparison of four biological nanopores reveals the relationship between ion selectivity, nanopore shape, composition of the nanopore surface, and electro-osmotic flow. Guanidinium ions are also found to produce anion selectivity and a giant electro-osmotic flow in solid-state nanopores via the same mechanism. Our sticky-ion approach to generate electro-osmotic flow can have numerous applications in controlling molecular transport at the nanoscale and for detection, identification, and sequencing of individual proteins.

Cooperativity and Folding Kinetics in a Multidomain Protein with Interwoven Chain Topology.

Although a large percentage of eukaryotic proteomes consist of proteins with multiple domains, not much is known about their assembly mechanism, especially those with intricate native state architectures. Some have a complex topology in which the structural elements along the sequence are interwoven in such a manner that the domains cannot be separated by cutting at any location along the sequence. Such proteins are multiply connected multidomain proteins (MMPs) with the three-domain (NMP, LID, and CORE) phosphotransferase enzyme adenylate kinase (ADK) being an example. We devised a coarse-grained model to simulate ADK folding initiated by changing either the temperature or guanidinium chloride (GdmCl) concentration. The simulations reproduce the experimentally measured melting temperatures (associated with two equilibrium transitions), FRET efficiency as a function of GdmCl concentration, and the folding times quantitatively. Although the NMP domain orders independently, cooperative interactions between the LID and the CORE domains are required for complete assembly of the enzyme. Kinetic simulations show that, on the collapse time scale, multiple interconnected metastable states are populated, attesting to the folding heterogeneity. The network of kinetically connected states reveals that the CORE domain folds only after the NMP and LID domains, reflecting the interwoven nature of the chain topology.

The denatured state of HIV-1 protease under native conditions.

The denatured state of several proteins has been shown to display transient structures that are relevant for folding, stability, and aggregation. To detect them by nuclear magnetic resonance (NMR) spectroscopy, the denatured state must be stabilized by chemical agents or changes in temperature. This makes the environment different from that experienced in biologically relevant processes. Using high‐resolution heteronuclear NMR spectroscopy, we have characterized several denatured states of a monomeric variant of HIV‐1 protease, which is natively structured in water, induced by different concentrations of urea, guanidinium chloride, and acetic acid. We have extrapolated the chemical shifts and the relaxation parameters to the denaturant‐free denatured state at native conditions, showing that they converge to the same values. Subsequently, we characterized the conformational properties of this biologically relevant denatured state under native conditions by advanced molecular dynamics simulations and validated the results by comparison to experimental data. We show that the denatured state of HIV‐1 protease under native conditions displays rich patterns of transient native and non‐native structures, which could be of relevance to its guidance through a complex folding process.

Stable polymer bilayers for protein channel recordings at high guanidinium chloride concentrations

The use of chaotropic reagents is common in biophysical characterization of biomolecules. When the study involves transmembrane protein channels, the stability of the protein channel and supporting bilayer membrane must be considered. In this letter, we show that planar bilayers composed of poly(1,2-butadiene)-b-poly(ethylene oxide) diblock copolymer are stable and leak-free at high guanidinium chloride concentrations, in contrast to diphytanoyl phosphatidylcholine bilayers, which exhibit deleterious leakage under similar conditions. Furthermore, insertion and functional analysis of channels such as α-hemolysin and MspA are straightforward in these polymer membranes. Finally, we demonstrate that α-hemolysin channels maintain their structural integrity at 2 M guanidinium chloride concentrations using blunt DNA hairpins as molecular reporters.

Guanidinium binding to proteins: The intriguing effects on the D1 and D2 domains of Thermotoga maritima Arginine Binding Protein and a comprehensive analysis of the Protein Data Bank

Thermotoga maritima Arginine Binding Protein has been extensively characterized because of its peculiar features and its possible use as a biosensor. In this characterization, deletion of the C-terminal helix to obtain the monomeric protein TmArgBP20–233 and dissection of the monomer in its two domains, D1 and D2, have been performed. In the present study the stability of these three forms against guanidinium chloride is investigated by means of circular dichroism and differential scanning calorimetry measurements. All three proteins show a high conformational stability; moreover, D1 shows an unusual behavior in the presence of low concentrations of guanidinium chloride. This finding has led us to investigate a possible binding interaction by means of isothermal titration calorimetry and X-ray crystallography; the results indicate that D1 is able to bind the guanidinium ion (GuH+), due to its similarity with the arginine terminal moiety. The analysis of the structural and dynamic properties of the D1-GuH+ complex indicates that the protein binds the ligand through multiple and diversified interactions. An exhaustive survey of the binding modes of GuH+ to proteins indicates that this is a rather common feature. These observations provide interesting insights into the effects that GuH+ is able to induce in protein structures.

