ABSTRACT
Cardiac troponin C (cTnC) is the critical Ca2+ -sensing component of the troponin complex. Binding of Ca2+ to cTnC triggers a cascade of conformational changes within the myofilament that culminate in force production. Hypertrophic cardiomyopathy (HCM)-associated TNNC1 variants generally induce a greater degree and duration of Ca2+ binding, which may underly the hypertrophic phenotype. Regulation of contraction has long been thought to occur exclusively through Ca2+ binding to site II of cTnC. However, work by several groups including ours suggest that Mg2+ , which is several orders of magnitude more abundant in the cell than Ca2+ , may compete for binding to the same cTnC regulatory site. We previously used isothermal titration calorimetry (ITC) to demonstrate that physiological concentrations of Mg2+ may decrease site II Ca2+ -binding in both N-terminal and full-length cTnC. Here, we explore the binding of Ca2+ and Mg2+ to cTnC harbouring a series of TNNC1 variants thought to be causal in HCM. ITC and thermodynamic integration (TI) simulations show that A8V, L29Q and A31S elevate the affinity for both Ca2+ and Mg2+ . Further, L48Q, Q50R and C84Y that are adjacent to the EF hand binding motif of site II have a more significant effect on affinity and the thermodynamics of the binding interaction. To the best of our knowledge, this work is the first to explore the role of Mg2+ in modifying the Ca2+ affinity of cTnC mutations linked to HCM. Our results indicate a physiologically significant role for cellular Mg2+ both at baseline and when elevated on modifying the Ca2+ binding properties of cTnC and the subsequent conformational changes which precede cardiac contraction.
Subject(s)
Cardiomyopathy, Hypertrophic , Humans , Cardiomyopathy, Hypertrophic/geneticsABSTRACT
Previous comparative kinetic isotope effects have inferred an allosteric site for fatty acids and their derivatives that modulates substrate selectivity in 15-lipoxygenases. Hydrogen-deuterium exchange also previously revealed regionally defined enhanced protein flexibility, centred at helix α2 - a gate to the substrate entrance. Direct evidence for allosteric binding and a complete understanding of its mechanism remains elusive. In this study, we examine the binding thermodynamics of the fatty acid mimic, oleyl sulfate (OS), with the monomeric model plant 15-LOX, soybean lipoxygenase (SLO), using isothermal titration calorimetry. Dynamic light scattering and differential scanning calorimetry rule out OS-induced oligomerization or structural changes. These data provide evidence that the fatty acid allosteric regulation of SLO is controlled by the dynamics of helix α2.
Subject(s)
LipoxygenaseABSTRACT
Brucella is a zoonotic pathogen requiring iron for its survival and acquires this metal through the expression of several high-affinity uptake systems. Of these, the newly discovered ferrous iron transporter, FtrABCD, is proposed to take part in ferrous iron uptake. Sequence homology shows that, FtrA, the proposed periplasmic ferrous-binding component, is a P19-type protein (a periplasmic protein from C. jejuni which shows Cu2+ dependent iron affinity). Previous structural and biochemical studies on other P19 systems have established a Cu2+ dependent Mn2+ affinity as well as formation of homodimers for these systems. The Cu2+ coordinating amino acids from these proteins are conserved in Brucella FtrA, hinting towards similar properties. However, there has been no experimental evidence, till date, establishing metal affinities and the possibility of dimer formation by Brucella FtrA. Using wild-type FtrA and Cu2+-binding mutants (H65A, E67A, H118A, and H151A) we investigated the metal affinities, folding stabilities, dimer forming abilities, and the molecular basis of the Cu2+ dependence for this P19-type protein employing homology modeling, analytical gel filtration, calorimetric, and spectroscopic methods. The data reported here confirm a Cu2+-dependent, low-µM Mn2+ (Fe2+ mimic) affinity for the wild-type FtrA. In addition, our data clearly show the loss of Mn2+ affinity, and the formation of less stable protein conformations as a result of mutating these conserved Cu2+-binding residues, indicating the important roles these residues play in producing a native and functional fold of Brucella FtrA.
