Hydrogen Exchange Experiments Research Articles

Heat-induced structural and chemical changes to a computationally designed miniprotein.

The de novo design of miniprotein inhibitors has recently emerged as a new technology to create proteins that bind with high affinity to specific therapeutic targets. Their size, ease of expression, and apparent high stability makes them excellent candidates for a new class of protein drugs. However, beyond circular dichroism melts and hydrogen/deuterium exchange experiments, little is known about their dynamics, especially at the elevated temperatures they seemingly tolerate quite well. To address that and gain insight for future designs, we have focused on identifying unintended and previously overlooked heat-induced structural and chemical changes in a particularly stable model miniprotein, EHEE_rd2_0005. Nuclear magnetic resonance (NMR) studies suggest the presence of dynamics on multiple time and temperature scales. Transiently elevating the temperature results in spontaneous chemical deamidation visible in the NMR spectra, which we validate using both capillary electrophoresis and mass spectrometry (MS) experiments. High temperatures also result in greatly accelerated intrinsic rates of hydrogen exchange and signal loss in NMR heteronuclear single quantum coherence spectra from local unfolding. These losses are in excellent agreement with both room temperature hydrogen exchange experiments and hydrogen bond disruption in replica exchange molecular dynamics simulations. Our analysis reveals important principles for future miniprotein designs and the potential for high stability to result in long-lived alternate conformational states.

Improved Repeat Protein Stability by Combined Consensus and Computational Protein Design.

High protein stability is an important feature for proteins used as therapeutics, as diagnostics, and in basic research. We have previously employed consensus design to engineer optimized Armadillo repeat proteins (ArmRPs) for sequence-specific recognition of linear epitopes with a modular binding mode. These designed ArmRPs (dArmRPs) feature high stability and are composed of M-type internal repeats that are flanked by N- and C-terminal capping repeats that protect the hydrophobic core from solvent exposure. While the overall stability of the designed ArmRPs is remarkably high, subsequent biochemical and biophysical experiments revealed that the N-capping repeat assumes a partially unfolded, solvent-accessible conformation for a small fraction of time that renders it vulnerable to proteolysis and aggregation. To overcome this problem, we have designed new N-caps starting from an M-type internal repeat using the Rosetta software. The superior stability of the computationally refined models was experimentally verified by circular dichroism and nuclear magnetic resonance spectroscopy. A crystal structure of a dArmRP containing the novel N-cap revealed that the enhanced stability correlates with an improved packing of this N-cap onto the hydrophobic core of the dArmRP. Hydrogen exchange experiments further show that the level of local unfolding of the N-cap is reduced by several orders of magnitude, resulting in increased resistance to proteolysis and weakened aggregation. As a first application of the novel N-cap, we determined the solution structure of a dArmRP with four internal repeats, which was previously impeded by the instability of the original N-cap.

Role of key protein residues involved in DinB DNA damage specificity: an in silico study

Y‐family DNA polymerases are a class of error‐prone polymerases able to perform DNA translesion synthesis. E. coli DNA Polymerase IV (also called DinB from the name of the gene) is a Y‐family DNA polymerase implicated in stress‐induced mutagenesis. As other DNA polymerases, DinB has the general architecture of a right hand, with palm, thumb, and fingers subdomains. In addition, Y‐family polymerases have an extra domain, namely the “little finger” or polymerase‐associated domain (PAD) and have a more solvent accessible active‐site than their high‐fidelity replicative counterparts. Kinetics studies have shown that DinB is particularly efficient at bypassing damaged guanosines, such as the N2‐furfuryl‐dG, and even more efficient in lesion bypass on damaged DNA than in replication of undamaged DNA. Hydrogen exchange experiments have suggested that the protein undergoes a conformational change in the presence of certain DNA damages on the template strand, but not others, implying a possible mechanism of bypass discrimination. However, the exact nature of such movement is not well characterized. Using molecular dynamics (MD) simulations we explored different DNA/protein/incoming nucleotides combinations to dissect the protein conformational change. Specifically, we analyzed a lesion that the protein can bypass (N2‐furfuryl‐dG) and one that instead the protein does not bypass well (O6‐Methylated dG). Our MD simulations revealed that there are some key residues in the fingers domain that are important switches for the conformational change. For example, R35 assumes a characteristic position when the protein is in a catalytically competent state, while it blocks the active site when the protein is not able to bypass the lesion. This result is in agreement with previous studies which showed that mutations of the R35 residue alter the protein specificity. Other areas of DinB which seem to undergo large movement reside in the PAD domain. This domain, which makes contact with the DNA major groove, has been implicated in DNA damage specificity, so it makes sense that residues on this domain assume different conformations, depending on the type of lesions present on the templating strand. A discussion of possible roles for some of the residues on the PAD will be presented.

