[17] Theoretical aspects of isothermal titration calorimetry

Indyk, Lawrence; Fisher, Harvey F.

doi:10.1016/s0076-6879(98)95048-0

Cited by 139 publications

(134 citation statements)

References 2 publications

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“…Heat flow traces obtained by ITC measurements were integrated, corrected for background, and analyzed using Origin Scripts provided by Microcal (Northampton, MA) using the expressions for the binding of titrant to identical and non-interacting sites (43)(44)(45). When necessary, non-linear least squares routines accommodated the simultaneous analysis of two or more data sets obtained with a staggered injection sequence.…”

Section: Kinetics Of Peptidyl Substrate Cleavage In the Presence Of Nmentioning

confidence: 99%

Nematode Anticoagulant Protein c2 Reveals a Site on Factor Xa That Is Important for Macromolecular Substrate Binding to Human Prothrombinase

Buddai¹,

Toulokhonova²,

Bergum³

et al. 2002

Journal of Biological Chemistry

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The binding of recombinant nematode anticoagulant protein c2 (NAPc2) to either factor X or Xa is a requisite step in the pathway for the potent inhibition of VIIa tissue factor. We have used NAPc2 as a tight binding probe of human Xa to investigate protein substrate recognition by the human prothrombinase complex. NAPc2 binds with high affinity (K d ϳ1 nM) to both X and Xa in a way that does not require or occlude the active site of the enzyme. In contrast, NAPc2 is a tight binding, competitive inhibitor of protein substrate cleavage by human Xa incorporated into prothrombinase with saturating concentrations of membranes and Va. By fluorescence binding studies we show that NAPc2 does not interfere with the assembly of human prothrombinase. These are properties expected of an inhibitor that blocks protein substrate recognition by targeting extended macromolecular recognition sites (exosites) on the enzyme complex. A weaker interaction (K d ‫؍‬ 260-500 nM) observed between NAPc2 and bovine X was restored to a high affinity one in a recombinant chimeric bovine X derivative containing 25 residues from the COOH terminus of the proteinase domain of human X. This region implicated in binding NAPc2 is spatially adjacent to a site previously identified as a potential exosite. Despite the weaker interaction with bovine Xa, NAPc2 was a tight binding competitive inhibitor of protein substrate cleavage by bovine prothrombinase as well. Extended enzymic surfaces elucidated with exosite-directed probes, such as NAPc2, may define a unique region of factor Xa that is modulated following its assembly into prothrombinase and in turn determines the binding specificity of the enzyme complex for its protein substrate.The proteolytic activation of prothrombin is catalyzed by the prothrombinase complex of coagulation (2-5). Prothrombinase assembles through membrane-dependent interactions between the serine proteinase, factor Xa, and the protein cofactor, factor Va (2, 3). Although solution-phase Xa is a competent enzyme, its incorporation into prothrombinase yields a profound increase in the rate of thrombin formation (2, 3).Prothrombin is the only known protein substrate cleaved efficiently by prothrombinase (2, 6). Such stringent selectivity is not evident in the action of factor Xa on oligopeptidyl substrates, nor is the rate of peptidyl substrate hydrolysis significantly enhanced upon assembly of factor Xa into prothrombinase (7,8). Thus, the narrow and defined specificity of prothrombinase toward its protein substrate is unlikely to be solely explained by the specific recognition of residues surrounding the scissile bond by the active site of factor Xa within the enzyme complex.Mechanistic studies of bovine prothrombinase function have borne out this suggestion (9 -11). A series of studies indicate that recognition of the biological substrate is achieved through stepwise interactions of the protein substrate at an extended macromolecular recognition site (exosite) in prothrombinase removed from the catalytic site of Xa, followed b...

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Section: Kinetics Of Peptidyl Substrate Cleavage In the Presence Of Nmentioning

confidence: 99%

Nematode Anticoagulant Protein c2 Reveals a Site on Factor Xa That Is Important for Macromolecular Substrate Binding to Human Prothrombinase

Buddai¹,

Toulokhonova²,

Bergum³

et al. 2002

Journal of Biological Chemistry

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“…In the work presented here, the Microcal Origin program (MicroCal Inc.) was used. A detailed discussion of this equations is presented by Indyk and Fisher (28).…”

