David L. Nelson and Michael M.

Cox

Lehninger Principles of Biochemistry

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

O2 by Hemoglobin in blood
Hemoglobin in red blood cells (RBC)
6-9 m in diameter Biconcave disks Hemocytoblasts (procursor stem cell) RBC No nucleus, mitochondria, ER 120 days 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 (141 residue each); 2 (146 residue each) Fewer than half of and subunit identical 3D structure very similar Mb similar to both Helix-naming : Hb lacks D helix

Strong interaction between unlike subunits

1 1; 2 2 (>30 residues) Though Urea treatment dimer 1 2; 2 1 (>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 deoxyhemoglobin T: tense; R: relaxed T stabilized by a greater number of ion at 1 2; 2 1

interfaces

onic interactions

onic interactions between alfa beta

O2 binds THb

Hb changes its conformation

slides each other narrow the pocket Some ionic bonds broken Some new ionic ones formed

Max Perutz Some aa of heme during T triggered and become R Porphyrin slightly puckered Protured on F8 His During 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) others binding in the same protein Modulator > or < active ; so it meains activator or inhibator If modulator = ligand interaction is homotropic If modulator ligand interaction is heterotropic Some proteins have 2 or more; two types exist Hb O2 is both ligand and activating homotropic modulator subunit-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

[PLn]

Ka=

[P][L]n

[PLn] Ka= [P][L]n

Expression of [L]n = rearrange 1- Take log of both log sides [L]n = Kd [L]n+Kd

1-

= n log [L] logKd

Hill equation

n Where Kd=[L]0.5

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

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

If nH=1 not cooperative If nH>1 positive cooperative Hemoglobin + cooperative Ones binding affects others binding Therotical limit nH=n means everywhere is saturated nH<1 negative ccoperative .... Veryt rare

Adaptation of Hill equation 1-

n = n log pO2 nlogP0.5

log

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

Red blood cell
By carbonic anhydrase

CO2 + H2OH+ +HCO3

Lower pH nsoluble, bobble formation

Bohr effect: effect of pH and CO2

H+ and CO2

concentration on binding and Only 40% of H and 15-20% of CO by Hb releasing of Oxygen

+ 2

Rest of H+ :in HCO3 ; Rest of CO2:by HCO3 and CO2 Binding of H+ and CO2 to Hb H+ and CO2 Affinity of O2 for Hb O2 release inversely related of that of O2 H+ and CO2 release out Affinity of O2 for Hb O2 binds

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 subunit ion pairs AspFG1 T state stabilizes protonated His HC3 abnormal high pKa in T

Unprotonated

R state

Tissues Lungs

7.2 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 Uluda (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 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

Oxygen Regulation by 2,3-bisphosphoglycerate

when person at sea level
lung: almost 100% saturated lung: almost 90% saturated tissue: ~60% saturated

when person at 4500 mt

normally found on hemoglobin what it does: to reduce its affinity to O2 where it binds: a site far from O2 binding site. function: to adapt us to sharp pO2 changes [BPG] in blood

result:

Normally , BODY INCREASE [BPG]

lung: almost 85% saturated

tissue: ~45% saturated

[BPG] in blood
to cells: ~40% released released AGAIN to cells: ~30% released

BUT SUPPOSE NO CHANGE [BPG] [BPG] : Hb AFFINITY TO IN OXYGEN

Hypoxia: a disorder because of inadequate lung, circulation n fetus: in steade of , it has, 22 ..< BPG affinity but >that of O2 O2 for its mother Hb receive

Sickle Cell Anemia

a genetic disease producing sickel cell Hb Normal Hb is HbA ; Diseased Hb is HbS HbS HbA insoluble while deoxyginated soluble while deoxyginated aggregate into tubular fibers

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

HbS charge=HbAs charge -2 So HbS abnormally interaction each them

SCA

painful disease 50% of 10-15gr/100mL of normal value

SCA patients hemoglobin

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

Child with SCA die early

Malaria Before Columbus

Origins of the Sickle Cell Gene

Italy

Greece Albania

Turkey

India

Senegal Benin
CAR

Egypt

Arabian ArabianPeninsula Peninsula

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 body Immune Response: A response against foreigners Cells of immune system leukocytes (WBC)
Macrophages and lymphocytes

Immune Response
Humoral Immune System
In fluid, Special weapons used Antibodies, Immunoglobulins, Ig
B-lymphocytes (B-cells) Ig binds foreign ones (virus, bacteria, protein ..)

Cellular Immune System

Cells, their self fight 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 Igs 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 hapten immunogenic. Developed antibody to the hapten can bind its free form

PUTTING IT ALL TOGETHER

APC

T helper cell

Activated T helper cell

Cytotoxic T cell (Tc) Antibodies

Memory Tc

Effector Tc

Lysis

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) 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 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 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 antigenbinding sites

Different types: IgE, IgA, IgG, IgD, IgM Major class one IgG
Y-shaped Mr 150000 2 heavy and 2 light chains

Fab: antigenbinding fragment

Fc: crystalizable F

light chain

hinges Gerald edelman & Rondey porter

Heavy chain

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

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

Each type of Ig
IgA; and in all

characteristics type of heavy chains

IgD; IgE, IgG, IgM

2 diffeent light chain 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 B lympohcytes IgD: not konown function

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 charged positivly

Induced fit in the binding of an antigen to IgG

HIV antigen

2residues of Heavy of IgG

Light of IgG

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 recognising a different epitope.

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

This technique developed by

Where we use it
Affinity Chromotography ELISA Immunoblot assay

http://www.medschool.lsuhsc.edu/Microbiology/mmip/2003mip/C lasses99/Laboratory/laboratory.htm

Actin, Myosin & Motor proteins

Motor 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

Contraction of muscles Migration of organelles Rotation of bacterail flagella Movement of some proteins around DNA

How Muscle contracte

Two proteins
Myosin Actin

Myosin:

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/an imations/actin_myosin/actin_myosin.swf

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