Slow Exchange Research Articles

A method for selective 19 F-labeling absent of probe sequestration (SLAPS).

Fluorine (19F) offers several distinct advantages for biomolecular nuclear magnetic resonance spectroscopy such as no background signal, 100% natural abundance, high sensitivity, and a large chemical shift range. Exogenous cysteine‐reactive 19F‐probes have proven especially indispensable for characterizing large, challenging systems that are less amenable to other isotopic labeling strategies such as G protein‐coupled receptors. As fluorine linewidths are inherently broad, limiting reactions with offsite cysteines is critical for spectral simplification and accurate deconvolution of component peaks—especially when analyzing systems with intermediate to slow timescale conformational exchange. Here, we uncovered noncovalent probe sequestration by detergent proteomicelles as a second source of offsite labeling when using the popular 19F‐probe BTFMA (2‐bromo‐N‐(4‐[trifluoromethyl]phenyl)acetamide). The chemical shift and relaxation rates of these unreacted 19F‐BTFMA molecules are insufficient to distinguish them from protein‐conjugates, but they can be easily identified using mass spectrometry. We present a simple four‐step protocol for Selective Labeling Absent of Probe Sequestration (SLAPS): physically disrupt cell membranes in the absence of detergent, incubate membranes with cysteine‐reactive 19F‐BTFMA, remove excess unreacted 19F‐BTFMA molecules via ultracentrifugation, and finally solubilize in the detergent of choice. Our approach builds upon the in‐membrane chemical modification method with the addition of one crucial step: removal of unreacted 19F‐probes by ultracentrifugation prior to detergent solubilization. SLAPS is broadly applicable to other lipophilic cysteine‐reactive probes and membrane protein classes solubilized in detergent micelles or lipid mimetics.

Not so fast: Identifying and remediating slow and imprecise cryptocurrency exchange data

Using raw tick-level trade data from 17 major cryptocurrency exchanges, we show that heterogeneity in matching engines can affect the computation of various liquidity and trading metrics. Using simple analytical techniques, we generate an algorithm to identify exchanges with slow matching engines or imprecise timestamps. Having identified problematic exchanges, we propose tractable techniques which can remediate the bias in metrics generated by problematic exchanges. Our techniques and exchange classifications are useful for academic and industry-based users of cryptocurrency exchange data to identify and remediate problematic trade-level data.

The Myth of The Annular Lipids.

In the early 1970s, the existence of a "lipid annulus" stably surrounding the individual intrinsic protein molecules was proposed by several authors. They referred to a number of lipid molecules in slow exchange with the bulk lipid in the bilayer, i.e., more or less protein-bound, and more ordered than the bulk lipid. The annular lipids would control enzyme activity. This idea was uncritically accepted by most scientists working with intrinsic membrane proteins at the time, so that the idea operated like a myth in the field. However, in the following decade, hard spectroscopic and biochemical evidence showed that the proposed annular lipids were not immobilized for a sufficiently long time to influence enzyme or transporter activity, nor were they ordered by the protein. Surprisingly, forty years later, the myth survives, and the term 'annular lipid' is still in use, in a different, but even more illogical sense.

Rapid and reliable RNA resonance assignment by combining chemical and enzymatic stable isotope labeling

In this work a rapid RNA assignment approach by combining chemical and enzymatic 13C and 15N stable isotope labeling is introduced. We exemplify the assignment strategy for imino N1H1 purine and N3H3 pyrimidine and aromatic C6H6 pyrimidine, C8H8 purine and C2H2 adenine resonances for a non-coding RNA comprising 66 nucleotides. The assignment strategy is based on position specific labeling by chemical solid phase synthesis and dilute stable isotope 13C/15N-labeling by mixing labeled and commercially available unlabeled RNA phosphoramidites. The assignment process is further facilitated by nucleotide specific labeling using T7 RNA polymerase in vitro transcription with in house produced atom specific 13C labeled ribonucleotide triphosphates. The approach is fast with a total NMR measurement time of only 22 h and also competitive in terms of costs as compared to the standard methodology relying on in vitro transcription using 2H, 15N and 13C/15N uniformly labeled ribonucleotide triphosphates. Furthermore, the assignment procedure revealed a slow exchange process on the NMR chemical shift time scale in the 66 nt non-coding RNA with possible biological implications in the regulation of bacterial competence.