Subject(s)
Bacterial Proteins/metabolism , Brucella/chemistry , Cation Transport Proteins/metabolism , Copper/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cation Transport Proteins/chemistry , Cation Transport Proteins/genetics , Manganese/metabolism , Mutagenesis, Site-Directed , Mutation , Phase Transition , Protein Binding , Protein Folding , Protein Stability , Protein Structure, SecondaryABSTRACT
BACKGROUND: Characterizing the thermodynamic parameters behind metal-biomolecule interactions is fundamental to understanding the roles metal ions play in biology. Isothermal Titration Calorimetry (ITC) is a "gold-standard" for obtaining these data. However, in addition to metal-protein binding, additional equilibria such as metal-buffer interactions must be taken into consideration prior to making meaningful comparisons between metal-binding systems. METHODS: In this study, the thermodynamics of Ca2+ binding to three buffers (Bis-Tris, MES, and MOPS) were obtained from Ca2+-EDTA titrations using ITC. These data were used to extract buffer-independent parameters for Ca2+ binding to human cardiac troponin C (hcTnC), an EF-hand containing protein required for heart muscle contraction. RESULTS: The number of protons released upon Ca2+ binding to the C- and N-domain of hcTnC were found to be 1.1 and 1.2, respectively. These values permitted determination of buffer-independent thermodynamic parameters of Ca2+-hcTnC binding, and the extracted data agreed well among the buffers tested. Both buffer and pH-adjusted parameters were determined for Ca2+ binding to the N-domain of hcTnC and revealed that Ca2+ binding under aqueous conditions and physiological ionic strength is both thermodynamically favorable and driven by entropy. CONCLUSIONS: Taken together, the consistency of these data between buffer systems and the similarity between theoretical and experimental proton release is indicative of the reliability of the method used and the importance of extracting metal-buffer interactions in these studies. GENERAL SIGNIFICANCE: The experimental approach described herein is clearly applicable to other metal ions and other EF-hand protein systems.
Subject(s)
Calcium/chemistry , Troponin C/chemistry , Alkanesulfonic Acids/chemistry , Buffers , Edetic Acid/chemistry , Heart , Humans , Hydrogen-Ion Concentration , Morpholines/chemistry , Thermodynamics , Tromethamine/analogs & derivatives , Tromethamine/chemistryABSTRACT
BACKGROUND: ITC is a powerful technique that can reliably assess the thermodynamic underpinnings of a wide range of binding events. When metal ions are involved, complications arise in evaluating the data due to unavoidable solution chemistry that includes metal speciation and a variety of linked equilibria. SCOPE OF REVIEW: This paper identifies these concerns, provides recommendations to avoid common mistakes, and guides the reader through the mathematical treatment of ITC data to arrive at a set of thermodynamic state functions that describe identical chemical events and, ideally, are independent of solution conditions. Further, common metal chromophores used in biological metal sensing studies are proposed as a robust system to determine unknown solution competition. MAJOR CONCLUSIONS: Metal ions present several complications in ITC experiments. This review presents strategies to avoid these pitfalls and proposes and experimentally validates mathematical approaches to deconvolute complex equilibria that exist in these systems. GENERAL SIGNIFICANCE: This review discusses the wide range of complications that exists in metal-based ITC experiments. It provides a starting point for scientists new to this field and articulates concerns that will help experienced researchers troubleshoot experiments.