Dual protease type XIII/pepsin digestion offers superior resolution and overlap for the analysis of histone tails by HX-MS

The N-terminal regions of histone proteins (tails) are dynamic elements that protrude from the nucleosome and are involved in many aspects of chromatin organization. Their epigenetic role is well-established, and post-translational modifications (PTMs) present on these regions contribute to transcriptional regulation. While hydrogen/deuterium exchange mass spectrometry (HX-MS) is well-suited for the analysis of dynamic structures, it has seldom been employed to analyze histones due to the poor N-terminal coverage obtained using pepsin. Here, we test the applicability of a dual protease type XIII/pepsin digestion column to this class of proteins. We optimize online digestion conditions using the H4 monomer, and extend the method to the analysis of histones in monomeric states and nucleosome core particles (NCPs). We show that the dual protease column generates many short and overlapping N-terminal peptides. We evaluate our method by performing hydrogen exchange experiments of NCPs for different time points and present full coverage of the tails at excellent resolution. We further employ electron transfer dissociation and showcase an unprecedented degree of overlap across multiple peptides that is several fold higher than previously reported methods. The method we report here may be readily applied to the HX-MS investigation of histone dynamics and to the footprints of histone binding proteins on nucleosomes.

Secondary structure and topology of the transmembrane domain of Syndecan-2 in detergent micelles.

Syndecans are single-span membrane proteins playing important roles in cell-cell and cell-matrix interactions. The transmembrane domain of syndecans is critical for signal transduction across the cell membrane. Here, the structure of the transmembrane domain of syndecan-2 in detergent micelles was investigated using solution NMR spectroscopy. Backbone resonance assignment was obtained, and NMR studies show that the transmembrane domain forms a helix in detergent micelles, which is also supported by the hydrogen and deuterium exchange experiment. A study of the dynamics revealed the rigid structure of the transmembrane domain formed in solution, and paramagnetic relaxation enhancement defined the topology of the transmembrane domain in detergent micelles. This structural analysis may facilitate a better understanding of the role of the syndecan-2 transmembrane domain in signal transduction.

Reference Parameters for Protein Hydrogen Exchange Rates.

The analysis of many hydrogen exchange (HX) experiments depends on knowledge of exchange rates expected for the unstructured protein under the same conditions. We present here some minor adjustments to previously calibrated values and a stringent test of their accuracy. Graphical Abstract ᅟ.

Molecular Dynamics studies of DNA polymerase kappa, a human protein involved in translesion synthesis

Y family DNA polymerases are a class of enzymes characterized by their ability to copy damaged DNA in a process known as translesion synthesis. They do not eliminate the damage per se, but at least they are able to bypass a stalled replication, avoiding cell death. These polymerases bypass a wide variety of DNA damage, including thymine‐thymine dimers, inter‐strand DNA crosslinks, and other damage caused by reactive oxygen species. Unfortunately, they do not replicate the DNA with high fidelity and some polymerases are more error prone than others. In addition, the kinds of lesions that this group of enzymes can bypass are dependent on the enzyme itself, despite the general architectural similarities of all these enzymes. Human DNA polymerase kappa (pol k) is a member of the Y‐family of DNA polymerases involved in translesion synthesis in humans. Kinetics students have shown that this enzyme can bypass minor groove lesions such as O6‐methylated guanine (O‐metG) but not major groove lesions such as N2‐furfuryl‐guanine (N‐fG). Hydrogen exchange experiments have suggested solvent accessibility changes in various systems, hinting at a mechanism of conformational change upon substrate binding. Through molecular dynamics simulations, we tested the validity of the conformational change hypothesis by comparing the structural changes undergone by pol k in the presence of different templating lesions and with correct and incorrect incoming nucleotides. In addition, binary systems, comprised of just the DNA and the protein without the incoming nucleotide, were examined. Our results show that pol k does not attain a close, catalytically competent state in both the binary and the O‐metG systems, as evidenced by the few contacts between the protein and the incoming nucleotides. We have also identified key residues important for the stabilization of the incoming nucleotide as well as formulated a hypothesis to why the O‐metG lesion does not get bypassed as efficiently as other damages.Support or Funding InformationThis work was supported in part by the Rose M. Badgeley Residuary Charitable Trust Award and the Colette Mahoney Award to BSB.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Overview of Relaxation Dispersion NMR Spectroscopy to Study Protein Dynamics and Protein-Ligand Interactions.

