Cyr61/CCN1 Displays High-Affinity Binding to the Somatomedin B 1–44 Domain of Vitronectin

Background

Cyr61 is a member of the CCN (Cyr61, connective tissue growth, NOV) family of extracellular-associated (matricellular) proteins that present four distinct functional modules, namely insulin-like growth factor binding protein (IGFBP), von Willebrand factor type C (vWF), thrombospondin type 1 (TSP), and C-terminal growth factor cysteine knot (CT) domain. While heparin sulphate proteoglycans reportedly mediate the interaction of Cyr61 with the matrix and cell surface, the role of other extracellular associated proteins has not been revealed.

Methods and Findings

In this report, surface plasmon resonance (SPR) experiments and solid-phase binding assays demonstrate that recombinant Cyr61 interacts with immobilized monomeric or multimeric vitronectin (VTNC) with K_D in the nanomolar range. Notably, the binding site for Cyr61 was identified as the somatomedin B domain (SMTB ^1–44) of VTNC, which mediates its interaction with PAI-1, uPAR, and integrin αvβ3. Accordingly, PAI-1 outcompetes Cyr61 for binding to immobilized SMTB ^1–44, and Cyr61 attenuates uPAR-mediated U937 adhesion to VTNC. In contrast, isothermal titration calorimetry shows that Cyr61 does not display high-affinity binding for SMTB ^1-44 in solution. Nevertheless, competitive ELISA revealed that multimeric VTNC, heat-modified monomeric VTNC, or SMTB ^1–44 at high concentrations attenuate Cyr61 binding to immobilized VTNC, while monomeric VTNC was ineffective. Therefore, immobilization of VTNC exposes cryptic epitopes that recognize Cyr61 with high affinity, as reported for a number of antibodies, β-endorphin, and other molecules.

Conclusions

The finding that Cyr61 interacts with the SMTB ^1–44 domain suggests that VTNC represent a point of anchorage for CCN family members to the matrix. Results are discussed in the context of the role of CCN and VTNC in matrix biology and angiogenesis.     

Activation of the B Cell Receptor Leads to Increased Membrane Proximity of the Igα Cytoplasmic Domain

Binding of antigen to the B cell receptor (BCR) induces conformational changes in BCR''s cytoplasmic domains that are concomitant with phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs). Recently, reversible folding of the CD3ε and ξ chain ITAMs into the plasma membrane has been suggested to regulate T cell receptor signaling. Here we show that the Igα and Igβ cytoplasmic domains of the BCR do not associate with plasma membrane in resting B cells. However, antigen binding and ITAM phosphorylation specifically increased membrane proximity of Igα, but not Igβ. Thus, BCR activation is accompanied by asymmetric conformational changes, possibly promoting the binding of Igα and Igβ to differently localized signaling complexes.     

Inhibition of F1-ATPase Rotational Catalysis by the Carboxyl-terminal Domain of the ? Subunit

Escherichia coli ATP synthase (F₀F₁) couples catalysis and proton transport through subunit rotation. The ϵ subunit, an endogenous inhibitor, lowers F₁-ATPase activity by decreasing the rotation speed and extending the duration of the inhibited state (Sekiya, M., Hosokawa, H., Nakanishi-Matsui, M., Al-Shawi, M. K., Nakamoto, R. K., and Futai, M. (2010) Single molecule behavior of inhibited and active states of Escherichia coli ATP synthase F₁ rotation. J. Biol. Chem. 285, 42058–42067). In this study, we constructed a series of ϵ subunits truncated successively from the carboxyl-terminal domain (helix 1/loop 2/helix 2) and examined their effects on rotational catalysis (ATPase activity, average rotation rate, and duration of inhibited state). As expected, the ϵ subunit lacking helix 2 caused about ½-fold reduced inhibition, and that without loop 2/helix 2 or helix 1/loop 2/helix 2 showed a further reduced effect. Substitution of ϵSer¹⁰⁸ in loop 2 and ϵTyr¹¹⁴ in helix 2, which possibly interact with the β and γ subunits, respectively, decreased the inhibitory effect. These results suggest that the carboxyl-terminal domain of the ϵ subunit plays a pivotal role in the inhibition of F₁ rotation through interaction with other subunits.     

