Lehninger Principles of BiochemistryFourth Edition

David L. Nelson and Michael M. Cox

Fourth Edition

Chapter 5:

Protein Function

Copyright © 2004 by W. H. Freeman & Company

A remind� Ligand: binding molecule to a protein� Binding site: site of a protein that a ligand binds� Mostly they are specific� Proteins are flexible: changing in conformatin� iduced fit: ligand result in a conformational change on � iduced fit: ligand result in a conformational change on

protein’s binding site so it fits the binding site more tightly.

� Interactions may be regulated.� Enzyme-catalytic or active site (binding site)-substrate

(ligand)

Reversible binding of L to Protein� Essential for cellular respiration

� O2 poorly soluble

� Impossible to transport by simple diffusion

� Reversible binding not to any aa

Only metals: iron, copper� Only metals: iron, copper

� Free iron�dangerous� reactive oxygen(-OH)�damage of DNA other macromol

O2 by Hemoglobin in blood

� Hemoglobin in red blood cells (RBC)� 6-9 µm in diameter� Biconcave disks� Hemocytoblasts (procursor stem cell)�RBC� No nucleus, mitochondria, ER� 120 days� Carry hemoglobin in dissolved in cytosol� Carry hemoglobin in dissolved in cytosol� Hb in lungs�96% saturated with O2

� Hb in tissues�64% saturated with O2

� 6.5 mL of O2 gas at atmosphoric pressure

� Interaction subunit�conformational change�alter affinity for O2

� Myoglobin�relatively insensitive to small concentration of dissolved O2

Hb Subunit Similar to Myoglobin� Hb�Mr:64500� Rouphly spherical� 5.5 nm

� Tetrameric�4 heme prosthetic group� 2 types of globin� 2 types of globin

� 2 β (141 residue each); 2α (146 residue each)� Fewer than half of β and α subunit� identical� 3D structure �very similar� Mb�similar to both� Helix-naming : Hbα lacks D helix

� Strong interaction between unlike subunits

�α1�β1; α2�β2 (>30 residues)

�Though Urea treatment�dimer αβ

�α1�β2; α2�β1 (>19 residues)

� Interaction

�Mostly� hydrophobic

� Some: Hydrogen

�A few: ionic

Hb conformation when O2 binding� Two conformation of Hb

� R State� T State

� R state� > affinity to O2

� R � > stable while oxygenated

� T � > stable while deoxygenated :predominant conformation of � T � > stable while deoxygenated :predominant conformation of deoxyhemoglobin

� T: tense; R: relaxed

� T stabilized by a greater number of ion at α1�β2; α2�β1 interfaces

Đonic interactions

Đonic interactions between alfa beta

� O2 binds THb� Hb changes its conformation� R� αβ slides each other�narrow the pocket

� Some ionic bonds broken

� Some new ionic ones formed

Max PerutzSome aa of heme during T triggered and become RPorphyrin�slightly puckeredProtured on F8 HisDuring F; planar (after O2)

Hb cooperatively O2 binding� O2 binds to Hb when pO2 ~ 13.3 kPa

� O2 release when pO2 ~ 4 kPa (Not anyother can do that)

� How does Hb do that?

� A transition stage�S :sigmoid binding curve� Only multisubunits wth multibinding site have SBC

� O2 bind to one SU� affinity of other SU change; increase

A sigmoid (cooperative) binding curve:

HB as an Allosteric protein

Allosteric protein: binding of an ligand (called modulator)�other’s binding in the same proteinModulator � > or << active ; so it meains activatoror inhibatorinhibatorIf modulator = ligand � interaction is homotropicIf modulator ≠ ligand � interaction is heterotropic

Some proteins have 2 or more; two types exist

Hb� O2 is both ligand and activating homotropic modulatorsubunit-subunit interaction�transmitted

If A sigmoid binding curve exist�cooperative binding-YES

At first; the sites less affinity

ligand

binds

Become

more stable

Qusntitatively Cooperative Ligand

Binding

� 1910, Archibald Hill�first cooperative binding of oxygen

P + nL ↔ PLn

+ ↔

Ka=[PLn]