Proteins and nucleic acids are central to all biological processes. NMR spectroscopy has proven to be excellent for studying the dynamics of these macromolecules over various timescales. Relaxation rates and heteronuclear nuclear Overhauser-effect values can resolve motion on pico- to nanosecond timescales, residual dipolar couplings provide information on submicro- to millisecond timescales, and even slower dynamics over seconds to hours can be resolved by hydrogen-exchange experiments. Relaxation dispersion experiments are especially valuable because they resolve motion on micro- to millisecond timescales, encompassing biomolecular motions associated with ligand binding, enzymatic catalysis, and domain-domain opening. These experiments provide structural, kinetic, and thermodynamic information on "invisible" excited conformational states. Relaxation dispersion can be applied not only to single biomolecules but also to protein-ligand complexes to study the kinetics and thermodynamics of association and dissociation. We review recent developments in relaxation dispersion methodology, outline the R1ρ relaxation dispersion experiment, and discuss application to biomolecular interactions. © 2018 by John Wiley & Sons, Inc.

On Hydride Diffusion in Transition Metal Perovskite Oxyhydrides Investigated via Deuterium Exchange

Perovskite oxyhydrides may find diverse applications, ranging from catalysis, topochemical synthesis to solid state ionics, but the understanding of their hydride transport behavior has remained limited. Here, gaseous hydrogen exchange and release experiments were analyzed using the Kissinger method to estimate the activation energy (Ea) for H/D exchange and H2 release in BaTiO3–xHx (x = 0.35–0.60) and LaSrCoO3H0.70. It is revealed that, for each BaTiO3–xHx at a given hydride concentration (x), both H/D exchange and H2 release experiments provide similar Ea values. For BaTiO3–xHx with different x, the obtained Ea values significantly decrease with increasing x until around 0.4; beyond 0.4, it becomes nearly constant (200–220 kJ mol–1). This observation suggests that the diffusion process in the low hydride concentration (x < 0.4) includes oxide as well as hydride diffusion, whereas, for 0.4 < x (<0.75), only hydride migrates, with second-nearest-neighbor (2NN) jumps as a rate-determining process, which is...

Empirical Method To Accurately Determine Peptide-Averaged Protection Factors from Hydrogen Exchange MS Data

Amide hydrogen exchange experiments measured by mass spectrometry have become commonplace to study protein structural dynamics; however, the underdetermined nature of these measurements render extraction of exchange rates unreliable at the level of individual peptides. This prevents orthogonal verification of results and severely limits interpretation of the data. This work describes an easy-to-implement empirical method to determine the change in an observed rate constant or the average change in multiple rate constants as compared to some reference condition. This allows direct empirical computation of the average protection factor (PF) for peptides in isolation requiring no knowledge of actual rate constants themselves. Benchmarking the method by comparison of average peptide PFs with site-resolved NMR-derived PFs demonstrates high reliability and accuracy. This empirical method provides the first universally reliable strategy for recovering subglobal structural physics from individual peptides and, in doing so, standardizes the hydrogen exchange experiments measured by bottom-up mass spectrometry (HX MS), simplifies interpretation, and facilitates clear communication of the results.

Aberrant increase of NMR signal in hydrogen exchange experiments. Observation and explanation

Hydrogen exchange (HX) NMR spectroscopy is widely used for monitoring structure, stability and dynamics of proteins at the level of individual residues. The stochastic replacement of protons by deuterons typically leads to an exponential decrease of the NMR signals. However, an unusual signal increase was observed in HX of several amides for T4 lysozyme L99A. This effect can be attributed to peak sharpening as a result of reduced dipolar relaxation from proximal amide protons that experience more rapid hydrogen/deuterium (H/D) exchange. The behavior was specifically observed at the termini of secondary structure elements, where large differences in protection against H/D exchange are observed. This effect is expected to be more widespread in NMR HX studies, and is important for the accurate determination of protection factors.