The N-terminal Flanking Region of the A1 Domain Regulates the Force-dependent Binding of von Willebrand Factor to Platelet Glycoprotein Ibα

Binding of platelet glycoprotein Ibα (GPIbα) to von Willebrand factor (VWF) initiates platelet adhesion to disrupted vascular surface under arterial blood flow. Flow exerts forces on the platelet that are transmitted to VWF-GPIbα bonds, which regulate their dissociation. Mutations in VWF and/or GPIbα may alter the mechanical regulation of platelet adhesion to cause hemostatic defects as found in patients with von Willebrand disease (VWD). Using a biomembrane force probe, we observed biphasic force-decelerated (catch) and force-accelerated (slip) dissociation of GPIbα from VWF. The VWF A1 domain that contains the N-terminal flanking sequence Gln¹²³⁸–Glu¹²⁶⁰ (1238-A1) formed triphasic slip-catch-slip bonds with GPIbα. By comparison, using a short form of A1 that deletes this sequence (1261-A1) abolished the catch bond, destabilizing its binding to GPIbα at high forces. Importantly, shear-dependent platelet rolling velocities on these VWF ligands in a flow chamber system mirrored the force-dependent single-bond lifetimes. Adding the Gln¹²³⁸–Glu¹²⁶⁰ peptide, which interacted with GPIbα and 1261-A1 but not 1238-A1, to whole blood decreased platelet attachment under shear stress. Soluble Gln¹²³⁸–Glu¹²⁶⁰ reduced the lifetimes of GPIbα bonds with VWF and 1238-A1 but rescued the catch bond of GPIbα with 1261-A1. A type 2B VWD 1238-A1 mutation eliminated the catch bond by prolonging lifetimes at low forces, a type 2M VWD 1238-A1 mutation shifted the respective slip-catch and catch-slip transition points to higher forces, whereas a platelet type VWD GPIbα mutation enhanced the bond lifetime in the entire force regime. These data reveal the structural determinants of VWF activation by hemodynamic force of the circulation.     

Mutation in the M1 Domain of the Acetylcholine Receptor α Subunit Decreases the Rate of Agonist Dissociation

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) α subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing αN217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC₅₀ to lower agonist concentrations for αN217K. The apparent affinity of ACh binding, measured by competition against the rate of ¹²⁵I-α-bungarotoxin binding, is also enhanced 20-fold by αN217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the β, ε, or δ subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.     

An MHC-I Cytoplasmic Domain/HIV-1 Nef Fusion Protein Binds Directly to the μ Subunit of the AP-1 Endosomal Coat Complex

Background

The down-regulation of the major histocompatibility complex class I (MHC-I) from the surface of infected cells by the Nef proteins of primate immunodeficiency viruses likely contributes to pathogenesis by providing evasion of cell-mediated immunity. HIV-1 Nef-induced down-regulation involves endosomal trafficking and a cooperative interaction between the cytoplasmic domain (CD) of MHC-I, Nef, and the clathrin adaptor protein complex-1 (AP-1). The CD of MHC-I contains a key tyrosine within the sequence YSQA that is required for down-regulation by Nef, but this sequence does not conform to the canonical AP-binding tyrosine-based motif Yxxφ, which mediates binding to the medium (μ) subunits of AP complexes. We previously proposed that Nef allows the MHC-I CD to bind the μ subunit of AP-1 (μ1) as if it contained a Yxxφmotif.