Section: Purification Of Hemocyanin Male European Lobsters [Hmentioning

confidence: 99%

Binding of Urate and Caffeine to Hemocyanin of the Lobster Homarus vulgaris (E.) As Studied by Isothermal Titration Calorimetry

et al. 2000

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Hemocyanin serves as an oxygen carrier in the hemolymph of the European lobster Homarus vulgaris. The oxygen binding behavior of the pigment is modulated by metabolic effectors such as lactate and urate. Urate and caffeine binding to 12-meric hemocyanin (H. vulgaris) was studied using isothermal titration calorimetry (ITC). Binding isotherms were determined for fully oxygenated hemocyanin between pH 7.55 and 8.15. No pH dependence of the binding parameters could be found for either effector. Since the magnitude of the Bohr effect depends on the urate concentration, the absence of any pH dependence of urate and caffeine binding to oxygenated hemocyanin suggests two conformations of the pigment under deoxygenated conditions. Urate binds to two identical binding sites (n = 2) each with a microscopic binding constant K of 8500 M-1 and an enthalpy change ΔH° of −32.3 kcal mol-1. Caffeine binds cooperatively to hemocyanin with two microscopic binding constants: K 1 = 14 100 M-1 and K 2 = 40 400 M-1. The corresponding enthalpy changes in binding are as follows: ΔH°1 = −23.3 kcal mol-1 and ΔH°2 = −27.1 kcal mol-1. The comparison of urate and caffeine binding to the oxygenated pigment indicates the existence of two protein conformations for oxygen-saturated hemocyanin. Since effector binding is not influenced by protons, four different conformations are required to create a convincing explanation for caffeine and urate binding curves. This was predicted earlier on the basis of the analysis of oxygen binding to lobster hemocyanin, employing the nesting model.

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“…and was titrated with 30 injections of ligand (2 or 5 mL) at 3-min intervals in a VP-ITC calorimeter (MicroCal). Thermograms were analyzed using origin software supplied with the calorimeter [27,28]. Measurements were performed at 5, 15, and 25 8C.…”

Section: Isothermal Titration Calorimetrymentioning

confidence: 99%

Large-scale expression and thermodynamic characterization of a glutamate receptor agonist-binding domain

Madden¹,

Abele²,

Andersson³

et al. 2000

Eur J Biochem

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The ionotropic glutamate receptors (GluR) are the primary mediators of excitatory synaptic transmission in the brain. GluR agonist binding has been localized to an extracellular domain whose core is homologous to the bacterial periplasmic binding proteins (PBP). We have established routine, baculovirus-mediated expression of a complete ligand-binding domain construct at the 10-L scale, yielding 10±40 milligrams of purified protein. This construct contains peptides that lie outside the PBP-homologous core and that connect the domain core to the transmembrane domains of the channel and to the N-terminal`X'-domain. These linker peptides have been implicated in modulating channel physiology. Such extended constructs have proven difficult to express in bacteria, but the protein described here is stable and monomeric. Isothermal titration calorimetry reveals that glutamate binding to the domain involves a substantial heat capacity change and that at physiological temperatures, the reaction is both entropically and enthalpically favorable.Keywords: glutamate receptor ion channels; calorimetry; baculovirus/insect-cell expression; protein purification; ligand-binding thermodynamics.Glutamate receptor ion channels (GluR) are responsible for most excitatory synaptic communication in the central nervous system. They play an important role in the regulation of synaptic strength and are involved in a number of neuropathological processes, including post-traumatic neuronal damage, epilepsy and neurodegenerative diseases. GluR can be divided into three subclasses based on their pharmacological selectivity for the agonists a-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA), kainate and N-methyl-d-aspartate (NMDA) (reviewed in [1]). Like many ion channels, the GluR exhibit desensitization [2], the kinetics and extent of which depend on the agonist used.The GluR ligand-binding domain (`S1S2') is formed by two extracellular peptide sequences, known as S1 and S2, each approximately 150 amino acids long [3±5]. S1 is located N-terminal to the first transmembrane domain, and S2 is between the second and third transmembrane domains. The core of the ligand-binding domain, consisting of 80±85% of S1S2, exhibits homology to the prokaryotic periplasmic binding proteins (PBP). The PBP bind their ligands between two lobes, trapping them by a`Venus flytrap'-style cleft closure[6] (reviewed in [7]), although more restricted conformational changes are also observed, particularly among oligomeric members of the PBP family [8,9]. Sequences lacking PBP homology are located at both ends of the S1 and S2 peptides and connect the core of the domain to the`X'-domain located at the N-terminus and to the three transmembrane domains. These peptides have been implicated in modulating the electrophysiological characteristics of the channel and include the`flip/flop' alternative splice sequence important for desensitization kinetics [10±12].A fusion protein, in which the complete S1 and S2 sequences of the AMPA receptor GluRD are linked, reproduces the...

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[17] Theoretical aspects of isothermal titration calorimetry

Cited by 139 publications

References 2 publications

Nematode Anticoagulant Protein c2 Reveals a Site on Factor Xa That Is Important for Macromolecular Substrate Binding to Human Prothrombinase

Nematode Anticoagulant Protein c2 Reveals a Site on Factor Xa That Is Important for Macromolecular Substrate Binding to Human Prothrombinase

Binding of Urate and Caffeine to Hemocyanin of the Lobster Homarus vulgaris (E.) As Studied by Isothermal Titration Calorimetry

Large-scale expression and thermodynamic characterization of a glutamate receptor agonist-binding domain

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