In silico exploration of the mechanism of translesion synthesis by DinB

In prokaryotic organisms, environmental stress elicits the up‐regulation of a variety of genes involved in the so‐called SOS response. One major aspect of this response is the increase of mutation frequency. Some of the mutations may confer unique characteristic to the organism that will be beneficial for adapting to the environment, therefore accelerating evolution during stress. Y‐family DNA polymerases are a class of error‐prone polymerases able to perform DNA translesion synthesis. E. coli DNA Polymerase IV (also called DinB from the name of the gene) is a Y‐family DNA polymerase implicated in stress‐induced mutagenesis. As other DNA polymerases, DinB has the general architecture of a right hand, with palm, thumb and fingers subdomains. In addition, Y‐family polymerases have an extra domain, namely the “little fingers” and have a more solvent accessible active‐site than their high‐fidelity replicative counterparts. Kinetics studies have shown that DinB is particularly efficient at bypassing damaged guanosines, such as the N2‐furfuryl‐dG, and even more efficient in lesion bypass on damaged DNA than in replication of undamaged DNA. Recent hydrogen exchange experiments have suggested that the protein undergoes a conformational change in the presence of certain DNA damages on the templating strand, but not others, implying a possible mechanism of bypass discrimination. However, the exact nature of such movement is not well characterized. Using molecular dynamics (MD) simulations we explored different DNA/protein/incoming nucleotides combinations to dissect the protein conformational change. We were also able to identify the important residues implicated in this process. All systems were prepared starting with the available crystal structure, solvated and equilibrated using CHARMM‐GUI. MD simulations were run for 10 ns with the NAMD simulation package and multiple runs were done for the same system to ensure reproducibility of the data.Support or Funding InformationThis work was partly funded by the Rose M. Badgeley Residuary Charitable Trust.

Folding Simulations of an α-Helical Hairpin Motif αtα with Residue-Specific Force Fields.

α-Helical hairpin (two-helix bundle) is a structure motif composed of two interacting helices connected by a turn or a short loop. It is an important model for protein folding studies, filling the gap between isolated α-helix and larger all-α domains. Here, we present, for the first time, successful folding simulations of an α-helical hairpin. Our RSFF1 and RSFF2 force fields give very similar predicted structures of this αtα peptide, which is in good agreement with its NMR structure. Our simulations also give site-specific stability of α-helix formation in good agreement with amide hydrogen exchange experiments. Combining the folding free energy landscapes and analyses of structures sampled in five different ranges of the fraction of native contacts (Q), a folding mechanism of αtα is proposed. The most stable sites of Q9-E15 in helix-1 and E24-A30 in helix-2 close to the loop region act as the folding initiation sites. The formation of interhelix side-chain contacts also initiates near the loop region, but some residues in the central parts of the two helices also form contacts quite early. The two termini fold at a final stage, and the loop region remains flexible during the whole folding process. This mechanism is similar to the "zipping out" pathway of β-hairpin folding.

(19)F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy.

In comparison to proteins and protein complexes, the size of RNA amenable to NMR studies is limited despite the development of new isotopic labeling strategies including deuteration and ligation of differentially labeled RNAs. Due to the restricted chemical shift dispersion in only four different nucleotides spectral resolution remains limited in larger RNAs. Labeling RNAs with the NMR-active nucleus (19)F has previously been introduced for small RNAs up to 40 nucleotides (nt). In the presented work, we study the natural occurring RNA aptamer domain of the guanine-sensing riboswitch comprising 73 nucleotides from Bacillus subtilis. The work includes protocols for improved in vitro transcription of 2-fluoroadenosine-5'-triphosphat (2F-ATP) using the mutant P266L of the T7 RNA polymerase. Our NMR analysis shows that the secondary and tertiary structure of the riboswitch is fully maintained and that the specific binding of the cognate ligand hypoxanthine is not impaired by the introduction of the (19)F isotope. The thermal stability of the (19)F-labeled riboswitch is not altered compared to the unmodified sequence, but local base pair stabilities, as measured by hydrogen exchange experiments, are modulated. The characteristic change in the chemical shift of the imino resonances detected in a (1)H,(15)N-HSQC allow the identification of Watson-Crick base paired uridine signals and the (19)F resonances can be used as reporters for tertiary and secondary structure transitions, confirming the potential of (19)F-labeling even for sizeable RNAs in the range of 70 nucleotides.

Network representation of conformational transitions between hidden intermediates of Rd-apocytochrome b562.