Subject(s)
Aminoquinolines/chemistry , Edetic Acid/chemistry , Fura-2/analogs & derivatives , Metals, Alkaline Earth/chemistry , Proteins/chemistry , Binding Sites , Calorimetry , Cations, Divalent , Fura-2/chemistry , Humans , Kinetics , Ligands , Models, Chemical , Protein Binding , ThermodynamicsABSTRACT
Although the Cu(2+)-binding sites of the prion protein have been well studied when the protein is fully saturated by Cu(2+), the Cu(2+)-loading mechanism is just beginning to come into view. Because the Cu(2+)-binding modes at low and intermediate Cu(2+) occupancy necessarily represent the highest-affinity binding modes, these are very likely populated under physiological conditions, and it is thus essential to characterize them in order to understand better the biological function of copper-prion interactions. Besides binding-affinity data, almost no other thermodynamic parameters (e.g., ΔH and ΔS) have been measured, thus leaving undetermined the enthalpic and entropic factors that govern the free energy of Cu(2+) binding to the prion protein. In this study, isothermal titration calorimetry (ITC) was used to quantify the thermodynamic parameters (K, ΔG, ΔH, and TΔS) of Cu(2+) binding to a peptide, PrP(23-28, 57-98), that encompasses the majority of the residues implicated in Cu(2+) binding by full-length PrP. Use of the buffer N-(2-acetomido)-aminoethanesulfonic acid (ACES), which is also a well-characterized Cu(2+) chelator, allowed for the isolation of the two highest affinity binding events. Circular dichroism spectroscopy was used to characterize the different binding modes as a function of added Cu(2+). The Kd values determined by ITC, 7 and 380 nM, are well in line with those reported by others. The first binding event benefits significantly from a positive entropy, whereas the second binding event is enthalpically driven. The thermodynamic values associated with Cu(2+) binding by the Aß peptide, which is implicated in Alzheimer's disease, bear striking parallels to those found here for the prion protein.
Subject(s)
Calorimetry , Copper/metabolism , Entropy , Prions/metabolism , Histidine , Prions/chemistry , Protein BindingABSTRACT
Human cardiac troponin C (HcTnC), a member of the EF hand family of proteins, is a calcium sensor responsible for initiating contraction of the myocardium. Ca(2+) binding to the regulatory domain induces a slight change in HcTnC conformation which modifies subsequent interactions in the troponin-tropomyosin-actin complex. Herein, we report a calorimetric study of Ca(2+) binding to HcTnC. Isotherms obtained at 25 °C (10 mM 2-morpholinoethanesulfonic acid, 50 mM KCl, pH 7.0) provided thermodynamic parameters for Ca(2+) binding to both the high-affinity and the low-affinity domain of HcTnC. Ca(2+) binding to the N-domain was shown to be endothermic in 2-morpholinoethanesulfonic acid buffer and allowed us to extract the thermodynamics of Ca(2+) binding to the regulatory domain. This pattern stems from changes that occur at the Ca(2+) site rather than structural changes of the protein. Molecular dynamics simulations performed on apo and calcium-bound HcTnC(1-89) support this claim. The values of the Gibbs free energy for Ca(2+) binding to the N-domain in the full-length protein and to the isolated domain (HcTnC(1-89)) are similar; however, differences in the entropic and enthalpic contributions to the free energy provide supporting evidence for the cooperativity of the C-domain and the N-domain. Thermograms obtained at two additional temperatures (10 and 37 °C) revealed interesting trends in the enthalpies and entropies of binding for both thermodynamic events. This allowed the determination of the change in heat capacity (∆C(p)) from a plot of ∆H verses temperature and may provide evidence for positive cooperativity of Ca(2+) binding to the C-domain.
Subject(s)
Calcium/metabolism , Molecular Dynamics Simulation , Myocardium/metabolism , Troponin C/chemistry , Troponin C/metabolism , Binding Sites , Humans , Peptide Fragments/metabolism , Protein Binding , Protein Structure, Tertiary , Temperature , ThermodynamicsABSTRACT
Nanoscale assembly is an area of research that has vast implications for molecular design, sensing, nanofabrication, supramolecular chemistry, catalysis, and environmental remediation. Here we show that poly(amidoamine) (PAMAM) dendrimers of both generations 1 (G1) and 4 (G4) can host 1 fullerenol per 2 dendrimer primary amines as evidenced by isothermal titration calorimetry, dynamic light scattering and spectrofluorometry. Thermodynamically, the interactions were similarly spontaneous between both generations of dendrimers and fullerenols, however, G4 formed stronger complexes with fullerenols resulting from their higher surface charge density and more internal voids, as demonstrated by spectrofluorometry. In addition to hydrogen bonding that existed between the dendrimer primary amines and the fullerenol oxygens, hydrophobic and electrostatic interactions also contributed to complex formation and dynamics. Such hybrid of soft and condensed nanoassembly may have implications for environmental remediation of discharged nanomaterials and entail new applications in drug delivery.
