Chapter 5.1: Protein Function - Reversible Binding of

Protein to a Ligand

CHEM 7784

Biochemistry

Professor Bensley

CHAPTER 5.1 Reversible Binding of Protein to a

Ligand

– Reversible binding of ligands– Structure of myoglobin and hemoglobin– Origin of cooperativity in hemoglobin

Today’s Objectives - To learn and understand:

Functions of Globular Proteins

• Storage of ions and molecules – myoglobin, ferritin

• Transport of ions and molecules – hemoglobin, serotonin transporter

• Defense against pathogens – antibodies, cytokines

• Muscle contraction – actin, myosin

• Biological catalysis – chymotrypsin, lysozyme

Binding: Quantitative Description• Consider a process in which a ligand (L) binds reversibly to a

site in the protein (P)

• The equilibrium composition is characterized by the equilibrium constant Ka

+

ka

kdPLP

L

d

aa k

kK

]L[]P[

]PL[

Binding: Analysis in Terms of the Bound Fraction

• In practice, we can often determine the fraction of occupied binding sites

• Substituting [PL] with Ka[L][P], we’ll

eliminate [PL]

• Eliminating [P] and rearranging gives the result in terms of equilibrium association constant:

• In terms of the more commonly used equilibrium dissociation constant:

]P[PL][

]PL[

]P[]P][L[

]P][L[

a

a

K

K

aK1

]L[

]L[

dK

]L[

]L[

Binding: Graphical Analysis

• The fraction of bound sites depends on the free ligand concentration and Kd

• In a typical experiment, ligand concentration is the known independent variable

• Kd can be determined graphically or via

least-squares regression

dK

]L[

]L[

[L] [L]total

Specificity: Lock-and-Key Model

•“Lock and Key” model by Emil Fischer (1894) assumes that complementary surfaces are preformed.

+

Specificity: Induced Fit

• Conformational changes may occur upon ligand binding (Daniel Koshland in 1958). – This adaptation is called the induced fit. – Induced fit allows for tighter binding of the ligand– Induced fit can increase the affinity of the protein for

a second ligand

• Both the ligand and the protein can change their conformations

+

Myoglobin/Hemoglobin

• First protein structures determined

• Oxygen carriers

• Hemoglobin: transports O2 from lungs to tissues

• Myoglobin: O2 storage protein

Mb and Hb Subunits Structurally Similar

• 8 alpha-helices• Contain heme group

myoglobin

hemoglobin

• Mb monomeric protein• Hb heterotetramer (α2β2)

Heme Group

Structure of Myoglobin

Hemoglobin

Oxygen Binding Curves

•Mb has hyberbolic O2 binding curve

•Mb binds O2 tightly. Releases at very low pO2

•Hb has sigmoidal O2 binding curve

•Hb high affinity for O2 at high pO2 (lungs)

•Hb low affinity for O2 at low pO2 (tissues)

Oxygen Binding Curve

Oxygen Binding Curve

O2 Binding to Hb shows Positive Cooperativity

• Hb binds four O2 molecules

• O2 affinity increases as each O2 molecule binds

• Increased affinity due to conformation change• Deoxygenated form = T (tense) form = low affinity• Oxygenated form = R (relaxed) form = high affinity

O2 Binding to Hb shows Positive Cooperativity

Conformational Change is Triggered by Oxygen Binding

Video on Hemoglobin

Allosteric Interactions

• Allosteric interaction occurs when specific molecules bind a protein and modulate activity

• Allosteric modulators or allosteric effectors• Bind reversibly to site separate from functional

binding or active site• Modulation of activity occurs through change in

protein conformation

• 2,3 bisphosphoglycerate (BPG), CO2 and protons are allosteric effectors of Hb binding of O2

Regulation of O2 Binding by

2,3-Bisphospho-

glycerate