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Exam II Learning Objectives BIOC 384

Exam II Learning Objectives BIOC 384

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Exam II Learning Objectives
Exam II Learning Objectives

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BIOC 384: EXAM II LEARNING OBJECTIVES
 
 2
Lecture XIII
 
Myoglobin, Hemoglobin 
Terminology: ligand. Fractional saturation prosthetic group, cooperativity, protomer, binding site, allosteric (allosteric site, allosteric effector, allosteric regulation).
Term Definition
Ligand An ion or molecule (usually) small that is bound by another molecule (usually large).Fractional Saturation Fraction of total binding sites on protein occupied by ligand, signified by Y.Calculated by formula:
=
[
!"
]
!"
!
[
!
]
=
[
!
]
!
!
!
[
!
]
.Prosthetic Group Metal ion or organic or metallo-organic compound other than an amino acid that’stightly bound to a protein (binding is covalent or tight non-covalent), required orthe protein’s function/activity.

 
+


 

 Cooperativity Affinity of binding sites is increased (positive cooperativity) or decreased(negative cooperativity) upon the binding of a ligand to a binding site. Basically,it is binding of a ligand to 1 binding site causes the effects of the properties (in theform of binding affinities) of other binding sites (on other subunits) of the sameprotein molecule.Protomer Structural unit of an oligomeric protein, can consist of one or several subunitsthat assemble to form an oligomer. For example, hemoglobin is a dimer of two
αβ
 protomers.Binding Site Site where specific, typically noncavalent interactions between molecularsurfaces occur. The essence of protein function/action is BINDING (recognition of and interaction with other molecules). It is dependent on three major factors:
-
 
Shape complementarity: Requiring lots of van der Waals interactions
-
 
Chemical complementarity: Requiring hydrogen bonds and ionicinteractions
-
 
Hydrophobic effect: Hydrophobic ligand minimizes exposure to water by  binding in hydrophobic site in protein. Allosteric (Allosteric site,allosteric effector, allostericregulation)Binding of ligand to one site on protein molecule affects binding properties/affinities of another site on same protein molecule.Homotropic implies that the interacting sites all bind to the same ligand.Heterotropic effects imply interacting sites binding to the same ligand. Allosteric Site: Site other than the protein’s active site. Allosteric Effector/Modulator: Ligands that spur allosteric effects. They canaffect the equilibrium between the T and R states. Allosteric Regulation: Regulation of an enzyme or other protein by binding aneffector molecule at the protein’s allosteric site. Allosteric activators enhance theprotein’s activity, while allosteric inhibitors decrease the protein’s activity.
Briefly describe the tertiary structure of Mb and Hb subunits (the “globin fold”),explain how the helices are designated, and the roles of the proximal and distal Hisresidues in heme and oxygen binding.
Myoglobin and hemoglobin subunits are similar in polypeptide sequence and contain a heme, and their overallstructure of subunits are similar. More notably, they have a similar tertiary structure, yet a differentquaternary structure. Myoglobin is a monomeric protein consisting of a single polypeptide chain with 153amino acid residues, while hemoglobin is heterotetrameric, consisting of two
α
 (141 AA residues) and two
β
chains (146 AA residues). The quaternary structureof hemoglobin confers then allosteric properties. Myoglobin can bind to oxygen as well as release. Hemoglobin’s function in vertebrate erythrocytes is to transportO
2
form lungs to tissues and transport CO
2
from tissues to lungs. Myoglobin ismainly the storage element within muscle tissue. It binds to O
2
delivered to tissue by blood and stored until needed as terminal electron acceptor for energy 
 
 3metabolism, and is the main intracellular transport for oxygen. To be a potent carrier, it needs to be able tocarry as well as release its ligand. The proximal and distal histidine residues in the heme group are typically themain point of interest in myoglobin and hemoglobin. They play a role in the cooperativity of hemoglobin andassociated metal complex.Remember that hemoglobin, as a structure, is very compact, with almost no empty space inside. It is mostly (~75%)
α
-helical, the rest mostly 
β
turns and loops (at the surface). There are 8 helices, designated A-H from Nto C terminus. The helices at the surface are amphipathic and the helices are termined by proline. The polar R-groups are mostly at the surface, except HIS (F8)/fifth ligand (proximal) and HIS (E7)-near sixth coordinationsite but distal. As a quaternary structure it is a tetramer with two
α
and two
β
subunits in adult (and different atdifferent stage of life). Noncovalent bonds stabilize this structure. The
α
 /
β
interactions dominate.
 Write a general protein-ligand binding/dissociation reaction in both the associationand dissociation directions. What is the mathematical relationship between theassociation and dissociation equilibrium constants?
 We know that the general reaction for ligand binding is:
[
]
+
[
]
[

]
. From there we can determine theequilibrium expressions in both the association and dissociation directions. From there, we also know that theassociation constant is the reciprocal of the dissociation constant.
Direction Equilibrium Expression Binding 
Strong Weak 
 Association Constant
!
=
[

]
!"
[
]
!"
[
]
!"
 Large SmallDissociation Constant
!
=
[
]
!"
[
]
!"
[

]
!"
=
1
!
 Small Large
Describe how and where in the structure of Mb and Hb O
2
binds, including roles of  protein functional groups and heme, and the oxidation state of the heme Fe requiredfor O
2
binding.
Remember that myoglobin is the oxygen storage protein (a single subunit) in muscle.It binds to oxygen in muscle cells and kept until needed. Hemoglobin is the oxygentransport protein in the blood. It circulates in red blood cells, and binds to oxygen inthe lungs and releases it to oxygen-requiring tissues. The affinity for oxygen ismodulated, involving tight binding in lungs, but easy release when needed in tissues.The modulation relies on the allosteric properties of hemoglobin.Oxygen binds to the heme group of hemoglobin and myoglobin. Heme is known also asiron protoporphyrin IX, with the bound Fe
2+
iron. Each iron has six coordination sites,one of which can be occupied by O
2
. Note that the iron acts as a prosthetic group topick up the oxygen. If iron was a lone actor, it would be immediately reactive and spurgreater damage to the organism, particularly humans.
Know how to interpret the O
2
binding curves [Y (fractional saturation) vs. pO
2
], i.e.NON cooperative ligand binding to a protein or cooperative ligand binding andexplain what is meant by cooperativity. On both curves, indicate the value of P
50
, the pO
2
at which fractional saturation of protein with O
2
is 0.5. In what part of thecooperative binding curve (what part of the [ligand] concentration range) is protein predominantly in the low binding affinity conformation, and in what part of theligand concentration range is the predominant form the high binding affinity conformation?
 We can utilize the association and dissociation equilibria to our advantage infractional saturation. Fractional saturation (signified as Y or
θ
) is the fraction of total binding sites on protein occupied by ligand. We can determine the fraction saturation by the following formula:
=
!"#$"#%
 
!"#$!
 
!""#$%&'
!"!#$
 
!"#$"#%
 
!"#$!
=
[
!"
]
!"
!
!
=
[
!
]
!
!
!
[
!
]
.
This shows that as[ligand] is added, more proteins are bound to ligand. When the fraction saturation isexactly 0.5, the concentration of ligand will be equal to the dissociation constant
(
=
!
)
.Myoglobin binding to oxygen follows the same protein ligand interactions. However, since O
2
is a gad it is moreconvenient to utilize partial pressures of oxygen (in the gas phase above the solution), than concentrations of O
2
 

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