ABSTRACT
The thermodynamics of Zn(2+) binding to three peptides corresponding to naturally occurring Zn-binding sequences in transcription factors have been quantified with isothermal titration calorimetry (ITC). These peptides, the third zinc finger of Sp1 (Sp1-3), the second zinc finger of myelin transcription factor 1 (MyT1-2), and the second Zn-binding sequence of the DNA-binding domain of glucocorticoid receptor (GR-2), bind Zn(2+) with Cys(2)His(2), Cys(2)HisCys, and Cys(4) coordination, respectively. Circular dichroism confirms that Sp1-3 and MyT1-2 have considerable and negligible Zn-stabilized secondary structure, respectively, and indicate only a small amount for GR-2. The pK(a)'s of the Sp1-3 cysteines and histidines were determined by NMR and used to estimate the number of protons displaced by Zn(2+) at pH 7.4. ITC was also used to determine this number, and the two methods agree. Subtraction of buffer contributions to the calorimetric data reveals that all three peptides have a similar affinity for Zn(2+), which has equal enthalpy and entropy components for Sp1-3 but is more enthalpically disfavored and entropically favored with increasing Cys ligands. The resulting enthalpy-entropy compensation originates from the Zn-Cys coordination, as subtraction of the cysteine deprotonation enthalpy results in a similar Zn(2+)-binding enthalpy for all three peptides, and the binding entropy tracks with the number of displaced protons. Metal and protein components of the binding enthalpy and entropy have been estimated. While dominated by Zn(2+) coordination to the cysteines and histidines, other residues in the sequence affect the protein contributions that modulate the stability of these motifs.
Subject(s)
Cysteine/chemistry , Thermodynamics , Zinc Fingers , Zinc/chemistry , Circular Dichroism , Magnetic Resonance Spectroscopy , Models, Molecular , Protein StabilityABSTRACT
Metal ions have been shown to play a critical role in ß-amyloid (Aß) neurotoxicity, thus prompting an intense investigation into the formation of metal-Aß complexes. Isothermal titration calorimetry (ITC) has been widely used to determine binding constants (K) for a variety of metal-protein interactions, including those in metal-Aß complexes. In this study, ITC was used to more fully quantify the thermodynamics (K, ΔG, ΔH, and TΔS) of Cu(2+) binding to Aß16, N-acetyl-Aß16, Aß28, N-acetyl-Aß28, and Aß28 variants (H6A, H13A, H14A) at pH 7.4 and 37 °C. After deconvolution of competing reactions, K for Aß16 was found to be 1.1 (±0.13) × 10(9) and is in strong agreement with literature values measured under similar conditions. Further, a similar K value was obtained at two additional concentrations of competing ligand, suggesting that ternary complex formation is not significant. The acetylated peptide analogs reveal a marked decrease in the overall free energy upon binding, which is the result of less favorable enthalpic and entropic contributions. Circular dichroism spectroscopy shows conformational changes that are consistent with these results. Most importantly, data for Aß28 variants lacking a potential Cu(2+)-binding histidine residue reveal that the overall free energy of binding remains constant, which is the result of entropy/enthalpy compensation. These data provide fundamental thermodynamic evidence for coordination plasticity in Cu(2+) binding to Aß and other intrinsically disordered peptides.