Anion Responsive Molecular Switch Based on a Doubly Strapped Calix[4]pyrrole

AbstractA calix[4]pyrrole receptor bearing two proximally meso‐meso linking isophthaloyl straps displays open and closed states depending on the calix[4]pyrrole conformation. In the crystal structures and in non‐polar solvent, the calix[4]pyrrole adopts open 1,3‐alternate conformation with straps on the sides. Anion binding triggers a closed state of the receptor providing two types of interactions with an aromatic benzoate guest: hydrogen bonds from the pyrrolic NH groups and π⋅⋅⋅π interactions from the phenyl groups of the straps. Slow exchange dynamics was observed on the NMR timescale indicating that benzoate, acetate and chloride anions, which bind with relatively low affinity get kinetically trapped in the receptor as insects in a Venus flytrap.

Quantitating Diffusion Enhancement in Pore Hierarchies.

A microporous continuum traversed by a set of mutually perpendicular channels is considered to be a model for a hierarchically porous system of the mesoporous zeolite type. Transient profiles of molecular uptake as determined by kinetic Monte Carlo (kMC) simulation are found to be in excellent agreement with the result attained by the application of the two-region model (the Kärger model) of molecular diffusion. In particular, it is found that, in the two limiting cases referred to as fast exchange and slow exchange, there exist two simple analytical expressions for the rate of molecular uptake and hence for the quantification of transport enhancement in comparison with the purely microporous adsorbent. In the general case, transport enhancement is simply recognized by the reciprocal addition of the expressions in the two limiting cases.

The study on contamination of bottom sediments from the Odra River estuary (SW Baltic Sea) by tributyltin using environmetric methods

We present the first comprehensive study on the occurrence of tributyltin (TBT) in the Odra River estuary (SW Baltic Sea) that encompasses both densely populated and urbanized agglomeration Szczecin city, and sparsely populated biosphere reserves “Natura 2000”. Relationship between TBT and physicochemical parameters of bottom sediments such as granulometry total organic carbon (TOC), total nitrogen (TN), acid volatile sulfide (AVS), As, and metals: Ba, Cd, Co, Cr, Cu, Fe, Hg, Ni, Mn, Mo, Pb, Sn, and Zn was investigated in 120 samples collected in 2017 and 2018. The highest TBT concentrations were over 3000 ng g−1 (dry weight). They were observed in samples collected in the vicinity of the ship maintenance zones of the Szczecin city. Despite the EU ban on its use since 2003, TBT is still present in the environment. Environmetrics analyses such as correlation, cluster, and principal component analysis of obtained results revealed that the main source of sediments contamination by TBT, metalloids, and metals is likely related to the maritime industry: shipyards, ship maintenance as well as ports and marines. TBT is still present in the bottom sediments because of its emission to the environment with dust and paint chips formed during sandblasting cleaning of ship surfaces. The pollutant is further transported with water current to remote localization in the Szczecin Lagoon. Slow water exchange between the Szczecin Lagoon and the Baltic Sea favors accumulation of pollutants in the lagoon sediments. Therefore, it is necessary to implement environmentally friendly methods into ship maintenance and management of the materials from dredged waterways, harbors, and marinas.

Dilational rheological properties of 2, 5-dialkyl benzene sulfonates at air-water and decane-water interfaces

The dilational properties of two anionic surfactants 5–6–4 and 5–6-t4 (2, 5-dialkyl benzene sulfonates) at the air-water and decane-water interfaces were investigated using the drop shape analysis method. The effects of aging time, oscillating frequency, bulk concentration, and surface/interfacial pressure on the dilational elasticity and dilational viscosity were explored. The results show that the slow process and diffusion exchange process control the nature of surface/interfacial films at low and high concentrations, respectively. At low concentrations, the strong interaction between the branched long alkyl chains dominates at the surface. At high concentrations, the surface elasticity of 5–6–4 is higher because of the slower diffusion-exchange process. The insertion of oil molecules disrupts the interactions between branched long alkyl chains and simultaneously enhances the diffusion-exchange process, resulting in lower maximum interfacial dilational elasticity and viscosity for both 5–6–4 and 5–6-t4 molecules. The rigid isobutyl group endows the 5–6-t4 molecule with higher dilational elasticity and viscosity at the interface.

Microsecond MD Simulations of the Plexin-B1 RBD: N-H Probability Density as Descriptor of Structural Dynamics, Dimerization-Related Conformational Entropy, and Transient Dimer Asymmetry.