Kinetic Stability of Long-Lived Human Lens γ-Crystallins and Their Isolated Double Greek Key Domains

The γ-crystallins of the eye lens nucleus are among the longest-lived proteins in the human body. Synthesized in utero, they must remain folded and soluble throughout adulthood to maintain lens transparency and avoid cataracts. γD- and γS-crystallin are two major monomeric crystallins of the human lens. γD-crystallin is concentrated in the oldest lens fiber cells, the lens nucleus, whereas γS-crystallin is concentrated in the younger cells of the lens cortex. The kinetic stability parameters of these two-domain proteins and their isolated domains were determined and compared. Kinetic unfolding experiments monitored by fluorescence spectroscopy in varying concentrations of guanidinium chloride were used to extrapolate unfolding rate constants and half-lives of the crystallins in the absence of the denaturant. Consistent with their long lifespans in the lens, extrapolated half-lives for the initial unfolding step were on the timescale of years. Both proteins’ isolated N-terminal domains were less kinetically stable than their respective C-terminal domains at denaturant concentrations predicted to disrupt the domain interface, but at low denaturant concentrations, the relative kinetic stabilities were reversed. Cataract-associated aggregation has been shown to proceed from partially unfolded intermediates in these proteins; their extreme kinetic stability likely evolved to protect the lens from the initiation of aggregation reactions. Our findings indicate that the domain interface is the source of significant kinetic stability. The gene duplication and fusion event that produced the modern two-domain architecture of vertebrate lens crystallins may be the origin of their high kinetic as well as thermodynamic stability.

Investigation of the role of central metal ion of Cyathus bulleri laccase 1 using guanidinium chloride-induced denaturation.

The structure and folding/unfolding kinetics of Cyathus bulleri laccase 1 (Lcc1), expressed in Pichia pastoris, were analyzed by spectroscopic methods to understand the role of central metal ion. Far UV CD structure analysis revealed major β-sheet and minor α helical segments present in the Lcc1. A significant change in the spectrum of Lcc1, indicative of unfolding of secondary structures, was observed with increasing concentrations of guanidinium chloride (GdnHCl) during Trp fluorescence, absorption and CD measurements. A similar trend was also noticed for enzyme activity with respect to GdnHCl concentrations. To establish the role of copper ion in the catalytic activity of laccase, a complete removal of copper was carried out and addition of copper was found to restore the structure and activity during the refolding process. The apo form was also reconstituted by addition of zinc ion which restored nearly 84% of enzyme activity, indicating non-essential role of copper in maintaining conformation and activity. Structural studies and inductively coupled plasma mass spectrometry data supported these observations.

Isolated domains of recombinant human apo-metallothionein 1A are folded at neutral pH: a denaturant and heat-induced unfolding study using ESI-MS.

Metallothioneins (MTs) are characterized by their high metal loading capacity, small molecular weight, and abundant cysteine residues. It has long been thought that metal-free, or apo-MT peptides were unstructured and only adopted as a distinct conformation upon forming the metal clusters, described as metal-induced folding. More recent studies have suggested that the presence of a globular, yet loosely defined structure actually exists that can be disrupted or unfolded. Residue modification and ion-mobility ESI (IM-ESI)-MS have been used to examine this unusual unfolding process. The structure of apo-MT plays a critical role as the starting point in the flexible metalation pathways that can accommodate numerous soft metals. ESI-MS measurements of the product species formed following the cysteine alkylation of the isolated domain fragments of recombinant human apo-MT 1A with n-ethylmaleimide (NEM) were used in the present study to monitor the denaturant- and heat-induced unfolding at physiological pH. The results indicate that these apo-MT fragments adopt distinct structures at neutral pH that react co-operatively with NEM when folded and non-cooperatively when heated or exposed to high concentrations of the denaturant guanidinium chloride (GdmCl). From these studies, we can conclude that at neutral pH, the domain fragments are folded into globular structures where some of the free cysteine residues are buried within the core and are stabilized by hydrogen bonds. Metalation therefore, must take place from the folded conformation.

