Name: how equilibrium binding constant affects binding
Uploaded: 2023-04-30T18:28:41.000Z
Duration: 2 min 18 s
Channel: JoVE

בתא, חלבונים מתנגשים באופן אקראי עם מולקולות אחרות, כגון חלבונים אחרים, חומצות גרעין וליגנדות של מולקולות קטנות.
אם הקישור אינו ספציפי, מעט אינטראקציות לא קוולנטיות, בין המולקולות, גורמות לקשר קצר.
אם ליגנד מסוים נקשר לחלבון, הוא יוצר אינטראקציות לא קוולנטיות, נרחבות, לאורך משטחים משלימים. קומפלקסים כאלה יציבים, נשארים קשורים במשך זמן רב, לפני ניתוק.
עוצמתה של אינטראקציית קישור מדווחת במונחים של קבוע שיווי המשקל שלה, Kb, המכונה גם קבוע הקשירה או האסוציאציה. 
Kb ניתן לחישוב על פי היחס בין ריכוז קומפלקס החלבון-ליגנד על ריכוזי החלבון והליגנד הבלתי קשורים הנמצאים בשיווי משקל. 
בהתחשב בקשר שלו עם שינוי האנרגיה החופשית עקב הקשירה, Kb גדול פירושו ירידה גדולה בדלתא G, מה שמצביע על זיקה חזקה בין חלבון לליגנד.
שני תהליכים מתחרים חשובים לקשירת ליגנד חלבון: הקשר של חלבון וליגנד ליצירת קומפלקס ודיסוציאציה של הקומפלקס למגיבים.
קבוע האסוציאציה, kon, הוא מדד למספר אירועי הקישור בשנייה בין חלבון לליגנד שלו; ניתן להשתמש בו כדי לחשב את קצב קשירת הליגנד לחלבון בריכוז נתון. 
לעומת זאת, koff הוא מדד למספר אירועי הדיסוציאציה; ניתן להשתמש בו כדי לחשב כמה מהר הקומפלקס מתפרק.
כאשר קצב ההתאגדות שווה לקצב הדיסוציאציה, מגיעים לשיווי משקל, שבו הריכוזים נטו של תוצרים ומגיבים נשארים קבועים.
כך, בשיווי משקל, kעל פי מכפלה של ריכוזי שיווי המשקל של החלבון והליגנד שווה kפעמים ריכוז שיווי המשקל של קומפלקס החלבון-ליגנד.
סידור מחדש של ביטוי זה מראה גם שהיחס בין kon ל-koff שווה ל-Kb בשיווי משקל.

<ol>
	<li>פולארד, תומס ד. "מדריך למבחני כריכה פשוטים ואינפורמטיביים". ביולוגיה מולקולרית של התא 21, מס '23 (2010): 4061-4067.</li>
	<li>הולם, אדוארד סי ומייק א. טרבת'יק. "מבחני כריכת ליגנד בשיווי משקל: אימות ופרשנות". כתב העת הבריטי לפרמקולוגיה 161, מס '6 (2010): 1219-1237.</li>
	<li>מדינה-פרנקו, חוסה ל., אוסקר מנדז-לוסיו וקרינה מרטינז-מאיורגה. "יחסי הגומלין בין מודלים מולקולריים וכימואינפורמטיקה לאפיון אינטראקציות חלבון-ליגנד וחלבון-חלבון לגילוי תרופות." בתוך חידושים בכימיה של חלבונים ובביולוגיה מבנית, כרך 96, עמ' 1-37. עיתונות אקדמית, 2014.</li>
</ol>

קבוע שיווי המשקל וחוזק הקשירה

The Equilibrium Binding Constant and Binding Strength

<div style="direction: rtl;">

קבוע הקשירה של שיווי המשקל (K_b) מכמת את חוזקה של אינטראקציה בין חלבון לליגנד. ניתן לחשב K_b באופן הבא כאשר התגובה נמצאת בשיווי משקל:

<img alt="Eq1" src="/files/ftp_upload/11513/Equation 1.png" style="text-align: center; max-width: 100%;">

כאשר P ו-L הם החלבון והליגנד הבלתי קשורים, בהתאמה, ו-PL הוא קומפלקס החלבון-ליגנד.