Amide-bond equilibrium probability density, Peq = exp(-u) (u, local potential), and associated conformational entropy, Sk = -∫Peq (ln Peq) dΩ ─ln ∫dΩ, are derived for the Rho GTPase binding domain of Plexin-B1 (RBD) as monomer and dimer from 1 μs MD simulations. The objective is to elucidate the effect of dimerization on the dynamic structure of the RBD. Dispersed (peaked) Peq functions indicate "flexibility" ("rigidity"; the respective concepts are used below in this context). The L1 and L3 loops are throughout highly flexible, the L2 loop and the secondary structure elements are generally rigid, and the L4 loop is flexible in the monomer and rigid in the dimer. Overall, many residues are more flexible in the dimer. These features, and their implications, are discussed. Unexpectedly, we find that monomer unit 1 of the dimer (in short, d1) is unusually flexible, whereas monomer unit 2 (in short, d2) is as rigid as the RBD monomer. This is revealed due to their engagement in slow-to-intermediate conformational exchange detected previously by 15N relaxation experiments. Such motions occur with rates on the order of 103-104 s-1; hence, they cannot be completely sampled over the course of 1 μs simulation. However, the extent to which rigid d2 is affected is small enough to enable physically relevant analysis. The entropy difference between d2 and the monomer yields an entropic contribution of -7 ± 0.7 kJ/mol to the free energy of RBD dimerization. In previous work aimed at similar objectives we used 50-100 ns MD simulations. Those results and the present result differ considerably. In summary, bond-vector Peq functions derived directly from long MD simulations are useful descriptors of protein structural dynamics and provide accurate conformational entropy. Within the scope of slow conformational exchange, they can be useful, even in the presence of incomplete sampling.

Microsecond MD Simulations of the Plexin-B1 RBD: 2. N-H Probability Densities and Conformational Entropy in Ligand-Free, Rac1-Bound, and Dimer RBD.

Orientational probability densities, Peq = exp(-u) (u, local potential), of bond-vectors in proteins provide information on structural flexibility. The related conformational entropy, Sk = -∫Peq(ln Peq)dΩ - ln ∫dΩ, provides the entropic contribution to the free energy of the physical/biological process studied. We have developed a new method for deriving Peq and Sk from MD simulations, using the N-H bond as probe. Recently we used it to study the dimerization of the Rho GTPase binding domain of Plexin-B1 (RBD). Here we use it to study RBD binding to the small GTPase Rac1. In both cases 1 μs MD simulations have been employed. The RBD has the ubiquitin fold with four mostly long loops. L3 is associated with GTPase binding, L4 with RBD dimerization, L2 participates in interdomain interactions, and L1 has not been associated with function. We find that RBD-Rac1 binding renders L1, L3, and L4 more rigid and the turns β2/α1 and α2/β5 more flexible. By comparison, RBD dimerization renders L4 more rigid, and the α-helices, the β-strands, and L2 more flexible. The rigidity of L1 in RBDRAC is consistent with L1-L3 contacts seen in previous MD simulations. The analysis of the L3-loop reveals two states of distinct flexibility which we associate with involvement in slow conformational exchange processes differing in their rates. Overall, the N-H bonds make an unfavorable entropic contribution of (5.9 ± 0.9) kJ/mol to the free energy of RBD-Rac1 binding; they were found to make a favorably contribution of (-7.0 ± 0.7) kJ/mol to the free energy of RBD dimerization. In summary, the present study provides a new perspective on the impact of Rac1 binding and dimerization on the flexibility characteristics of the RBD. Further studies are stimulated by the results of this work.

Exchange of Adsorbed Pb(II) at the Rutile Surface: Rates and Mechanisms.

The dynamics of Pb(II) at mineral surfaces affect its mobility in the environment. Pb(II) forms inner- and outer-sphere complexes on mineral surfaces, and this adsorbed pool often represents a large portion of the bioaccessible Pb in contaminated soils. To assess the lability of this potentially reactive adsorbed Pb(II) pool at metal oxide surfaces, we performed Pb(II) isotope exchange measurements between dissolved Pb(II) enriched in 207Pb and natural isotopic abundance Pb(II) adsorbed to rutile at pH 5, 6, and 7. We find that ∼95% of the adsorbed lead is exchangeable. An initially fast exchange (<1 h) is followed by a slower exchange that occurs on a time scale of hours to days. Pb LIII-edge extended X-ray absorption fine structure spectra indicate that similar binding mechanisms are present at all pH values and Pb(II) loadings, implying that differences in exchange rates across the pH range examined are not attributable to changes in the coordination environment. The slower exchange at pH 5 may be associated with interparticle and intraparticle diffusion resulting from particle aggregation. These findings demonstrate that the dissolved Pb(II) pool can be rapidly replenished by adsorbed Pb(II) if this pool is drawn down incrementally by biological uptake or a shift in chemical conditions.