Counteraction ability of TMAO toward different denaturing agents.

Differential scanning calorimetry measurements performed on RNase A in aqueous binary solutions containing different concentrations of urea, tetramethylurea, guanidinium chloride, and guanidinium thiocyanate, and in aqueous ternary solutions, containing the same denaturants plus 1 M trimethylamine N-oxide, TMAO, demonstrate that the latter has a general counteracting ability at pH 7.0, but not at pH 4.0. Experimental data rule out the idea that counteraction originates from direct interactions between TMAO molecules and denaturing agents. A rationalization is provided on the basis of a theoretical approach grounded on the solvent-excluded volume effect, whose magnitude depends on the density of aqueous solutions.

Glutamate Induced Thermal Equilibrium Intermediate and Counteracting Effect on Chemical Denaturation of Proteins.

When organisms are subjected to stress conditions, one of their adaptive responses is accumulation of small organic molecules called osmolytes. These osmolytes affect the structure and stability of the biological macromolecules including proteins. The present study examines the effect of a negatively charged amino acid osmolyte, glutamate (Glu), on two model proteins, ribonuclease A (RNase A) and α-lactalbumin (α-LA), which have positive and negative surface charges at pH 7, respectively. These proteins follow two-state unfolding transitions during both heat and chemical induced denaturation processes. The addition of Glu stabilizes the proteins against temperature and induces an early equilibrium intermediate during unfolding. The stability is found to be enthalpy-driven, and the free energy of stabilization is more for α-LA compared to RNase A. The decrease in the partial molar volume and compressibility of both of the proteins in the presence of Glu suggests that the proteins attain a more compact state through surface hydration which could provide a more stable conformation. This is also supported by molecule dynamic simulation studies which demonstrate that the water density around the proteins is increased upon the addition of Glu. Further, the intermediates could be completely destabilized by lower concentrations (∼0.5 M) of guanidinium chloride and salt. However, urea subverts the Glu-induced intermediate formed by α-LA, whereas it only slightly destabilizes in the case of RNase A which has a positive surface charge and could possess charge-charge interactions with Glu. This suggests that, apart from hydration, columbic interactions might also contribute to the stability of the intermediate. Gdm-induced denaturation of RNase A and α-LA in the absence and the presence of Glu at different temperatures was carried out. These results also show the Glu-induced stabilization of both of the proteins; however, all of the unfolding transitions followed two-state transitions during chemical denaturation. The extent of stability exerted by Glu is higher for RNase A at higher temperature, whereas it provides more stability for α-LA at lower temperature. Thus, the experiments indicate that Glu induces a thermal equilibrium intermediate and increases the thermodynamic stability of proteins irrespective of their surface charges. The extent of stability varies between the proteins in a temperature-dependent manner.

Thermal and chemical denaturation of Colocasia esculenta tuber agglutinin from α2β2 to unfolded state

The major tuber storage protein of Colocasia esculenta, is a monocot mannose-binding, widely used dietary lectin, containing two polypeptides of 12.0 and 12.4 kDa. By both gel filtration and dynamic light scattering at pH 7.2, the lectin has a α2β2 form of apparent molecular mass of 48.2 kDa and a hydrodynamic radius of 6.1 ± .2 nm; however, at pH 3, it migrates as αβ and has a reduced hydrodynamic radius of 4.6 ± .3 nm. Our circular dichroism spectroscopy studies show that the lectin retains approximately 100% of its secondary structure between pH 2–8, going down to ~90% for extreme acidic/alkaline conditions. The fluorescence emission maxima of 346 to 350 nm for pH 4 to 10 show that the tryptophan residues are relatively exposed. The unfolding is a simple two-state process, N4 ↔ 4U, as seen in our denaturation scan profiles. These denaturation profiles, monitored separately by fluorescence, far-UV CD, and near-UV CD, are completely super imposable. Analyses of these profiles provide an estimate of several thermodynamic parameters at each guanidinium chloride concentration, including the melting temperature Tg, which is 346.9 K in 0 M, but lowers to 321.8 K in 3.6 M. Dimeric and tetrameric interfaces observed in the crystal structure for the same protein are used to rationalize solution data in some detail.