מכיוון שכמות הליגנד הקשור קשורה גם לקצב הקישור של הליגנד, ניסויים יכולים גם לקבוע את K_b על ידי בחינת שיעורי האסוציאציה של חלבון-ליגנד (K_on) והדיסוציאציה (K_off) באמצעות היחס הבא:

<img alt="Eq1" src="/files/ftp_upload/11513/Equation 2.png" style="text-align: center; max-width: 100%;">

לפיכך, נעשה שימוש בשתי קטגוריות של מבחני חיבור לקביעת קבוע הקישור של שיווי המשקל - אלו המודדים ריכוזי שיווי משקל ואלו המודדים את הקינטיקה של תגובה. במקרה, התגובה חייבת להיות בשיווי משקל בזמן המדידה.

שיטת קביעת ריכוזי שיווי המשקל תלויה ברגישות הרצויה ובקלות זיהוי האות. מסיבות אלה, מבחנים ספקטרוסקופיים נמצאים בשימוש הנפוץ ביותר. בניסויים אלה, התגובה מייצרת שינוי ספיגה של מגיב או מוצר באורך גל נתון, הניתן לזיהוי באמצעות ספקטרופוטומטר UV-Vis. לחלופין, המגיב או המוצר יכולים להיות מתויגים עם בדיקה ניאון או עשויים להכיל פלואורופור מהותי. לאחר מכן, ניתן למדוד את התקדמות התגובה מהשינוי בקרינה. מבחנים אלה מבוצעים על ידי שינוי ריכוזי מגיב אחד בזמן ששאר הניסוי נשמר קבוע. לאחר מכן ניתן לצייר את התוצאות ולנתח אותן בשיטות שונות של התאמת עקומה.

אינטראקציות בין חלבונים וליגנים נחקרות גם באמצעות מגוון טכניקות ביוכימיות וספקטרוסקופיות. ניתוח מבני, באמצעות קריסטלוגרפיה של קרני רנטגן וספקטרוסקופיה NMR, מסייע בחיזוי אינטראקציות חלבון-ליגנד באמצעות הדמיות מולקולריות. גישות תיאורטיות וחישוביות, כגון מחקרי עגינה של חלבון-ליגנד, נמצאות בשימוש נרחב כדי לאפיין את המיקום והאינטראקציות של ליגנים מולקולות קטנות, כולל מועמדים לתרופות. עיצוב תרופות בעזרת מחשב הוא אלטרנטיבה מהירה ובעלות נמוכה להאצת קצב בדיקות התרופות הקונבנציונליות של ניסוי וטעייה.

</div>

קבוע קשירת שיווי המשקל (Kb) מכמת את עוצמת האינטראקציה חלבון-ליגנד. Kb ניתן לחשב כדלקמן כאשר התגובה היא ב ...

the-equilibrium-binding-constant-and-binding-strength

Education

JoVE Core

Cell Biology

ביולוגיה של התא

Unit 1

יחידה 1

Energy and Catalysis

אנרגיה וקטליזה

KEY TERMS AND CONCEPTS

מונחים ומושגים מרכזיים

how equilibrium binding constant affects binding

The equilibrium binding constant describes how strongly two molecules bind at equilibrium. It is a useful way to compare binding strength in molecular interactions.

This topic focuses on how binding is measured and what the equilibrium binding constant means. A higher value indicates stronger binding, while a lower value indicates weaker binding. Understanding this constant helps explain how tightly molecules stay together under equilibrium conditions.

The page also connects the binding constant to real binding strength. It shows why this value matters when comparing different molecular pairs. For high school students, it provides a clear link between a chemistry concept and molecular behavior.

How Equilibrium Binding Constant Affects Binding

how-equilibrium-binding-constant-affects-binding

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the equilibrium binding constant and binding strength

The equilibrium binding constant describes how strongly two molecules bind at equilibrium. It is a useful way to compare binding strength in molecular interactions. This topic focuses on how binding is measured and what the equilibrium binding constant means. A higher value indicates stronger binding, while a lower value indicates weaker binding. Understanding this constant helps explain how tightly molecules stay together under equilibrium conditions. The page also connects the binding...

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key terms and concepts

Learn how the equilibrium binding constant reflects binding strength and why higher or lower values matter in molecular interactions at equilibrium.

How Equilibrium Binding Constant Affects Binding | JoVE K12

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How Equilibrium Binding Constant Affects Binding

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