Mechanism of photo-ionic stoichiometry changes in SrTiO3

The impact of UV light (λ = 365 nm) on defect chemistry and composition of undoped SrTiO3 single crystals was investigated by means of impedance spectroscopy. In-plane conductivity measurements between 280 and 450 °C in air reveal a drastic increase of the bulk conductivity upon UV illumination. The measured time dependence of this increase is in accordance with oxygen chemical diffusion in SrTiO3. The corresponding photo-induced change in defect concentrations is caused by oxygen being pumped from the gas phase into the oxide under UV irradiation. This affects the entire SrTiO3 crystal rather than only the thin UV absorption zone and leads to very high oxygen chemical potentials with nominal oxygen pressures up to 106 bar. After switching the UV light off, the resulting conductivity increase relaxes extremely slowly due to slow surface exchange kinetics. A mechanistic model is introduced to explain the impact of UV light on the oxygen chemical potential as well as on the oxygen vacancy and hole/electron concentrations in semiconducting oxides, here SrTiO3. This model is based on the formation of oxygen quasi-chemical potentials in the illuminated region. It is discussed how the interplay of four kinetic parameters causes the observed time-dependent stoichiometry and thus conductivity changes.

Synoeca‐MP: New insights into its mechanism of action by using NMR and molecular dynamics simulations approach

AbstractSynoeca‐MP is a 14‐residue amidated peptide, belongs to the mastoparan family and it is found in the venom of the wasp Synoeca surinama and has antibacterial and antifungal activity. The low cytotoxicity of the peptide also makes it an excellent candidate for drug development. To better understand its selectivity and interaction with the membrane, the peptide behavior in membrane‐like environments was studied here and the peptide structure in SDS micelles was determined by NMR spectroscopy. The behavior of the peptide in hydrophobic media and in different pH ranges was studied by CD spectroscopy. The incorporation of residues into the anionic micelles was studied by hydrogen‐deuterium exchange. The peptide stability and insertion in the micelles was studied by molecular dynamics simulations. Synoeca‐MP, bound to SDS micelles, exhibits a partial α‐helix conformation, with the first five residues and the last two unfolded. H/D exchange showed that the peptide has a slow exchange rate. After 164 h, four residues had not yet completed H/D substitution, suggesting parallel alignment of the peptide with the micelle, mainly due to the hydrophobic interface. This may indicate a carpet interaction model of the peptide with micelles. The molecular simulation study of peptide showed that the peptide consists of a well‐folded alpha‐helix core and unfolded extremities, which are responsible for the nature of the peptide interaction. The biophysical analyses can improve the atomic understanding of the mode of action of the peptide and help in future improvements of the peptide for clinical usage.

Self‐Assembly of a Bis‐NHC Ligand and Coinage Metal Ions: Unprecedented Metal‐Driven Chemistry between the Tri‐ and Tetranuclear Species

The discrete multinuclear AgI -NHC complexes [Agn (1)n )](X)n ; (n=3 or 4, X=BF4 , PF6 ), synthesized from a bis-NHC ligand 1 and AgI ions via multiligand self-assembly, were observed to be in equilibrium between two forms (tri- and tetranuclear) in solution on the NMR timescale, which is unprecedented in CNHC -donor based chemistry. The existence of both the species was confirmed by various studies such as NMR (1 H, VT, 2D-DOSY) and ESI-MS analysis along with concentration studies. Importantly, replacing AgI with other coinage metals (AuI and CuI ) was found to alter the phenomenon entirely: a slow exchange from the tetra- to trinuclear species was noticed for the AuI complexes (4 a, b), whereas no such process was detected in case of the CuI complexes (5 a, b).

Self‐Assembly of a Bis‐NHC Ligand and Coinage Metal Ions: Unprecedented Metal‐Driven Chemistry between the Tri‐ and Tetranuclear Species

AbstractThe discrete multinuclear AgI−NHC complexes [Agn(1)n)](X)n; (n=3 or 4, X=BF4, PF6), synthesized from a bis‐NHC ligand 1 and AgI ions via multiligand self‐assembly, were observed to be in equilibrium between two forms (tri‐ and tetranuclear) in solution on the NMR timescale, which is unprecedented in CNHC‐donor based chemistry. The existence of both the species was confirmed by various studies such as NMR (1H, VT, 2D‐DOSY) and ESI‐MS analysis along with concentration studies. Importantly, replacing AgI with other coinage metals (AuI and CuI) was found to alter the phenomenon entirely: a slow exchange from the tetra‐ to trinuclear species was noticed for the AuI complexes (4 a, b), whereas no such process was detected in case of the CuI complexes (5 a, b).