Methods and Findings

Here, we show that a direct interaction between the MHC-I CD/Nef and μ1 plays a primary role in the down-regulation of MHC-I: GST pulldown assays using recombinant proteins indicated that most of the MHC-I CD and Nef residues that are required for the down-regulation in human cells contribute to direct interactions with a truncated version of μ1. Specifically, the tyrosine residue of the YSQA sequence in the MHC-I CD as well as Nef residues E62-65 and P78 each contributed to the interaction between MHC-I CD/Nef and μ1 in vitro, whereas Nef M20 had little to no role. Conversely, residues F172/D174 and V392/L395 of the binding pocket on μ1 for Yxxφ motifs were required for a robust interaction.

Conclusions

These data indicate that the MHC-I cytoplasmic domain, Nef, and the C-terminal two thirds of the μ subunit of AP-1 are sufficient to constitute a biologically relevant interaction. The data also reveal an unexpected role for a hydrophobic pocket in μ1 for interaction with MHC-I CD/Nef.     

Zn2+ Mediates High Affinity Binding of Heparin to the αC Domain of Fibrinogen

The nonspecific binding of heparin to plasma proteins compromises its anticoagulant activity by reducing the amount of heparin available to bind antithrombin. In addition, interaction of heparin with fibrin promotes formation of a ternary heparin-thrombin-fibrin complex that protects fibrin-bound thrombin from inhibition by the heparin-antithrombin complex. Previous studies have shown that heparin binds the E domain of fibrinogen. The current investigation examines the role of Zn²⁺ in this interaction because Zn²⁺ is released locally by platelets and both heparin and fibrinogen bind the cation, resulting in greater protection from inhibition by antithrombin. Zn²⁺ promotes heparin binding to fibrinogen, as determined by chromatography, fluorescence, and surface plasmon resonance. Compared with intact fibrinogen, there is reduced heparin binding to fragment X, a clottable plasmin degradation product of fibrinogen. A monoclonal antibody directed against a portion of the fibrinogen αC domain removed by plasmin attenuates binding of heparin to fibrinogen and a peptide analog of this region binds heparin in a Zn²⁺-dependent fashion. These results indicate that the αC domain of fibrinogen harbors a Zn²⁺-dependent heparin binding site. As a consequence, heparin-catalyzed inhibition of factor Xa by antithrombin is compromised by fibrinogen to a greater extent when Zn²⁺ is present. These results reveal the mechanism by which Zn²⁺ augments the capacity of fibrinogen to impair the anticoagulant activity of heparin.     

Inhibition of αIIbβ3 Ligand Binding by an αIIb Peptide that Clasps the Hybrid Domain to the βI Domain of β3

Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, ^p1YMESRADR⁸, corresponding to amino acids 313–320 of the β-ribbon extending from the β-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbβ3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the β-ribbon by forming a clasp restraining the β3 hybrid and βI domains in a closed conformation. The involvement of species-specific residues of the β3 hybrid domain (E356 and K384) and the β1 domain (E297) as well as an intrapeptide bond (^pE315-^pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.     

Open-Channel Structures of the Human Glycine Receptor α1 Full-Length Transmembrane Domain

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Molecular Analysis of the Prostacyclin Receptor’s Interaction with the PDZ1 Domain of Its Adaptor Protein PDZK1

The prostanoid prostacyclin, or prostaglandin I₂, plays an essential role in many aspects of cardiovascular disease. The actions of prostacyclin are mainly mediated through its activation of the prostacyclin receptor or, in short, the IP. In recent studies, the cytoplasmic carboxy-terminal domain of the IP was shown to bind several PDZ domains of the multi-PDZ adaptor PDZK1. The interaction between the two proteins was found to enhance cell surface expression of the IP and to be functionally important in promoting prostacyclin-induced endothelial cell migration and angiogenesis. To investigate the interaction of the IP with the first PDZ domain (PDZ1) of PDZK1, we generated a nine residue peptide (KK⁴¹¹IAACSLC⁴¹⁷) containing the seven carboxy-terminal amino acids of the IP and measured its binding affinity to a recombinant protein corresponding to PDZ1 by isothermal titration calorimetry. We determined that the IP interacts with PDZ1 with a binding affinity of 8.2 µM. Using the same technique, we also determined that the farnesylated form of carboxy-terminus of the IP does not bind to PDZ1. To understand the molecular basis of these findings, we solved the high resolution crystal structure of PDZ1 bound to a 7-residue peptide derived from the carboxy-terminus of the non-farnesylated form of IP (⁴¹¹IAACSLC⁴¹⁷). Analysis of the structure demonstrates a critical role for the three carboxy-terminal amino acids in establishing a strong interaction with PDZ1 and explains the inability of the farnesylated form of IP to interact with the PDZ1 domain of PDZK1 at least in vitro.     

