Раскрывающееся кальмодулина-связывающие белки

Summary

Кальмодулин (CAM) выпадающее анализ является эффективным способом для исследования взаимодействия СаМ с различными белками. Этот метод использует CAM-сефарозе шарики для эффективного и конкретного анализа CAM-связывающих белков. Это является важным инструментом для изучения СаМ сигнализации в функционирование клетки.

Abstract

Calcium (Ca²⁺) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca²⁺ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)^1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca²⁺ (Ca²⁺-CaM) as opposed to its Ca²⁺-free state (ApoCaM)³.

While most target proteins bind Ca²⁺-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin⁴, neurogranin (Ng)⁵, and certain myosins⁶. These proteins have been shown to play important roles in presynaptic function⁷, postsynaptic function⁸, and muscle contraction⁹, respectively. Their ability to bind and release CaM in the absence or presence of Ca²⁺ is pivotal in their function. In contrast, many proteins only bind Ca²⁺-CaM and require this binding for their activation. Examples include myosin light chain kinase¹⁰, Ca²⁺/CaM-dependent kinases (CaMKs)¹¹ and phosphatases (e.g. calcineurin)¹², and spectrin kinase¹³, which have a variety of direct and downstream effects¹⁴.

The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca²⁺-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca²⁺-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function^8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.

Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca²⁺, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca²⁺ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca²⁺/CaM signaling and proteins that interact with CaM.

Protocol

См. на рисунке 1 представлена ​​основная схема процедуры, начиная с гомогената. Расчетное время от подготовки клеточные экстракты для вымывания CAM-связанных белков составляет около шести-семи часов. 1. Ткань подготовки Inject органотипической срезах гиппокампа с вир…

Discussion

При условии, протокол использует CAM-сефарозе бусин для расследования Ca ^{2 +-зависимость} CAM-связывающих белков. Многие белки связываются камеры в Ca ^{2 +-зависимым} образом. Эти взаимодействия имеют большое значение, учитывая количество CAM-связывающих белков и их важную роль во мно?…

Materials

Product	Company	Catalogue number	Notes
Calmodulin-Sepharose beads	GE Healthcare	17-0529-01
Anti-CamKII alpha	Sigma-Aldrich	C6974
Anti-neurogranin	Millipore	07-425
Gel Loading Pipet Tips	Fisher	02-707-138	Use for aspiration of supernatants
Microcentrifuge tubes (2.0 mL)	Fisher	05-408-146	Use for all steps involving calmodulin-sepharose beads