Human and Bacterial Toll-Interleukin Receptor Domains Exhibit Distinct Dynamic Features and Functions.

Toll-interleukin receptor (TIR) domains have emerged as critical players involved in innate immune signaling in humans but are also expressed as potential virulence factors within multiple pathogenic bacteria. However, there has been a shortage of structural studies aimed at elucidating atomic resolution details with respect to their interactions, potentially owing to their dynamic nature. Here, we used a combination of biophysical and biochemical studies to reveal the dynamic behavior and functional interactions of a panel of both bacterial TIR-containing proteins and mammalian receptor TIR domains. Regarding dynamics, all three bacterial TIR domains studied here exhibited an inherent exchange that led to severe resonance line-broadening, revealing their intrinsic dynamic nature on the intermediate NMR timescale. In contrast, the three mammalian TIR domains studied here exhibited a range in terms of their dynamic exchange that spans multiple timescales. Functionally, only the bacterial TIR domains were catalytic towards the cleavage of NAD+, despite the conservation of the catalytic nucleophile on human TIR domains. Our development of NMR-based catalytic assays allowed us to further identify differences in product formation for gram-positive versus gram-negative bacterial TIR domains. Differences in oligomeric interactions were also revealed, whereby bacterial TIR domains self-associated solely through their attached coil-coil domains, in contrast to the mammalian TIR domains that formed homodimers and heterodimers through reactive cysteines. Finally, we provide the first atomic-resolution studies of a bacterial coil-coil domain and provide the first atomic model of the TIR domain from a human anti-inflammatory IL-1R8 protein that undergoes a slow inherent exchange.

En Route to Stabilized Compact Conformations of Single-Chain Polymeric Nanoparticles in Complex Media.

Precise control over the folding pathways of polypeptides using a combination of noncovalent and covalent interactions has evolved into a wide range of functional proteins with a perfectly defined 3D conformation. Inspired hereby, we develop a series of amphiphilic copolymers designed to form compact, stable, and structured single-chain polymeric nanoparticles (SCPNs) of defined size, even in competitive conditions. The SCPNs are formed through a combination of noncovalent interactions (hydrophobic and hydrogen-bonding interactions) and covalent intramolecular cross-linking using a light-induced [2 + 2] cycloaddition. By comparing different self-assembly pathways of the nanoparticles, we show that, like for proteins in nature, the order of events matters. When covalent cross-links are formed prior to the folding via hydrophobic and supramolecular interactions, larger particles with less structured interiors are formed. In contrast, when the copolymers first fold via hydrophobic and hydrogen-bonding interactions into compact conformations, followed by covalent cross-links, good control over the size of the SCPNs and microstructure of the hydrophobic interior is achieved. Such a structured SCPN can stabilize the solvatochromic dye benzene-1,3,5-tricarboxamide–Nile Red via molecular recognition for short periods of time in complex media, while showing slow exchange dynamics with the surrounding complex media at longer time scales. The SCPNs show good biocompatibility with cells and can carry cargo into the lysosomal compartments of the cells. Our study highlights the importance of control over the folding pathway in the design of stable SCPNs, which is an important step forward in their application as noncovalent drug or catalyst carriers in biological settings.

Atomic View of Aqueous Cyclosporine A: Unpacking a Decades-Old Mystery.

An atomic view of a main aqueous conformation of cyclosporine A (CycA), an important 11-amino-acid macrocyclic immunosuppressant, is reported. For decades, it has been a grand challenge to determine the conformation of free CycA in an aqueous-like solution given its poor water solubility. Using a combination of X-ray and single-crystal neutron diffraction, we unambiguously resolve a unique conformer (A1) with a novel cis-amide between residues 11 and 1 and two water ligands that stabilize hydrogen bond networks. NMR spectroscopy and titration experiments indicate that the novel conformer is as abundant as the closed conformer in 90/10 (v/v) methanol/water and is the main conformer at 10/90 methanol/water. Five other conformers were also detected in 90/10 methanol/water, one in slow exchange with A1, another one in slow exchange with the closed form and three minor ones, one of which contains two cis amides Abu2-Sar3 and MeBmt1-MeVal11. These conformers help better understand the wide spectrum of membrane permeability observed for CycA analogues and, to some extent, the binding of CycA to protein targets.