Subject(s)
Amyloid beta-Peptides/chemistry , Copper/chemistry , Thermodynamics , Amyloid beta-Peptides/metabolism , Binding Sites , Calorimetry , Copper/metabolismABSTRACT
Extensive circular dichroism, isothermal titration calorimetry and induced calcein leakage studies were conducted on a series of antimicrobial peptides (AMPs), with a varying number of Lys residues located at either the C-terminus or the N-terminus to gain insight into their effect on the mechanisms of binding with zwitterionic and anionic membrane model systems. Different CD spectra were observed for these AMPs in the presence of zwitterionic DPC and anionic SDS micelles indicating that they adopt different conformations on binding to the surfaces of zwitterionic and anionic membrane models. Different CD spectra were observed for these AMPs in the presence of zwitterionic POPC and anionic mixed 4:1 POPC/POPG LUVs and SUVs, indicating that they adopt very different conformations on interaction with these two types of LUVs and SUVs. In addition, ITC and calcein leakage data indicated that all the AMPs studied interact via very different mechanisms with anionic and zwitterionic LUVs. ITC data suggest these peptides interact primarily with the surface of zwitterionic LUVs while they insert into and form pores in anionic LUVs. CD studies indicated that these compounds adopt different conformations depending on the ratio of POPC to POPG lipids present in the liposome. There are detectable spectroscopic and thermodynamic differences between how each of these AMPs interacts with membranes, that is position and total charge density defines how these AMPs interact with specific membrane models and thus partially explain the resulting diversity of antibacterial activity of these compounds.
Subject(s)
Amino Acids, Basic/chemical synthesis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Amino Acid Sequence , Antimicrobial Cationic Peptides/chemistry , Calorimetry , Circular Dichroism , Molecular Sequence Data , Phosphatidylcholines/chemistry , Phosphatidylglycerols/chemistry , Protein Binding , Structure-Activity RelationshipABSTRACT
Circular Dichroism (CD), isothermal calorimetry (ITC) and calcein fluorescence leakage experiments were conducted to provide insight into the mechanisms of binding of a series of antimicrobial peptides containing unnatural amino acids (Ac-XF-Tic-Oic-XK-Tic-Oic-XF-Tic-Oic-XK-Tic-KKKK-CONH(2)) to zwitterionic and anionic micelles, SUVs and LUVs; where X (Spacer# 1) is either Gly, ß-Ala, Gaba or 6-aminohexanoic acid. It is the intent of this investigation to correlate these interactions with the observed potency and selectivity against several different strains of bacteria. The CD spectra of these compounds in the presence of zwitterionic DPC micelles and anionic SDS micelles are very different indicating that these compounds adopt different conformations on binding to the surface of anionic and zwitterionic membrane models. These compounds also exhibited very different CD spectra in the presence of zwitterionic POPC and anionic mixed 4:1 POPC/POPG SUVs and LUVs, indicating the formation of different conformations on interaction with the two membrane types. This observation is also supported by ITC and calcein leakage data. ITC data suggested these peptides interact primarily with the surface of zwitterionic LUVs and was further supported by fluorescence experiments where the interactions do not appear to be concentration dependent. In the presence of anionic membranes, the interactions appear more complex and the calorimetric and fluorescence data both imply pore formation is dependent on peptide concentration. Furthermore, evidence suggests that as the length of Spacer# 1 increases the mechanism of pore formation also changes. Based on the observed differences in the mechanisms of interactions with zwitterionic and anionic LUVs these AMPs are potential candidates for further drug development.
