आदेश में बाध्यकारी तंत्र निर्धारित परिवर्तनीय एकाग्रता इज़ोटेर्माल अनुमापन calorimetry डेटासेट संग्रह
Summary
आईटीसी अपने मेजबान के लिए एक ligand के बंधन के अध्ययन के लिए एक शक्तिशाली उपकरण है. जटिल प्रणालियों में, तथापि, कई मॉडल डेटा को समान रूप से अच्छी तरह से फिट हो सकती है. विधि यहाँ वर्णित करने के लिए एक जटिल प्रणालियों के लिए उपयुक्त बाध्यकारी मॉडल को स्पष्ट और इसी thermodynamic मापदंडों निकालने का मतलब प्रदान करता है.
Abstract
Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host macromolecule. ITC has some advantages over common spectroscopic approaches for studying host/ligand interactions. For example, the heat released or absorbed when the two components interact is directly measured and does not require any exogenous reporters. Thus the binding enthalpy and the association constant (Ka) are directly obtained from ITC data, and can be used to compute the entropic contribution. Moreover, the shape of the isotherm is dependent on the c-value and the mechanistic model involved. The c-value is defined as c = n[P]tKa, where [P]t is the protein concentration, and n is the number of ligand binding sites within the host. In many cases, multiple binding sites for a given ligand are non-equivalent and ITC allows the characterization of the thermodynamic binding parameters for each individual binding site. This however requires that the correct binding model be used. This choice can be problematic if different models can fit the same experimental data. We have previously shown that this problem can be circumvented by performing experiments at several c-values. The multiple isotherms obtained at different c-values are fit simultaneously to separate models. The correct model is next identified based on the goodness of fit across the entire variable-c dataset. This process is applied here to the aminoglycoside resistance-causing enzyme aminoglycoside N-6′-acetyltransferase-Ii (AAC(6′)-Ii). Although our methodology is applicable to any system, the necessity of this strategy is better demonstrated with a macromolecule-ligand system showing allostery or cooperativity, and when different binding models provide essentially identical fits to the same data. To our knowledge, there are no such systems commercially available. AAC(6′)-Ii, is a homo-dimer containing two active sites, showing cooperativity between the two subunits. However ITC data obtained at a single c-value can be fit equally well to at least two different models a two-sets-of-sites independent model and a two-site sequential (cooperative) model. Through varying the c-value as explained above, it was established that the correct binding model for AAC(6′)-Ii is a two-site sequential binding model. Herein, we describe the steps that must be taken when performing ITC experiments in order to obtain datasets suitable for variable-c analyses.
Protocol
1. शेयर समाधान की तैयारी शुद्ध ब्याज की macromolecule. (इस मामले में, एन 6'-acetyltransferase-II (AAC6'-II) aminoglycoside, कहीं और रिपोर्ट के रूप में अलग है 13 .) डायलिसिस बफर के 4 लीटर तैयार. (इस मामले में हम 25 4 मिमी इस्तेमाल किया – (2 hydroxyet…
Discussion
चर ग फिटिंग के इस विश्लेषणात्मक भाग पहले 10 में विस्तार से वर्णित किया गया है. यहाँ हम इस दृष्टिकोण के लिए चर ग उपयुक्त डेटासेट इकट्ठा करने के व्यावहारिक पहलुओं की रिपोर्ट. यह आवश्यक है कि सभी प्रोटी?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
यह काम स्वास्थ्य अनुसंधान संस्थान कनाडा (CIHR), राष्ट्रीय विज्ञान और इंजीनियरिंग अनुसंधान परिषद (NSERC), और एक CIHR प्रशिक्षण अनुदान छात्रवृत्ति (वामो के लिए) द्वारा समर्थित किया गया. हम प्रो AAC के लिए जेरार्ड डी. राइट (McMaster विश्वविद्यालय, कनाडा) (6)-II अभिव्यक्ति प्लाज्मिड धन्यवाद.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Acetyl coenzyme A (AcCoA) | Sigma-Aldrich | A2056 | ||
| 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) | Fisher | 7365-45-9 | ||
| ethylenediaminetetraacetic acid (EDTA) | Sigma-Aldrich | 431788 | ||
| Spectra/Por 2 Dialysis Tubing | Spectrum Labs | 132678 | ||
| Sterile Syringe Filter (0.2 μm) | VWR | 281445-477 | ||
| Cellulos Nitrate Membrane Filters (0.45 μm) | Whatman | 7184-004 | ||
| VP-ITC | MicroCal | VP-ITC | Microcalorimeter used for measurements | |
| ThermoVac | MicroCal | USB Thermo Vac | Temperature Controlled Degassing Station |
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Cite This Article
Freiburger, L. A., Mittermaier, A. K., Auclair, K. Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms. J. Vis. Exp. (50), e2529, doi:10.3791/2529 (2011).