Potentially toxic elements in the Gusinoye Lake (Republic of Buryatia, Russia).

Enterprises of fuel and energy complex are the main sources of carcinogenic and mutagenic potentially toxic elements (PTEs). Their particulate and gaseous emissions, wastes, and effluents enforce migration processes of PTEs in atmosphere, soil, and then in water bodies, creating a danger to the functioning of aquatic ecosystems. Vulnerable objects include water bodies with slow water exchange, due to the accumulation of PTEs in the bottom sediments and the threat of secondary contamination. Among them it is necessary to note cooling ponds of thermal power plants (TPP). In this study, the ecological and geochemical state of components of "water-suspended sediments-bottom sediments" aquatic system is assessed on the example of the Gusinoye Lake (the Republic of Buryatia), which is used as cooling pond for the largest in the region TPP, burning brown coal from local deposits. The Gusinoye Lake is the second largest lake in Buryatia after the Baikal Lake and a valuable source of water supply, place of recreation, and fish breeding. To study the distribution of PTEs in the waters of the lake, a network of 19 monitoring stations was deployed in the summer of 2019. In contrast to the previous researches, detailed studies of the central deep-water part of the lake were carried out. As a result, data on the concentration of PTEs in the dissolved and suspended forms and also in bottom sediments was obtained. To study the balance of chemicals in the lake, its main tributaries were surveyed, as well as the drainage channel of TPP. For the first time, for the Lake Gusinoye, bulk concentrations of As, Cd, Pb, Zn, Co, Mo, Cu, Sb, Cr, V, Mn, Sr, and Ag were determined using the inductively coupled plasma mass spectrometry. The highest concentrations of dissolved PTEs in the lake water are confined to the mouths of tributaries in the northern part of the lake. Dissolved Sr, Mo, and Cu in the lake exceed maximum permissible concentrations (MPCs) for fishery. The maximum concentrations of Sr and Cu are almost 3 MPCs and that of Mo is 22 MPCs near the TPP. The suspended sediments of the Gusinoye Lake are depleted of all studied PTEs as compared to the regional clarkes (natural abundances) of the lithosphere. Mn has the highest concentrations in the suspension, and its maximum concentration was due to the surface runoff from the solid waste landfill and an abandoned coal mine. Concentrations of Cu, Zn, Sr, Mo, and Pb in bottom sediments are higher than in suspended sediments, thus indicating the accumulation of pollutants. The maximum intensity of accumulation is characteristic of the deep central basin of the lake and its eastern part, where Sr, Ag, and Mo accumulate most intensively near the coal field. The main sources of PTE pollution are concentrated in the northern and northeastern parts of the lake catchment area. Significant amount of dissolved Mo, Sr, and As enter the lake with the waters of quarries and mines of the coal fields, the solid waste landfill of the town of Gusinoozyorsk, and northern rivers-tributaries, draining the dumps of the large Zagustaj coal field.

The time-zero HSQC method improves the linear free energy relationship of a polypeptide chain through the accurate measurement of residue-specific equilibrium constants.

EXSY (exchange spectroscopy) NMR provides the residue-specific equilibrium constants, K, and residue-specific kinetic rate constants, k, of a polypeptide chain in a two-state exchange in the slow exchange regime. A linear free energy relationship (LFER) discovered in a log k versus log K plot is considered to be a physicochemical basis for smooth folding and conformational changes of protein molecules. For accurate determination of the thermodynamic and kinetic parameters, the measurement bias arising from state-specific differences in the R1 and R2 relaxation rates of 1H and other nuclei in HSQC and EXSY experiments must be minimized. Here, we showed that the time-zero HSQC acquisition scheme (HSQC0) is effective for this purpose, in combination with a special analytical method (Π analysis) for EXSY. As an example, we applied the HSQC0 + Π method to the two-state exchange of nukacin ISK-1 in an aqueous solution. Nukacin ISK-1 is a 27-residue lantibiotic peptide containing three mono-sulfide linkages. The resultant bias-free residue-based LFER provided valuable insights into the transition state of the topological interconversion of nukacin ISK-1. We found that two amino acid residues were exceptions in the residue-based LFER relationship. We inferred that the two residues could adopt special conformations in the transition state, to allow the threading of some side chains through a ring structure formed by one of the mono-sulfide linkages. In this context, the two residues are a useful target for the manipulation of the physicochemical properties and biological activities of nukacin ISK-1.