The folding kinetics of Rd-apocytochrome b562 is two-state, but native-state hydrogen exchange experiments show that there are discrete partially unfolded (PUF) structures in equilibrium with the native state. These PUF structures are called hidden intermediates because they are not detected in kinetic experiments and they exist after the rate-limiting step. Structures of the mimics of hidden intermediates of Rd-apocytochrome b562 are resolved by NMR. Based upon their relative stability and structural features, the folding mechanism was proposed to follow a specific pathway (unfolded → rate-limiting transition state → PUF1 → PUF2 → native). Investigating the roles of equilibrium PUF structures in folding kinetics and their interrelationship not only deepens our understanding of the details of folding mechanism but also provides guides in protein design and prevention of misfolding. We performed molecular dynamics simulations starting from a hidden intermediate and the native state of Rd-apocytochrome b562 in explicit solvent, for a total of 37.18 μs mainly with Anton. We validated our simulations by detailed comparison with experimental data and other computations. We have verified that we sampled the post rate-limiting transition state region only. Markov state model was used to analyze the simulation results. We replace the specific pathway model with a network model. Transition-path theory was employed to calculate the net effective flux from the most unfolded state towards the most folded state in the network. The proposed sequential folding pathway via PUF1 then more stable, more native-like PUF2 is one of the routes in our network, but it is not dominant. The dominant path visits PUF2 without going through PUF1. There is also a route from PUF1 directly to the most folded state in the network without visiting PUF2. Our results indicate that the PUF states are not necessarily sequential in the folding. The major routes predicted in our network are testable by future experiments such as single molecule experiment.

PH dependence of conformational fluctuations of the protein backbone.

Proton binding equilibria (pK(a) values) of ionizable groups in proteins are exquisitely sensitive to their microenvironments. Apparent pK(a) values measured for individual ionizable residues with NMR spectroscopy are actually population-weighted averages of the pK(a) in different conformational microstates. NMR spectroscopy experiments with staphylococcal nuclease were used to test the hypothesis that pK(a) values of surface Glu and Asp residues are affected by pH-sensitive fluctuations of the backbone between folded and locally unfolded conformations. (15)N spin relaxation studies showed that as the pH decreases from the neutral into the acidic range the amplitudes of backbone fluctuations in the ps-ns timescale increase near carboxylic residues. Hydrogen exchange experiments suggested that backbone conformational fluctuations promoted by decreasing pH also reflect slower local or sub-global unfolding near carboxylic groups. This study has implications for structure-based pKa calculations: (1) The timescale of the backbone's response to ionization events in proteins can range from ps to ms, and even longer; (2) pH-sensitive fluctuations of the backbone can be localized to both the segment the ionizable residue is attached to or the one that occludes the ionizable group; (3) Structural perturbations are not necessarily propagated through Coulomb interactions; instead, local fluctuations appear to be coupled through the co-operativity inherent to elements of secondary structure and to networks of hydrogen bonds. These results are consistent with the idea that local conformational fluctuations and stabilities are important determinants of apparent pK(a) values of ionizable residues in proteins.

Measuring hydrogen exchange rates in invisible protein excited states.

Hydrogen exchange rates have become a valuable probe for studying the relationship between dynamics and structure and for dissecting the mechanism by which proteins fold to their native conformation. Typically measured rates correspond to averages over all protein states from which hydrogen exchange can occur. Here we describe a new NMR experiment based on chemical exchange saturation transfer that provides an avenue for obtaining uncontaminated, per-residue amide hydrogen exchange rates for interconverting native and invisible states so long as they can be separated on the basis of distinct (15)N chemical shifts. The approach is applied to the folding reaction of the Fyn SH3 domain that exchanges between a highly populated, NMR-visible native state and a conformationally excited, NMR-invisible state, corresponding to the unfolded ensemble. Excellent agreement between experimentally derived hydrogen exchange rates of the excited state at a pair of pHs is obtained, taking into account the expected dependence of exchange on pH. Extracted rates for the unfolded ensemble have been used to test hydrogen exchange predictions based on the primary protein sequence that are used in many analyses of solvent exchange rates, with a Pearson correlation coefficient of 0.84 obtained.

Evidence for the sequential folding mechanism in RNase H from an ensemble-based model.