Evolution of High-Affinity Peptide Probes to Detect the SH3 Domain of Cancer Biomarker BCR–ABL

The BCR–ABL fusion protein is closely associated with the pathological progression of chronic myelogenous leukaemia and some other myeloproliferative diseases, which has long been recognized as one of the most important cancer biomarkers in the tumor diagnosis community. The SH3 domain of BCR–ABL is a small, conserved protein module that specifically recognizes and binds proline-rich peptide fragments. In the current study, we used a synthetic strategy to discover new peptide probes with high affinity binding to the BCR–ABL SH3 domain. In the procedure, a sequence-based machine learning predictor was developed based on a set of affinity-known SH3 binders, and the predictor was then used to guide the evolutional optimization of numerous virtual peptides to enrich high binding potency for the SH3 domain. Subsequently, a evolved peptide population was generated, from which ten peptides with the highest affinity scores were selected and their interaction free energies with SH3 domain were characterized systematically using a combination of molecular dynamics simulation and binding free energy analysis. Consequently, four peptides were suggested as promising candidates and their affinities toward SH3 domain were assayed; two peptides, APTYTPPPPP and APTYAPPPPP, were identified to have potent binding capability with dissociation constants K _d of 3 and 8 μM, respectively. Further, the structural basis and energetic property of SH3 domain in complex with APTYTPPPPP were examined in detail, revealing a non-specific interaction in SH3–peptide recognition that should render a broad ligand spectrum for the domain.     

The Nonreceptor Protein Tyrosine Phosphatase PTP1B Binds to the Cytoplasmic Domain of N-Cadherin and Regulates the Cadherin–Actin Linkage

Cadherin-mediated adhesion depends on the association of its cytoplasmic domain with the actin-containing cytoskeleton. This interaction is mediated by a group of cytoplasmic proteins: α-and β- or γ- catenin. Phosphorylation of β-catenin on tyrosine residues plays a role in controlling this association and, therefore, cadherin function. Previous work from our laboratory suggested that a nonreceptor protein tyrosine phosphatase, bound to the cytoplasmic domain of N-cadherin, is responsible for removing tyrosine-bound phosphate residues from β-catenin, thus maintaining the cadherin–actin connection (Balsamo et al., 1996). Here we report the molecular cloning of the cadherin-associated tyrosine phosphatase and identify it as PTP1B. To definitively establish a causal relationship between the function of cadherin-bound PTP1B and cadherin-mediated adhesion, we tested the effect of expressing a catalytically inactive form of PTP1B in L cells constitutively expressing N-cadherin. We find that expression of the catalytically inactive PTP1B results in reduced cadherin-mediated adhesion. Furthermore, cadherin is uncoupled from its association with actin, and β-catenin shows increased phosphorylation on tyrosine residues when compared with parental cells or cells transfected with the wild-type PTP1B. Both the transfected wild-type and the mutant PTP1B are found associated with N-cadherin, and recombinant mutant PTP1B binds to N-cadherin in vitro, indicating that the catalytically inactive form acts as a dominant negative, displacing endogenous PTP1B, and rendering cadherin nonfunctional. Our results demonstrate a role for PTP1B in regulating cadherin-mediated cell adhesion.     

