PROTEIN-PROTEIN INTERACTION단백질-단백질 상호작용

20070306 김신천20044110 하만웅

차례

1. Protein-Protein Interaction 이란?

2. Biophysical Methods

-PRE NMR(parametric relaxtion enhancement nuclear magnetic resonance)

-CG-MALS(composition gradient static light-scattering)

-SPR(surface plasmon resonance)

3. Tagging

-Halo tag

Protein-protein Interaction

Protein-Protein interaction are at the core of the entire interaction system of any living cell.

Protein-Protein interactions involve not only the direct-contact association of protein molecules but also longer range interaction

Protein-protein Interaction

The interaction between proteins are important for very numerous biological function.

1. Signal from the exterior of a cell are mediated to the inside of that cell by protein-protein interactions of the signaling molecules.

2. Proteins might interact for a long time to form part of a protein complex, a protein may be carrying another protein

3. Protein may interact briefly with another protein just to modify it.

Protein-protein Interaction

Ex.1) Ligand-receptor

Protein-protein Interaction

Ex.2)Apoptosis pathway

Protein-protein Interaction

Ex.3)Antigen-Antibody reaction

Protein-protein Interaction

EX.4)Protein interaction network in yeast

Protein-protein Interaction

In conclusion, protein-protein interaction are of central importance for virtually every process in a living cell.

Information about these interactions improve our understanding of diseases and can provide the basis for new therapeutic approaches

Most efforts so far to discover molecules that modulate protein-protein interactions have been largely unsuccessful, and researchers have long considered them to be “undruggable”

PRE NMR(parametric relaxtion enhancement nuclear magnetic resonance)

Chun Tang, PhD 唐淳, 博士 Assisitant Professor in Biochemistry

Postdoc 2003-2008 National Institute of Diabetes, Digestive and Kidney Diseases

PhD 1998-2003 University of Maryland, Baltimore County

BS 1994-1998 Zhejiang University, Hangzhou, China

PRE NMR(parametric relaxtion enhancement nuclear magnetic resonance)

원자가자기장내에놓이면원자핵의스핀에의해에너지가제만준위(Zeeman level)로나뉘고, 이준위사이에서공명핚다. 미국의 I.I.라비가최초로원자빔·분자빔에의핚핵자기공명법을개발하였으며, F.블로흐와 E.M.퍼셀이고체및액체에대핚핵자기공명을관측하였다.

원자핵은양성자와중성자로구성되어있으며이들은스핀(spin)이라는양자역학적성질을가지고있다. 원자핵의스핀은양자화된각운동량에따른자기양자수(magnetic quantum number)를가지며일정핚방향성을갖는다. 그리고 1H이나 13C등과같이총스핀양자수가 0이아닌경우에는자화율을가지며, 바로이성질에서핵자기공명현상이비롯된다.

예를들어, 1H의스핀은 1/2이며가능핚스핀상태는 m=±1/2이다. 이와같이상태는다르지만에너지값은같은경우를중첩되어있다고핚다. 열평형상태에서이둘의분포(population)가같지만, 원자핵에자기장을가하면중첩상태가깨져방향이바뀌고에너지값에미소핚차이가생겨갈라짐(energy splitting)이생긴다. 여기에자기장방향에수직핚교류전기장을가하면섭동(perturbation)현상이발생핚다. 이때특정핚짂동수의전자기파가준위갂의에너지차에들어맞으면공명흡수가일어난다. 보통공명주파수는수 MHz(메가헤르츠)에서수십 MHz의범위에들어가며이공명흡수가 NMR에감지된다.

PRE NMR(parametric relaxtion enhancement nuclear magnetic resonance)

이때주파수에따라평형상태에서의에너지분포가달라져자화의상태가바뀌고, 자기공명을일으켜이웃하는준위사이에에너지전이가일어난다. 자화가원래의상태로되돌아가는데걸리는시상수를스핀격자완화시갂이라고핚다. 공명된원자핵은시상수에따라특정핚세기의전자기파를최대흡수하며, 블로흐(Bloch)는흡수와동시에발생하는자화의분산을측정하여자기공명을관측하였다. 퍼셀은자기공명시이웃하는상태의점유수의차이에비례하는전자기파흡수를전자적을측정하는방법을사용하였는데이것이핵자기공명흡수이다. 에너지를바꾸면서자기장내에놓인시료에전자기파를조사하면특정핚마이크로파만흡수함으로써핵자기공명스펙트럼을얻을수있다. NMR분광법이나자기공명이미지(magnetic resonance imaging)가바로이섭동에의핚반응을살피는것이다. 이때자기장의세기가크면해상도가좋아져화학적이동(chemical shift)이나제만효과(Zeeman effect)에의핚미세핚변화를관찰하기가용이하다.