The number of distinct protein folding pathways starting from an unfolded ensemble, and hence, the folding mechanism is an intricate function of protein size, sequence complexity, and stability conditions. This has traditionally been a contentious issue particularly because of the ensemble nature of conventional experiments that can mask the complexity of the underlying folding landscape. Recent hydrogen-exchange experiments combined with mass spectrometry (HX-MS) reveal that the folding of RNase H proceeds in a hierarchical fashion with distinct intermediates and following a single discrete path. In our current work, we provide computational evidence for this unique folding mechanism by employing a structure-based statistical mechanical model. Upon calibrating the energetic terms of the model with equilibrium measurements, we predict multiple intermediate states in the folding of RNase H that closely resemble experimental observations. Remarkably, a simplified landscape representation adequately captures the folding complexity and predicts the possibility of a well-defined sequence of folding events. We supplement the statistical model study with both explicit solvent molecular simulations of the helical units and electrostatic calculations to provide structural and energetic insights into the early and late stages of RNase H folding that hint at the frustrated nature of its folding landscape.

Temperature-dependent Kinetics Study for Hydrogen Exchange of Type I Antifreeze Protein from Winter Flounder

C) is not same with their biologically activetemperature (subzero degree). Because aqueous solutioncould be frozen at subzero degree, it is required that thesystematic NMR experiments should be carried out at vari-ous temperatures to understand physical properties of AFPat subzero degree. Here, to further understand the correlationbetween the dynamic properties and antifreeze activities oftype I AFPs, we performed NMR hydrogen exchange experi-ments on the winter flounder type I AFP (Fig. 1, referred toas wt-AFP) at temperatures from 1-23 °C.The winter flounder type I AFP and its mutants, A17L-and A20L-AFPs, were purchased from Cosmo Genetech Inc.(Seoul, Korea) and desalted using a Sephadex G-25 gelfiltration column. Each protein sample (5 mg) was dissolvedin 500 L of 90% H

Hydrogen Exchange Study of Winter Flounder Type I Antifreeze Protein

Antifreeze proteins (AFPs) are found in various organisms, such as fish, insects, plants, bacteria, and fungi, to promote survival at subzero temperatures by decreasing the freezing point of bodily fluids. AFPs, which are isolated from Arctic and Antarctic fish, comprise several structurally diverse classes of proteins and four classes of structurally independent proteins are identified: type I AFPs, alanine-rich αhelical proteins of 3.3 to 4.5 kDa; type II AFPs, cysteinerich globular proteins containing five disulfide bonds; type III AFPs, 6-kDa globular proteins; and Type IV AFPs, glutamateand glutamine-rich α-helical proteins. The type I AFPs are alanine-rich amphiphilic α-helical proteins found in winter flounder, yellowtail flounder, Alaskan plaice, shorthorn sculpin, and Arctic sculpin. The winter flounder AFP isoform, HPLC6, which is the most extensively studied AFP, contains three 11-amino acid repeats of the sequence Thr-X2-Asx-X7, where X is generally alanine. X-ray crystallographic studies revealed that the type I AFP is completely α-helical in conformation, with the exception of the last residue (R37), which adopts a 310helix. The hydroxyl and methyl groups of four threonine residues, particularly of the central two residues, T13 and T24, play key roles in the ice-binding properties of the type I AFP. For example, T13S/T24S mutations of the type I AFP caused a 90-100% loss in activity relative to wild-type AFP. The mutants, A17L and A21L, where the substitution lies adjacent to the Thr-rich face, caused a significant decrement of the thermal hysteresis activity, whereas the mutants A19L and A20L exhibited wild-type activity. It was reported that termini of the HPLC6 isoform possess greater helix-stabilizing ability compared to the HPLC2 isoform, leading to a 50% difference in the thermal hysteresis activity between these two AFPs. These results suggested that helical stabilization of the N and C termini is the critical component for antifreeze activity of the type I AFP. It was recently reported that flexibility of the C-terminal region causes a loss of thermal hysteresis activity because its dynamic nature strongly prevents binding to the ice surface. Here, to further understand the correlation between the dynamic properties and function of type I AFPs, we performed NMR hydrogen exchange experiments on the winter flounder type I AFP (HPLC6 isoform) (Fig. 1, referred to as wt-AFP). The hydrogen exchange rate constants for the two mutants, A17Land A20L-AFP (Fig. 1), which exhibit no and wild-type activities in thermal hysteresis respectively, are also determined. Comparison of the wtAFP and mutant AFPs can provide valuable insights into the thermal hysteresis mechanism of type I AFPs.