Destabilization of the Quaternary Structure of Bovine Heart Cytochrome c Oxidase upon Removal of Tightly Bound Cardiolipin

The quaternary structural stability of cardiolipin-containing (CcO(CL+)) versus CL-free cytochrome c oxidase (CcO(CL-)) was compared using structural perturbants as probes. Exposure to increasing concentrations of urea or guanidinium chloride causes sequential dissociation of five subunits from CcO(CL+) in the order VIa and VIb, followed by III and VIIa, and ultimately Vb. Removal of CL from CcO destabilizes the association of each of these five subunits with the core of CcO. Subunits VIa and VIb spontaneously dissociate from CcO(CL-) even in the absence of denaturant and are no longer present after purification of the CL-free 11-subunit complex by ion exchange chromatography. The other 11 subunits remain associated in a partially active complex, but the association of subunits III, VIIa, and Vb is weakened; i.e., the midpoints for the subunit dissociation curves are each shifted to a lower perturbant concentration (lower by 1.1-1.7 M urea; lower by 0.3-0.4 M GdmCl). This corresponds to a decrease of ∼9 kJ in the Gibbs free association energy for each of these subunits when CL is removed from CcO. With either CcO(CL+) or CcO(CL-), loss of enzymatic activity occurs coincident with dissociation of subunits III and VIIa. The loss of activity is irreversible, and reactivation of CcO(CL-) by exogenous CL occurs only if both subunits remain associated with the core of CcO. Inclusion of sulfate anions stabilizes the association of VIIa more than III, resulting in a slight separation of the urea-induced dissociation curves. In this case, activity loss correlates much better with dissociation of subunit VIIa than III. We conclude that (1) bound cardiolipin is an important stabilizing factor in the quaternary structure of CcO and (2) association of subunit VIIa (possibly together with subunit III) is critical for functional CL binding and full electron-transfer activity of CcO.

Insights into the interactions between enzyme and co-solvents: Stability and activity of stem bromelain

In present study, an attempt is made to elucidate the effects of various naturally occurring osmolytes and denaturants on BM at pH 7.0. The effects of the varying concentrations of glycerol, sorbitol, sucrose, trehalose, urea and guanidinium chloride (GdnHCl) on structure, stability and activity of BM are explored by fluorescence spectroscopy, circular dichroism (CD), UV–vis spectroscopy and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Our experimental observations reveal that glycerol and sorbitol are acting as stabilizers at all concentrations while sucrose and trehalose are found to be destabilizers at lower concentrations, however, acted as stabilizers at higher concentrations. On the other hand, urea and GdnHCl are denaturants except at lower concentrations. There is a direct relationship between activity and conformational stability as the activity data are found to be in accordance with conformational stability parameters (ΔGu, Tm, ΔCp) and BM profile on SDS-PAGE.

Concentration-dependent like-charge pairing of guanidinium ions and effect of guanidinium chloride on the structure and dynamics of water from all-atom molecular dynamics simulation

An all-atom molecular dynamics simulation shows concentration-dependent like-charge ion pairing of the guanidinium ion in an aqueous solution of guanidinium chloride. We have observed two types of like-charge ion pairing for guanidinium ions, namely, stacked ion pairs and solvent-separated ion pairs. Interestingly, both of these like-charge ion-pair formations are dependent on the concentration of guanidinium chloride in water. The probability of stacked like-charge ion-pair formation decreases, whereas, the probability of solvent-separated like-charge pairing increases as the concentration of guanidinium chloride increases, which is shown from radial distribution functions and is confirmed from the energy calculations. Besides like-charge ion-pair formation, we also investigated guanidinium chloride induced changes in water structure. Hydrogen-bond analysis indicates that guanidinium chloride does not alter the strict-hydrogen-bonding patterns of water, whereas, it breaks the bend-hydrogen bond and the non-hydrogen-bonding patterns. Tetrahedral order, nearest neighbor orientation, and distance distribution of water molecules around a probe water molecule show the extent of water structure distortion.