[P][L] n

Ka=[PLn]

[P][L] n

Expression of Ө

Ө =[L] n

[L] n+Kd

[L] nrearrange

Ө [L] n

Kd=

1-Ө

Take log of both sides

Ө n log [L] – logKd=1-Ө

log

Where Kd=[L] n0.5

Hill equation

� Hill plot: a plot of log[Ө/(1- Ө)] versus log[L]� Slop of the plot � slop of the n� However, experimental results shows

� # of binding sites� Đnteraction of binding sites

� Hill plot denoted as nH, Hill coefficient: degree of cooperativity

� If nH=1 � not cooperative

� If nH>1 � positive cooperative� If nH>1 � positive cooperative

� Hemoglobin �+ cooperative

� One’s binding affects others’ binding

� Therotical limit � nH=n means everywhere is saturated

� nH<1 � negative ccoperative .... Veryt rare

Adaptation of Hill equation

Ө n log pO2– nlogPn=1-Ө

log 0.5

Two model mechanism

T�R transition occur. HOW?

Two models

1. MWC model (concerted model)

2. Sequential model

All or none

Hemoglobin transport H+ & CO2

By carbonic anhydrase

Red blood cell

CO2 + H2O↔H+ +HCO3

By carbonic anhydrase

Lower pH

Đnsoluble, bobble formation

↔

�

Only 40% of H+ and 15-20% of CO2 by Hb

Rest of H+ :in HCO3 ; Rest of CO2:by HCO3 and CO2

Binding of H+ and CO2 to Hb ���� inversely related of that of O2

H+ and CO2

Bohr effect:Bohr effect: effect of pH and CO2

concentration on binding and releasing of Oxygen

H+ and CO2 release out↓ Affinity of O 2 for Hb ↑ O2 binds

H+ and CO2 ↑Affinity of O 2 for Hb ↓O2 release

Hb + O2↔ HbO2

In fact

HHb+ + O2↔ HbO2 + H+

O2 and H+ do not bind to the same site of Hb

BUT

H+ binding makes Hb T

H+ binds any but if it binds His HC3 in β subunit� ion pairs H+ binds any but if it binds His HC3 in β subunit� ion pairs AspFG1�T state � stabilizes protonated His HC3�abnormal high pKa in T

Unprotonated �R state

Tissues � 7.2

Lungs � 7.6

7.4�experimental hemoglobin binding

CO2 Binding� CO2 bind amino-terminal of globular PPC forming

carbaminohemoglobin (carbamino-terminal residue)

� H+ produced�Bohr effect

� T state stabilization�release of O2

2,3-bisphosphoglycerate (BPG)

� HbBPG + O2 F HbO2 + BPG

� BPG �bound Hb� BPG reduce the affinity of Hb to O2

� What is the benefit?� Seen while insuffient O2

� Example: If you go to Uludağ (over 3000 meters high), pO2 is 7 kPa, (at sea level..13 kPa). � Hb fill only 85% of its sites, (~ 95% at sea). BUT The pO2 in the tissues is the kPa). � Hb fill only 85% of its sites, (~ 95% at sea). BUT The pO2 in the tissues is the same � Hb will deliver only 30% of O2 (normally 40%)

� Then, your body make more BPG. �This bind in the tissues to Hb in the T state (it only binds to the T state) � the release of O2. �T state is stabilized� lowering the affinity of O2 when pO2 reaches the levels found in the tissues.

� This affects more the low affinity state than the high affinity state, and restores the delivery of O2 to normal levels of (delivery of 40% of the bound oxygen). you feel OK.

� The interaction between Hb and BPG is due to the formation of several salt bridges between the negatively charged groups in BPG and several positively charged amino acids that are present in the binding pocket between the a2b2 subunits.

