Chapter 7 Protein Function

Ligand --- a molecule bound reversibly by a protein

Binding site --- the site on protein to which a ligand binds

Induced fit --- the structure adaptation that occurs between protein and ligand

Substrate --- the molecule acted upon by enzymes

Catalytic/active site --- the substrate/ligand binding site

Ch 7 --- Noncatalytic functions of proteins

Fig. 7-1,2Heme

Reversible binding of a protein to a ligand: Oxygen-binding proteins

protoporphyrin

Fig. 7-3 The structure of myoglobulin

*a single binding site for O2

*78% helices (8)

*His93 or HisF8 (the 8th residue in helix F) binds to heme

*Bends between helices

Protein-ligand interactions can be described quantitatively

P + L PL Ka = [PL]/[P][L] Kd = [P][L]/[PL]

= (binding sites occupied)/(total binding sites) = [PL]/[PL] + [P] = [L]/([L] +1/Ka)

Fig. 7-4 Graphical representation of ligand binding

= 0.5 [L] = 1/Ka, or Kd

Dissociation constant, Kd = [P][L]/[PL]

P + L PL Ka = [PL]/[P][L]

= (binding sites occupied)/(total binding sites) = [PL]/[PL] + [P] = [L]/([L] +1/Ka) = [L]/([L] +Kd)

When [L] = Kd = 0.5 (half saturation) [L] = 9 Kd = 0.9Kd: the molar concentration of ligand at which half of the available ligand-binding sites are occupied

Kd , affinity ( ? )

When O2 binds to Mb

P50 = 0.26 kPa

= [L]/([L] +Kd)

= [O2]/([O2] + Kd) = [O2]/([O2] + [O2]0.5)

The concentration of a volatile substance in solution is always proportional to its partial pressure in the gas phase above the solution

= pO2/(pO2 + P50)

Protein structure affects how ligands bind

1. Steric effects2. Molecular motions/breathing in the structure

1: 20,000 1: 200

Oxygen is transported in blood by hemoglobin (Hb)

In arterial blood, Hb ~96% saturatedIn venous blood, Hb ~64% saturated

P50 = 0.26 kPa

Mb

Mb has only one subunit,as an oxygen-storage protein

Fig. 7-6 Comparison between Mb and Hb

Fig. 7-7 Comparison of aa between whale Mb and Hb,

A-H helices

Only 27 aa identical

Fig. 7-8 Dominant interactions between Hb subunits

>30 aa

19 aa (hydrophobic, H-bonds,affected strongly upon O2 binding)

Hb undergoes a structural change on binding oxygen

Fig. 7-10 The T(tense) R(relaxed) transition

Fig. 7-9 Some ion pairs that stabilize the T state of deoxyHb

Fig. 7-11 Changes in conformation near heme on O2 binding

Hb binds oxygen cooperatively

Fig. 7-12 A sigmoid (cooperative) binding curve

4 vs. 13.3 kPa

Mb – a singlesubunit protein

Hb – 4 subunits,an allosteric protein

Allosteric protein – a protein in which the binding of a ligand to one site affects the binding properties of another site on the same protein

allos --- otherstereos --- solid or shape

Homotropic interaction --- liagnd = modulator

Heterotropic interaction --- ligand = modulator

O2 --- as both a normal ligand and an activating homotropic modulator for Hb

Cooperative ligand binding can be described quantitatively

Dissociation constant, Kd = [P][L] n/[PLn]

P + nL PLn Ka = [PLn]/[P][L]n

= (binding sites occupied)/(total binding sites) = [L]n/([L]n +Kd)

= [L]n/Kd

Log{ = n log [L] – log Kd (Hill equation, 1910)

Log{ = n log pO2 – log P50

nH – the Hill coefficient (slope of Hill plot)<, =, > 1

Fig. 7-13

Two models suggest mechanisms for cooperative binding

Concerted (all-or-none), 1965 Sequential, 1966

Fig. 7-14

O2 binding to Hb is regulated by 2,3-bisphosphoglycerate (BPG)

HbBPG + O2 HbO2 +BPG4 1

[BPG] during hypoxia

Fig. 7-16

Fig. 7-17Binding of BPG to deoxyHb

T state

T

R

O2

++

BPG is negatively charged

Sickle-cell anemia is a molecular disease of Hb

Val6 mutates to Glu6 in two chains

Complementary interactions between proteins and ligands: The immune system and immunoglobulins

MHC (major histocompatibility complex)

all vertebrate cells macrophages, B cells

Structure of a human class I MHC protein

Recognized by T-cell receptor

Over view of the immune response to a viral infection

The structure of immunoglobulin G (IgG)

Binding of IgG to an antigen

Induced fit in the binding of an antigen to IgG

Heavy chain

Light chain Kd~10-10M

The Ab-Ag interaction is the basis fro a variety of important analytical procedures

Ployclonal vs. monoclonal Ab

ELISA (enzyme-linked immunosorbent assay)

Immunoblot assay (Western Blot)

Protein interactions modulated by chemical energy Actin, myosin, and molecular motors

Fig. 7-29 Myosin

S1

The major components of muscle

Fig. 7-29

Structure of skeletal muscle

relaxed

contracted

Muscle contraction

Molecular mechanism of muscle contraction

3~4 pN of forces, 5~10 nm movement/cycle