The Molecular Mechanism of Hsp100 Chaperone Inhibition by the Prion Curing Agent Guanidinium Chloride

The Hsp100 chaperones ClpB and Hsp104 utilize the energy from ATP hydrolysis to reactivate aggregated proteins in concert with the DnaK/Hsp70 chaperone system, thereby playing an important role in protein quality control. They belong to the family of AAA+ proteins (ATPases associated with various cellular activities), possess two nucleotide binding domains per monomer (NBD1 and NBD2), and oligomerize into hexameric ring complexes. Furthermore, Hsp104 is involved in yeast prion propagation and inheritance. It is well established that low concentrations of guanidinium chloride (GdmCl) inhibit the ATPase activity of Hsp104, leading to so called "prion curing," the loss of prion-related phenotypes. Here, we present mechanistic details about the Hsp100 chaperone inhibition by GdmCl using the Hsp104 homolog ClpB from Thermus thermophilus. Initially, we demonstrate that NBD1 of ClpB, which was previously considered inactive as a separately expressed construct, is a fully active ATPase on its own. Next, we show that only NBD1, but not NBD2, is affected by GdmCl. We present a crystal structure of ClpB NBD1 in complex with GdmCl and ADP, showing that the Gdm(+) ion binds specifically to the active site of NBD1. A conserved essential glutamate residue is involved in this interaction. Additionally, Gdm(+) interacts directly with the nucleotide, thereby increasing the nucleotide binding affinity of NBD1. We propose that both the interference with the essential glutamate and the modulation of nucleotide binding properties in NBD1 is responsible for the GdmCl-specific inhibition of Hsp100 chaperones.

N-Propanol Based Solubilization Buffer Enhances Refolding Yield of Inclusion Body Protein by Populating Intermediates to the Folding Pathway

Most of the times, high level expression of recombinant proteins in bacteria results in accumulation of recombinant proteins into inclusion body (IB) aggregates. To obtain native protein from these aggregates, it is necessary to solubilize these aggregates followed by refolding of solubilized protein by appropriate refolding method. Conventionally, high concentration of denaturant like urea or guanidinium chloride (GdmCl) is used for solubilization of inclusion bodies which often results into aggregation of protein during refolding process. In the present study we have evaluated a novel solubilization method using n-propanol in presence of low concentration of urea. n-Propanol based solubilization agent was compared with traditional solubilization agents like 8 M urea and 6 M GdmCl for solubilization efficiency, structure and stability of the solubilized model protein, recombinant human growth hormone (hGH). hGH IBs were found to be tough and were only solubilized efficiently in presence of high concentration of denaturants (8 M urea or 6 M GdmCl). 4 to 6 M n-propanol in combination with 2 M urea was sufficient for the efficient solubilization of hGH IBs. Aggregation during refolding was also studied and it was found that solubilization with n-propanol based buffer resulted into bioactive hGH without aggregation giving significantly higher refolding yield in comparison to those obtained with urea and GdmCl based buffers which resulted in aggregation of hGH during refolding. From the results obtained, it can be concluded that solubilization of hGH IBs in n-propanol based buffer results in a partially folded folding intermediate of hGH which readily folds into native form on dilution with reduced chances of protein getting into aggregation pathway.

High throughput testing of drug library substances and monoclonal antibodies for capacity to reduce formation of cystatin C dimers to identify candidates for treatment of hereditary cystatin C amyloid angiopathy

Objective: To establish a high-throughput system for testing the ability of drugs or monoclonal antibodies to reduce the in vitro formation of cystatin C dimers to identify substances potentially useful for treatment of patients with hereditary cystatin C amyloid angiopathy (HCCAA). Methods: Various combinations of incubation temperature, time period, guanidinium chloride concentration and concentration of cystatin C monomers were tested in low-volume formats to induce dimer formation of recombinant cystatin C. The extent of dimerization was analysed by gel filtration chromatography and agarose gel electrophoresis. Results: A high-throughput system based upon agarose gel electrophoresis was developed and used to test 1040 drugs in a clinical drug library for their capacity to reduce cystatin C dimer formation in vitro. Seventeen substances reducing dimer formation by more than 30% were identified. A similar system for testing the capacity of monoclonal antibodies against cystatin C to reduce the in vitro formation of cystatin C dimers was also developed and used to test a panel of 12 monoclonal antibodies. Seven antibodies reducing dimer formation by more than 30% were identified and the two most potent, Cyst28 and HCC3, reduced dimerization by 75 and 60%, respectively. Conclusion: We constructed a simple high-throughput system for testing the capacity of drugs and monoclonal antibodies to reduce the in vitro formation of cystatin C dimers and several candidates for treatment of HCCAA could be identified.