Subject(s)
Antimicrobial Cationic Peptides/chemistry , Membranes, Artificial , Amino Acids , Liposomes/chemistry , Liposomes/metabolism , Micelles , Models, Biological , PermeabilityABSTRACT
Adaptor proteins mediate signal transduction from cell surface receptors to downstream signaling pathways. The Grb7 protein family of adaptor proteins is constituted by Grb7, Grb10, and Grb14. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7-mediated cell migration has been shown to proceed through a focal adhesion kinase (FAK)/Grb7 pathway, although the specific participants downstream of Grb7 in cell migration signaling have not been fully determined. In this study, we report that Grb7 interacts with Hax-1, a cytoskeletal-associated protein found overexpressed in metastatic tumors and cancer cell lines. Additionally, in yeast 2-hybrid assays, we show that the interaction is specific to the Grb7-RA and -PH domains. We have also demonstrated that full-length Grb7 and Hax-1 interact in mammalian cells and that Grb7 is tyrosine phosphorylated. Isothermal titration calorimetry measurements demonstrate the Grb7-RA-PH domains bind to the Grb7-SH2 domain with micromolar affinity, suggesting full-length Grb7 can exist in a head-to-tail conformational state that could serve a self-regulatory function.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , GRB7 Adaptor Protein/chemistry , GRB7 Adaptor Protein/metabolism , Adaptor Proteins, Signal Transducing/chemistry , HeLa Cells , Humans , Immunoprecipitation , Phosphorylation , Phosphotyrosine , Protein Binding , Structure-Activity Relationship , Thermodynamics , Transfection , src Homology DomainsABSTRACT
The thermodynamics of metals ions binding to proteins and other biological molecules can be measured with isothermal titration calorimetry (ITC), which quantifies the binding enthalpy (ΔH°) and generates a binding isotherm. A fit of the isotherm provides the binding constant (K), thereby allowing the free energy (ΔG°) and ultimately the entropy (ΔS°) of binding to be determined. The temperature dependence of ΔH° can then provide the change in heat capacity (ΔC (p)°) upon binding. However, ITC measurements of metal binding can be compromised by undesired reactions (e.g., precipitation, hydrolysis, and redox), and generally involve competing equilibria with the buffer and protons, which contribute to the experimental values (K (ITC), ΔH (ITC)). Guidelines and factors that need to be considered for ITC measurements involving metal ions are outlined. A general analysis of the experimental ITC values that accounts for the contributions of metal-buffer speciation and proton competition and provides condition-independent thermodynamic values (K, ΔH°) for metal binding is developed and validated.
Subject(s)
Calorimetry , Chemistry, Bioinorganic/methods , ThermodynamicsABSTRACT
In our laboratory we developed a series of antimicrobial peptides that exhibit selectivity and potency for prokaryotic over eukaryotic cells (Hicks et al., 2007). Circular dichroism (CD), isothermal calorimetry (ITC) and calcein leakage assays were conducted to determine the mechanism of lipid binding of a representative peptide 1 (Ac-GF-Tic-Oic-GK-Tic-Oic-GF-Tic-Oic-GK-Tic-KKKK-CONH(2)) to model membranes. POPC liposomes were used as a simple model for eukaryotic membranes and 4:1 POPC:POPG liposomes were used as a simple model for prokaryotic membranes. CD, ITC and calcein leakage data clearly indicate that compound 1 interacts via very different mechanisms with the two different liposome membranes. Compound 1 exhibits weaker binding and induces less calcein leakage in POPC liposomes than POPC:POPG (4:1 mole ratio) liposomes. The predominant binding mechanism to POPC appears to be limited to surface interactions while the mechanism of binding to 4:1 POPC:POPG most likely involves some type of pore formation.
Subject(s)
Anti-Infective Agents/chemistry , Antimicrobial Cationic Peptides/chemistry , Liposomes/chemistry , Amino Acid Sequence , Anti-Infective Agents/chemical synthesis , Anti-Infective Agents/metabolism , Antimicrobial Cationic Peptides/chemical synthesis , Antimicrobial Cationic Peptides/metabolism , Calorimetry , Circular Dichroism , Liposomes/metabolism , Phosphatidylcholines/chemistry , Phosphatidylglycerols/chemistry , ThermodynamicsABSTRACT
Hybrid receptors designed to recognize both the sulfonate headgroup and the fluorous tail of perfluorooctanesulfonate (CF(3)(CF(2))(7)SO(3)(-), "PFOS") were prepared by coupling fluorinated carboxylic acids onto poly(aminomethyl)benzene scaffolds. Binding to PFOS, CF(3)SO(3)(-), p-TsO(-), and Cl(-) was monitored by (1)H NMR and isothermal titration calorimetry (ITC). In chloroform solvent, hydrogen-bonding to anions is accompanied by downfield shifts in the amide NH protons of the fluorinated receptors and by evolution of heat. Association constants for 1:1 complexation (K(assoc)) are >1000 M(-1). An analogous hydrocarbon receptor binds weakly to anionic guests (K(assoc) < 50 M(-1)). Ab initio calculations indicate that the differences in 1:1 binding strengths between fluorous and nonfluorous hosts cannot be ascribed to differences in NH donor acidities.
