lehninger principles of biochem ch5
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Biochemistry TextbookTRANSCRIPT
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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
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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)
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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
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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
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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
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� 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
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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
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Đonic interactions
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Đonic interactions between alfa beta
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� O2 binds THb� Hb changes its conformation� R� αβ slides each other�narrow the pocket
� Some ionic bonds broken
� Some new ionic ones formed
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Max PerutzSome aa of heme during T triggered and become RPorphyrin�slightly puckeredProtured on F8 HisDuring F; planar (after O2)
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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
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A sigmoid (cooperative) binding curve:
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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
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At first; the sites less affinity
ligand
binds
Become
more stable
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Qusntitatively Cooperative Ligand
Binding
� 1910, Archibald Hill�first cooperative binding of oxygen
P + nL ↔ PLn
+ ↔
Ka=[PLn]
[P][L] n
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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
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� 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
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Adaptation of Hill equation
Ө n log pO2– nlogPn=1-Ө
log 0.5
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Two model mechanism
T�R transition occur. HOW?
Two models
1. MWC model (concerted model)
2. Sequential model
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All or none
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Hemoglobin transport H+ & CO2
By carbonic anhydrase
Red blood cell
CO2 + H2O↔H+ +HCO3
By carbonic anhydrase
Lower pH
Đnsoluble, bobble formation
↔
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�
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
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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
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Tissues � 7.2
Lungs � 7.6
7.4�experimental hemoglobin binding
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CO2 Binding� CO2 bind amino-terminal of globular PPC forming
carbaminohemoglobin (carbamino-terminal residue)
� H+ produced�Bohr effect
� T state stabilization�release of O2
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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.
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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
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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
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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
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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
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Malaria Before Columbus
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IndiaItaly
GreeceAlbania
Turkey
Origins of the Sickle Cell Gene
Senegal
Benin
CAR
IndiaTurkey
Arabian PeninsulaArabian Peninsula
Egypt
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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
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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)
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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
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� 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
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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
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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
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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
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http://www-ermm.cbcu.cam.ac.uk/smc/swf001smc.htm
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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.
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http://www-ermm.cbcu.cam.ac.uk/smc/swf002smc.htm
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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
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light chainFab: antigen-
Heavy chain
light chain
hinges
Fab: antigen-binding fragment
Gerald edelman & Rondey porter
Fc: crystalizable F
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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;
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� 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
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Pentamer,
J chain (Mr 20000) found in IgM and IgA
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Macrophages have receptors to Fc portion of IgG
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IgE ���� role in the allergic response
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Antigen-Antibody Binding
�Specificty depending on chemical complementar�Location of charged, nonpolar, hydrogen binding groups etc; shape�Example: negative charged �positivly charged
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Induced fit in the binding of an antigen to IgG
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2residues of Heavy of IgG
Light of IgG
HIV antigen
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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.
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� Monoclonal antiobdies: identical antibodies. All are specific the same epitope
This technique developed by developed by
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Where we use it� Affinity Chromotography
� ELISA
� Immunoblot assay
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http://www.medschool.lsuhsc.edu/Microbiology/mmip/2003mip/Classes99/Laboratory/laboratory.htm
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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
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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
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Globular ATP hydrolyzed
Left handed, coiled coil
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http://www.wiley.com/college/pratt/0471393878/student/animations/actin_myosin/actin_myosin.swf
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http://msjensen.cehd.umn.edu/1135/Links/Animations/Flash/0011-swf_breakdown_of_a.swf
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