The Role of the Amino-Terminal β-Barrel Domain of the α and β Subunits in the Yeast F1-ATPase

The crystal structure of mitochondrial F₁-ATPase indicatesthat the and subunits fold into a structure defined by threedomains: the top -barrel domain, the middle nucleotide-binding domain,and the C-terminal -helix bundle domain (Abraham et al.1994); Bianchet et al., 1998). The -barrel domains of the and subunits form a crown structure at the top ofF₁, which was suggested to stabilize it (Abraham et al.1994). In this study. the role of the -barrel domain in the and subunits of the yeast Saccharomyces cerevisiae F₁,with regard to its folding and assembly, was investigated. The -barreldomains of yeast F₁ and subunits were expressedindividually and together in Escherichia coli. When expressedseperately, the -barrel domain of the subunit formed a largeaggregate structure, while the domain of the subunit waspredominately a monomer or dimer. However, coexpression of the -barreldomain of subunit domain. Furthermore, the two domains copurified incomplexes with the major portion of the complex found in a small molecularweight form. These results indicate that the -barrel domain of the and subunits interact specifically with each other and thatthese interactions prevent the aggregation of the -barrel domain of the subunit. These results mimic in vivo results and suggest thatthe interactions of the -barrel domains may be critical during thefolding and assembly of F₁.     

Crystal Structure of BamB Bound to a Periplasmic Domain Fragment of BamA,the Central Component of the β-Barrel Assembly Machine

The β-barrel assembly machinery (BAM) mediates folding and insertion of β-barrel outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria. BAM is a five-protein complex consisting of the β-barrel OMP BamA and lipoproteins BamB, -C, -D, and -E. High resolution structures of all the individual BAM subunits and a BamD-BamC complex have been determined. However, the overall complex architecture remains elusive. BamA is the central component of BAM and consists of a membrane-embedded β-barrel and a periplasmic domain with five polypeptide translocation-associated (POTRA) motifs thought to interact with the accessory lipoproteins. Here we report the crystal structure of a fusion between BamB and a POTRA3–5 fragment of BamA. Extended loops 13 and 17 protruding from one end of the BamB β-propeller contact the face of the POTRA3 β-sheet in BamA. The interface is stabilized by several hydrophobic contacts, a network of hydrogen bonds, and a cation-π interaction between BamA Tyr-255 and BamB Arg-195. Disruption of BamA-BamB binding by BamA Y255A and probing of the interface by disulfide bond cross-linking validate the physiological relevance of the observed interface. Furthermore, the structure is consistent with previously published mutagenesis studies. The periplasmic five-POTRA domain of BamA is flexible in solution due to hinge motions in the POTRA2–3 linker. Modeling BamB in complex with full-length BamA shows BamB binding at the POTRA2–3 hinge, suggesting a role in modulation of BamA flexibility and the conformational changes associated with OMP folding and insertion.     

On the Role of the SP1 Domain in HIV-1 Particle Assembly: a Molecular Switch?

Expression of a retroviral protein, Gag, in mammalian cells is sufficient for assembly of immature virus-like particles (VLPs). VLP assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We have investigated the role of SP1, a spacer between CA and NC in HIV-1 Gag, in VLP assembly. Mutational analysis showed that even subtle changes in the first 4 residues of SP1 destroy the ability of Gag to assemble correctly, frequently leading to formation of tubes or other misassembled structures rather than proper VLPs. We also studied the conformation of the CA-SP1 junction region in solution, using both molecular dynamics simulations and circular dichroism. Consonant with nuclear magnetic resonance (NMR) studies from other laboratories, we found that SP1 is nearly unstructured in aqueous solution but undergoes a concerted change to an α-helical conformation when the polarity of the environment is reduced by addition of dimethyl sulfoxide (DMSO), trifluoroethanol, or ethanol. Remarkably, such a coil-to-helix transition is also recapitulated in an aqueous medium at high peptide concentrations. The exquisite sensitivity of SP1 to mutational changes and its ability to undergo a concentration-dependent structural transition raise the possibility that SP1 could act as a molecular switch to prime HIV-1 Gag for VLP assembly. We suggest that changes in the local environment of SP1 when Gag oligomerizes on nucleic acid might trigger this switch.     