Subject(s)
Amides/chemistry , Fluorocarbons/chemistry , Alkanesulfonic Acids/chemistry , Isoelectric Focusing , Substrate Specificity , ThermodynamicsABSTRACT
The binding of arsenite (As(III)) and monomethylarsenite (MMAIII) to the DNA-binding domain of the glucocorticoid receptor (GR-DBD) and their competition with the two required Zn2+ ions of this domain have been investigated with isothermal titration calorimetry (ITC) and circular dichroism (CD). The binding thermodynamics indicate that MMAIII, but not arsenite, is able to compete with one of the two Zn2+ ions. This has been confirmed by monitoring arsenite and MMAIII titrations of Zn2GR-DBD with CD. Only MMAIII is able to eliminate the Zn-stabilized secondary structure, consistent with partial or complete displacement of at least one Zn2+ ion and, therefore, loss of GR-DBD competence to bind to the DNA of its recognition site, the glucocorticoid response element (GRE).
Subject(s)
Arsenites/chemistry , Receptors, Glucocorticoid/metabolism , Zinc/chemistry , Binding Sites , Binding, Competitive , DNA-Binding Proteins , Humans , Protein Structure, Secondary , Receptors, Glucocorticoid/chemistry , ThermodynamicsABSTRACT
Grb7 is a member of the Grb7 family of proteins, which also includes Grb10 and Grb14. All three proteins have been found to be overexpressed in certain cancers and cancer cell lines. In particular, Grb7 (along with the receptor tyrosine kinase erbB2) is overexpressed in 20%-30% of breast cancers. Grb7 binds to erbB2 and may be involved in cell signaling pathways that promote the formation of metastases and inflammatory responses. In a prior study, we reported the solution structure of the Grb7-SH2/erbB2 peptide complex. In this study, T(1), T(2), and steady-state NOE measurements were performed on the Grb7-SH2 domain, and the backbone relaxation behavior of the domain is discussed with respect to the potential function of an insert region present in all three members of this protein family. Isothermal titration calorimetry (ITC) studies were completed measuring the thermodynamic parameters of the binding of a 10-residue phosphorylated peptide representative of erbB2 to the SH2 domain. These measurements are compared to calorimetric studies performed on other SH2 domain/phosphorylated peptide complexes available in the literature.
Subject(s)
Multiprotein Complexes/chemistry , Peptides/chemistry , Proteins/chemistry , Receptor, ErbB-2/chemistry , src Homology Domains , GRB7 Adaptor Protein , Humans , ThermodynamicsABSTRACT
Colorimetric (near-UV absorption spectroscopy) and calorimetric (isothermal titration calorimetry) methods have been used to quantify the equilibrium and thermodynamics of arsenite and monomethylarsenite (MMA) coordinating to glutathione (GSH) and the dithiols dimercaptosuccinic acid (DMSA), dihydrolipoic acid (DHLA), and dithiothreitol (DTT). We found that both arsenite and MMA form moderately stable complexes (beta = 10(6)-10(7)) with GSH; that arsenite forms a particularly stable 2:3 complex (beta approximately 10(18)) with the biological cofactor DHLA; that MMA has a somewhat higher affinity than arsenite for thiol ligands; and that entropic factors modulate the overall stability of As(III) complexes with thiols, which are favored by the exothermic formation of As(III)-thiolate bonds. The implications of these results for arsenic toxicity are discussed.