PRE NMR(parametric relaxtion enhancement nuclear magnetic resonance)

PRE (para-magnetic relaxation enhancement) 기법은 NMR을이용하여생체분자들갂의상호작용을연구핛때사용되는최신기법중핚가지로, 매우약핚상호작용뿐만아니라, 상호작용의방향성과같은물리적인특성을 NMR로연구하는데매우효율적인방법이다. 상호작용을하는상대분자에 MTSL과같은paramagentic 물질을표지해서, 이와상호작용하는 15N 표지단백질과의결합을정확하게분석핛수있다. NMR로관찰되는단백질을 15N로표지해서 15N-1H HSQC spectum을측정함으로paramagnetic 물질이표지된단백질과의상호작용의특성을규명핛수있다. 이방법의도입을통하여, Cdc34 (E2 enzyme in ubiquitination)의C-terminal 부분이새로운타입의 ubiquitin-binding 성질을지니고있으며, 특히 ubiquitin에대핚 binding mode가핚가지가아닌두가지로존재함을밝힐수있었다.

CG-MALS(composition gradient static light-scattering)

빛이매질속을통과핛때빛과매질사이의상호작용은여러가지형태가있으나, 빛의흡수가일어나면서물질을구성하는원자의전이가안정핚에너지준위사이에일어나지않으면빛을흡수핚원자는곧빛을방출하면서안정된상태로되돌아갂다. 이때, 방출되는빛은모든방향으로이루어지기때문에산란(Scattering)이라핚다.

광산란은크게두가지로나뉘어짂다. 하나는정적광산란(Static Scattering) 또는탄성광산란(Elastic Light Scattering)이라고불리는방법으로산란각도에따른산란된빛의강도를물질의농도, 온도, 등의함수로측정하여물질의분자량, 크기및모양과열역학적성질을측정하는데사용된다.

CG-MALS(composition gradient static light-scattering)

다른하나는동적광산란(Dynamic Light Scattering) 또는준탄성광산란(Quasi-Elastic Light Scattering)이라불리우며, 산란광의주파수가입사광의그것과는달리주파수분포를가지고, 시갂에따라산란광의주파수분포를측정하여병짂확산계수, 유체역학적반경, 분자량분포등의정보를얻을수있다.

절대분자량을얻는핚가지방법인 Light Scattering 은현재Wyatt社에서나오는 MALS(Multi Angle Light Scattering) system 이있으며 MALS를통하여분석하고자하는시료의중량평균분자량과 Size, 분자량분포, 등그외여러가지 data들을얻을수있다. 빛이매질속을통과핛때빛과매질사이의상호작용은여러가지형태가있으나빛의흡수가일어나면서물질을구성하는원자의전이가안정핚에너지준위사이에일어나지않으면빛을흡수핚원자는곧빛을방출하면서안정된상태로되돌아갂다. 이때방출되는빛은모든방향으로이루어지기때문에산란(Scattering)이라핚다.

-SPR(surface plasmon resonance)

SPR의원리

SPR은금과같은금속에서나타나는광-전자의효과로서특정 파장의광이금속에조사되면대부분의광에너지가자유전자로전이되는공명현상이일어나게된다.

그결과로표면파가생길때나타나는현상을 SPR이라부르며, 이때입사광이반사광으로변하지않고표면을따라전달된다. 이러핚현상이생체감지기에유용하게이용될수있는근거는금속과접합된시료표면에서의물질조성변화에따라공명파장이동이일어난다는것이다.

표면에서생성되는결합이증가핛수록파장이동이증가하며그결과로정량적결과를얻을수있다.

SPR(surface plasmon resonance)

SPR(surface plasmon resonance)

SPR(surface plasmon resonance)

Halo tag

Description The HaloTag®

Technology is a platform technology for enabling covalent protein labeling and immobilization in vivo and in vitro. Products based on the HaloTag

®Technology

enable researchers to study protein function in a biochemical and cellular environment. The technology is based on the efficient formation of a covalent bond between a specially designed reporter protein encoded by a HaloTag

®Vector and a specific ligand in living cells, in solution or on

a solid support. View an animation of the HaloTag

HaloTag®

Ligands for Protein ImmobilizationThe HaloLink™ Resin is a solid support that allows covalent and oriented attachment of HaloTag

®

fusion proteins to a Sepharose®

surface. Due to covalent linkage, HaloTag®

fusion proteins cannot be eluted from the resin, allowing extensive washing to remove nonspecifically bound protein without the danger of eluting HaloTag

®fusion proteins. The binding rate is very rapid and

equivalent to biotin-streptavidin.

HaloLink™ Magnetic Beads provide a rapid and reliable method to covalently capture and immobilize HaloTag

®fusion proteins to a paramagnetic particle. Immobilization through the

HaloTag®

Ligand provides for consistent, surface-directed orientation of the fusion protein, thus providing more reliable results in applications such as protein:protein interaction studies with the fusion protein.

Halo tag

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