Oxygen Regulation by 2,3-bisphosphoglycerateOxygen Regulation

•normally found on hemoglobin

•what it does: to reduce its affinity to O2

• where it binds: a site far from O2

binding site.

lung: almost 100% saturated

tissue: ~60% saturated

when person at sea level

lung: almost 90% saturated result: ����

when person at 4500 mt

Normally , lung: almost 85%

•function: to adapt us to sharp pO2

changes[BPG] in blood

to cells: ~40% released

[BPG] in blood

to cells: ~30% released

BUT SUPPOSE NO CHANGE IN [BPG]

Normally , BODY

INCREASE[BPG]

lung: almost 85% saturated

tissue: ~45% saturated

to cells: ~40% released AGAIN

[BPG] ↑ : Hb ↓ AFFINITY TO OXYGEN

Hypoxia: a disorder because of inadequate lung, circulation

Đn fetus: in steade of β, γ it has, α2γ2 ..< BPG affinity but >that of O2 � receive O2 for its mother Hb

Sickle Cell Anemia Sickle Cell Anemia

a genetic disease producing sickel cell Hb

Normal Hb is HbA ; Diseased Hb is HbS

HbS� insoluble while deoxyginated� aggregate into tubular fibers

HbA �soluble while deoxyginated

HbS result from an aa substiution; Valine in stead of Glutamate at 6th positon of beta

Glu� charged so

HbS charge=HbA’s charge -2

So HbS abnormally interaction each them

SCA � painful diseaseSCA � painful disease

SCA patients’ hemoglobin �50% of 10-15gr/100mL of normal value

WHY? HbS is so fragile so repture easly�anemia (lack of blood)

Child with SCA die early

Malaria Before Columbus

IndiaItaly

GreeceAlbania

Turkey

Origins of the Sickle Cell Gene

Senegal

Benin

CAR

IndiaTurkey

Arabian PeninsulaArabian Peninsula

Egypt

Immunoglobulins & Immune System� A complex system that is responsible for distinguishing

us from everything foreign to us, and for protecting us against infections and foreign substances. The immune system works to seek and kill invaders.

� Immune system distinguish nonself ones and protect the bodybody

� Immune Response: A response against foreigners� Cells of immune system�leukocytes (WBC)

� Macrophages and lymphocytes

Immune ResponseImmune ResponseImmune ResponseImmune Response

� Humoral Immune System� In fluid, Special weapons used

� Antibodies, Immunoglobulins, Ig

� Cellular Immune System� Cells, their self fight

� Most important one�T Immunoglobulins, Ig� B-lymphocytes (B-cells)

� Ig binds foreign ones (virus, bacteria, protein ..)

� Most important one�T lymphocytes� 2 improtant types

� Cytotoxic T cells (TC)

� T Helper (TH)

IS distinguish self from nonself� Antibodies and receptors on lymphocytes distinguish self

from nonself� 100.000.000 (100 million) types Ig� Each with distinct binding specifity� Ig’s produced randomly� Ig’s produced randomly� Antigen: any molecule capable of eliciting an immune

responce � Virus, bacterial cell wall etc

� Epitope: the place of antigen that antibody binds

� No response to small molecules, products of celullar metabolism

� Mr<5,000 �not antigenic� Combine them � antigenic

1.Immunogen Hapten* and carrier** are bound together to form an immunogen.

Hapten: a small molecule not antigenic but when attached to a large molecule Carrier: an immunogenic substance that, when coupled to a hapten, renders the haptenimmunogenic.

Developed antibody to the hapten can bind its Developed antibody to the hapten can bind its free form

PUTTING IT ALL TOGETHER…

Activated T helper cell

APC T helper cell

Activated T helper cell

Cytotoxic T cell (Tc)

Memory T c Effector T c Lysis

Antibodies

MHC (Major histocompatibility

complex) Proteins

� 2 types� Class I MHC

� Class II MHC

� On the surface of cells and present the peptides of the digested proteins (inside or outside)digested proteins (inside or outside)

� Many aim: to find out nonself

Class I MHC� Surface protein� On vertebrate cells� 6 variants in one individual� But countless variant in each species� Function: display peptides derived from proteolytic � Function: display peptides derived from proteolytic

degradation� Recognation targets of T-cell receptors of Tc cells (cytotoxic T cell- killer

T cell) cellular immune system� To represent viral proteins� For organ transplantation, choose organ with the same 6 variants

http://www-ermm.cbcu.cam.ac.uk/smc/swf001smc.htm

Class II MHC

� With specialized cells:macrophages & B lymphocytes.