Collapse kinetics and chevron plots from simulations of denaturant-dependent folding of globular proteins

Quantitative description of how proteins fold under experimental conditions remains a challenging problem. Experiments often use urea and guanidinium chloride to study folding whereas the natural variable in simulations is temperature. To bridge the gap, we use the molecular transfer model that combines measured denaturant-dependent transfer free energies for the peptide group and amino acid residues, and a coarse-grained C(α)-side chain model for polypeptide chains to simulate the folding of src SH(3) domain. Stability of the native state decreases linearly as [C] (the concentration of guanidinium chloride) increases with the slope, m, that is in excellent agreement with experiments. Remarkably, the calculated folding rate at [C] = 0 is only 16-fold larger than the measured value. Most importantly ln k(obs) (k(obs) is the sum of folding and unfolding rates) as a function of [C] has the characteristic V (chevron) shape. In every folding trajectory, the times for reaching the native state, interactions stabilizing all the substructures, and global collapse coincide. The value of (m(f) is the slope of the folding arm of the chevron plot) is identical to the fraction of buried solvent accessible surface area in the structures of the transition state ensemble. In the dominant transition state, which does not vary significantly at low [C], the core of the protein and certain loops are structured. Besides solving the long-standing problem of computing the chevron plot, our work lays the foundation for incorporating denaturant effects in a physically transparent manner either in all-atom or coarse-grained simulations.

The CLN025 Decapeptide Retains a β-Hairpin Conformation in Urea and Guanidinium Chloride

The conformational stability of the β-hairpin miniprotein, CLN025, a variant of chignolin in which the N- and C-terminal glycines are replaced by tyrosines, in various concentrations of guanidinium chloride (GdmCl) and urea was examined by molecular dynamics simulations and electronic circular dichroism (ECD) spectropolarimetry. The peptide maintains its β-hairpin conformation in GdmCl and urea solutions. In GdmCl, Gly7 influences the turn to reduce the number of Asp3-Gly7 H-bonds and the Tyr1-Trp9 H-bond is lost. The structure of the peptide is less stable in 3 M GdmCl than in water or 6 M GdmCl, because the number of Asp3-Thr8 and Tyr1-Tyr10 H-bonds are reduced and the Tyr2 side chain moves away from the Pro4 and Trp9 side chains and toward the Tyr10 side chain. This reduces the number of Tyr2-Pro4 CH-π interactions and Tyr2-Trp9 and Tyr1-Tyr10 aromatic-aromatic (Ar-Ar) interactions and increases the number of Tyr2-Tyr10 Ar-Ar interactions. In 6 M GdmCl at 300 and 333 K, the number of Tyr1-Tyr10 and Asp3-Thr8 H-bonds increases, but fewer structures have Tyr2-Pro4 CH-π and Tyr1-Tyr10 and Tyr2-Trp9 Ar-Ar interactions. In urea, Gly7 is in a mixture of β-turn and random meander structures and the number of Asp3-Thr6 and Tyr1-Tyr10 H-bonds are reduced as are the number of Tyr2-Pro4 CH-π interactions and Tyr1-Tyr10 and Tyr2-Trp9 Ar-Ar interactions. In 4 M urea, a shorter turn places Gly7 into the β-sheet region and Tyr10 is pushed out into the solvent. In 8 M urea, the number of Asp3-Glu5 H-bonds is increased and the β-sheet is lost, but the electrostatic interaction between the charged termini is restored and a cation-π interaction between the indolyl ring of Trp9 and the positively charged N-terminus is formed. In 8 M urea at 333 K, the β-hairpin conformation is almost lost. The structure of CLN025 is stable, because the weakly polar interactions and H-bonds maintain the β-hairpin conformation in the various environments. CLN025 should not be considered a miniprotein, because it lacks a well-defined tertiary structure, it is resistant to denaturation, it does not have an increased heat capacity near its melting temperature, and the structures near and above the melting temperature retain a β-hairpin conformation.