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 bind s Mostly they are specific Proteins are flexible: changing in conformatin iduc iduced ed fit: fit: li and and res resul ultt in in a conf conform ormat atio iona nall chan chan e on on protein’s protein’s binding site si te so it fits the binding bind ing site more tightly. Interactions may be regulated. Enzyme-catalytic 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 irondangerous 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 arry em emog o n n sso ve n cy oso Hb in lungs96% saturated with O 2 Hb in tissues64% saturated with O 2
6.5 mL of O2 gas at atmosphoric pressure
Interaction subunit conformational conformational change alter affinity for O 2
Myoglobinrelativel relativelyy insensitive to small concentration of dissolved O2
Hb Subunit Similar to Myoglobin
HbMr:64500 Rouphly spherical 5.5 nm Tetrameric4 heme prosthetic group ypes o g o n
2 β (141 residue each); 2 α (146 residue each) Fewer than half of β and α subunit identical 3D structure very similar Mbsimilar to both Helix-naming : Hb α lacks D helix
Strong interaction between unlike subunits
α1β1; α2β2 (>30 residues)
Though Urea treatmentdimer αβ
α1β2; α2β1 (>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 sta e w e eoxygenate :pre om nant con ormat on o deoxyhemoglobin T: tense; R: relaxed T stabilized by a greater number of ion at α1β2; α2β1
interfaces
Đonic interactions
Đonic interactions between alfa beta
O2 binds THb Hb changes its conformation R
αβ slides each othernarrow the pocket Some ionic bonds broken Some new ionic ones formed
Max Perutz Some aa of heme during T triggered and become R Porphyrinslightly puckered Protured on F8 His During F; planar (after O 2)
Hb cooperatively O2 binding
O2 binds to Hb when pO 2 ~ 13.3 kPa O2 release when pO2 ~ 4 kPa (Not anyother can do that)
How does Hb do that? A transition stageS :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 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 Hillfirst cooperative binding of oxygen nL ↔ P + PLn
↔
+
[PLn] Ka=
[P][L]n
[PLn] Ka=
[P][L]n
Expression of Ө Ө=
[L]n [L]n+Kd
rearrange
Ө = 1- Ө Take log of both log sides
[L]n Kd
Ө = n log [L] – logKd 1- Ө
n Where Kd=[L]0.5
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 H
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
log
Ө n = n log pO2– nlogP0.5 1- Ө
Two model mechanism TR 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 + H2O↔H+ +HCO3 Lower pH Đ nsoluble, bobble formation
H+ and CO2
Bohr effect: effect of pH and CO 2 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 inversely related of that of O2
H+ and CO2 ↑ Affinity of O2 for Hb ↓ O2 release
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 +
AspFG1 T state stabilizes protonated His HC3abnormal 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+ producedBohr effect T state stabilizationrelease of O2
2,3-bisphosphoglycerate (BPG)
HbBPG + O2 F HbO2 + BPG BPG bound Hb BPG reduce the affinity of Hb to O 2 What is the benefit? Seen while insuffient O 2 Example: If you go to Uludağ (over 3000 meters high), pO2 is 7 kPa, (at sea level..13 . , ~ . 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
when person at 4500 mt
• normally found on hemoglobin
lung: almost 100% saturated
result: • where it binds: a site far from O
•what it does: to reduce its affinity to O 2
lung: almost 90% saturated
2
tissue: ~60% saturated
binding site.
Normally , BODY INCREASE [BPG]
saturated
• function: to adapt us to sharp pO 2
changes [BPG] in blood tissue: ~45% saturated
[BPG] in blood to cells: ~40% released released AGAIN to cells: ~30% released
BUT SUPPOSE NO CHANGE [BPG] [BPG] TO IN OXYGEN ↑ : Hb ↓ AFFINITY
Hypoxia: a disorder because of inadequate lung, circulation , γ it has, α 2γ Đ n fetus: in steade of β 2 ..< BPG affinity but >that of O2 receive O2 for its mother Hb
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 patients’ hemoglobin 50% of 10-15gr/100mL of normal value WHY? HbS is so fragile so repture easlyanemia (lack of blood) Child with SCA die early
Malaria Before Columbus
Origins of the Sickle Cell Gene
Italy
Greece Albania
Turkey
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 Immune Response: A response against foreigners Cells of immune systemleukocytes (WBC)
Macrophages and lymphocytes
Immune Response
Humoral Immune System In fluid, Special weapons used Antibodies, ,
B-lymphocytes (B-cells) Ig binds foreign ones (virus, bacteria, protein ..)
Cellular Immune System Cells, their self fight os mpor an one 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 ’ 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. eve ope anti o y to t e apten can in its free form
PUTTING IT ALL TOGETHER…
APC
T helper cell
Activated T hel er 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
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: dis la e tides derived from roteol tic degradation
Recognation targets of T-cell receptors of T c 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 oneIgG
Y-shaped Mr150000
binding fragment Fc: crystalizable F
hinges
Heavy chain
Gerald edelman & Rondey porter
Immunoglobulin o s: a eta (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 characteristics type of heavy chains
2 diffeent light chain
α IgA; ∆ IgD; Ε IgE, γ IgG, µIgM
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 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 positivly charged
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 , recognising a different epitope.
Monoclonal antiobdies: identical antibodies. All are specific the same epitope
This technique eve ope y
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
. 6 subunits 2 heavy;each220kD 4 light; each20 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