� MHC II �polymorphic like MHC I (12 variants)� Represent bacterail and parasitic organisms

Class II MHC protein-peptide complex binds targets of � Class II MHC protein-peptide complex binds targets of T-cell receptors of various helper T cell (TH).

� AIDS (no T-helper)� T-cells are produced carefully

� Most (95%) are eliminated because of recognation of self proteins in stead of nonself.

http://www-ermm.cbcu.cam.ac.uk/smc/swf002smc.htm

Antibodies with two identical antigen-

binding sites

� Different types: IgE, IgA, IgG, IgD, IgM

�� Major class oneMajor class one��������IgGIgG� Y-shaped

� Mr�150000

� 2 heavy and 2 light chains� 2 heavy and 2 light chains

light chainFab: antigen-

Heavy chain

light chain

hinges

Fab: antigen-binding fragment

Gerald edelman & Rondey porter

Fc: crystalizable F

Immunoglobulin folds: all beta

http://www.callutheran.edu/Academic_Programs/Departments/BioDev/omm/jmol/ig_div/start.html

folds: all beta (page: 142), 3 in each heavy; 1 in light;

� Each type of Ig �characteristics type of heavy chains � α� IgA; ∆� IgD; Ε� IgE, γ� IgG, µ�IgM

� 2 diffeent light chain� Κ and λ in all

� IgD and IgE � very simillar to IgG� IgM� both monomeric on cell surface or

pentameric secreted form� IgA: saliva, tears, milk (dimer or tetramer)� IgM: first immune response � IgG: second immune response� IgD: not konown function

B lympohcytes

Pentamer,

J chain (Mr 20000) found in IgM and IgA

Macrophages have receptors to Fc portion of IgG

IgE ���� role in the allergic response

Antigen-Antibody Binding

�Specificty depending on chemical complementar�Location of charged, nonpolar, hydrogen binding groups etc; shape�Example: negative charged �positivly charged

Induced fit in the binding of an antigen to IgG

2residues of Heavy of IgG

Light of IgG

HIV antigen

Usage of antigen-antibody interactions

in the development of techniques

� Two types of antibody preperations� Polyclonal and monoclonal anitbodies

� Polyclonal antibodies: antibodies that are derived from different B-cell lines and a mixture of immunoglobulin molecules secreted against a specific antigen, each molecules secreted against a specific antigen, each recognising a different epitope.

� Monoclonal antiobdies: identical antibodies. All are specific the same epitope

This technique developed by developed by

Where we use it� Affinity Chromotography

� ELISA

� Immunoblot assay

http://www.medschool.lsuhsc.edu/Microbiology/mmip/2003mip/Classes99/Laboratory/laboratory.htm

Actin, Myosin & Motor proteins

��Motor proteinsMotor proteins: Protein such as myosin or kinesin

that uses energy derived from ATP hydrolysis to propel itself along a protein filament.

� Use them to move

Motor proteins� Motor proteins

•Contraction of muscles

•Migration of organelles

•Rotation of bacterail flagella

•Movement of some proteins around DNA

How Muscle contracte� Two proteins

� Myosin

� Actin

� Myosin:� Mr: 540.000

80% of muscle� Mr: 540.000

� 6 subunits

� 2 heavy;each�220kD

� 4 light; each�20 kD

Globular ATP hydrolyzed

Left handed, coiled coil

http://www.wiley.com/college/pratt/0471393878/student/animations/actin_myosin/actin_myosin.swf

http://msjensen.cehd.umn.edu/1135/Links/Animations/Flash/0011-swf_breakdown_of_a.swf