Rational Improvement of Peptide Affinity to Human Pregnancy-Related Serine Protease HtrA3 PDZ Domain by Introducing a Halogen Bond to the Domain–Peptide Complex Interface

The pregnancy-related serine protease HtrA3 plays an important role in human placental development and has recently been recognized as a potential therapeutic target in the treatment of cancer. Previously, a C-terminal pentapeptide FGRWV^–COOH was identified to bind at the PDZ domain of HtrA3 with a moderate affinity. Here, based on the high-resolution complex crystal structure of HtrA3 PDZ domain with the pentapeptide ligand we successfully introduced a rationally designed halogen bond to the complex interface by substituting R₄-hydrogen atom of the indole moiety of peptide Trp_-1 residue with a halogen atom. High-level theoretical calculations suggested that bromine is the best choice that can render strong interaction energy for the halogen bond and can confer high affinity to the PDZ–peptide complex. Fluorescence spectroscopy characterizations revealed that the resulting R₄-brominated peptide (K _d = 0.15 ± 0.03 μM) exhibited 12-fold affinity improvement relative to its nonhalogenated counterpart (K _d = 1.8 ± 0.4 μM). In contrast, the PDZ-binding affinity of R₆-brominated peptide (K _d = 1.2 ± 0.1 μM), a negative control that was unable to form the halogen bond according to theoretical investigations, did not change substantially as compared to the nonhalogenated peptide.     

Forcing Switch from Short- to Intermediate- and Long-lived States of the αA Domain Generates LFA-1/ICAM-1 Catch Bonds

Binding of lymphocyte function-associated antigen-1 (LFA-1) to intercellular adhesion molecule-1 (ICAM-1) mediates leukocyte adhesion under force. Using a biomembrane force probe capable of measuring single bond interactions, we showed ICAM-1 binding to LFA-1 at different conformations, including the bent conformation with the lowest affinity. We quantify how force and conformations of LFA-1 regulate its kinetics with ICAM-1. At zero-force, on-rates were substantially changed by conditions that differentially favor a bent or extended LFA-1 with a closed or open headpiece; but off-rates were identical. With increasing force, LFA-1/ICAM-1 bond lifetimes (reciprocal off-rates) first increased (catch bonds) and then decreased (slip bonds). Three states with distinct off-rates were identified from lifetime distributions. Force shifted the associated fractions from the short- to intermediate- and long-lived states, producing catch bonds at low forces, but increased their off-rates exponentially, converting catch to slip bonds at high forces. An internal ligand antagonist that blocks pulling of the α₇-helix suppressed the intermediate-/long-lived states and eliminated catch bonds, revealing an internal catch bond between the αA and βA domains. These results elucidate an allosteric mechanism for the mechanochemistry of LFA-1/ICAM-1 binding.     

Pressure-Temperature Analysis of the Stability of the CTL9 Domain Reveals Hidden Intermediates

The observation of two-state unfolding for many small single-domain proteins by denaturants has led to speculation that protein sequences may have evolved to limit the population of partially folded states that could be detrimental to fitness. How such strong cooperativity arises from a multitude of individual interactions is not well understood. Here, we investigate the stability and folding cooperativity of the C-terminal domain of the ribosomal protein L9 in the pressure-temperature plane using site-specific NMR. In contrast to apparent cooperative unfolding detected with denaturant-induced and thermal-induced unfolding experiments and stopped-flow refolding studies at ambient pressure, NMR-detected pressure unfolding revealed significant deviation from two-state behavior, with a core region that was selectively destabilized by increasing temperature. Comparison of pressure-dependent NMR signals from both the folded and unfolded states revealed the population of at least one invisible excited state at atmospheric pressure. The core destabilizing cavity-creating I98A mutation apparently increased the cooperativity of the loss of folded-state peak intensity while also increasing the population of this invisible excited state present at atmospheric pressure. These observations highlight how local stability is subtly modulated by sequence to tune protein conformational landscapes and illustrate the ability of pressure- and temperature-dependent studies to reveal otherwise hidden states.