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PROTEIN FUNCTIONS. Introduction. Functions of many proteins involve the reversible binding of other molecules Ligand. Ligand binds at a site on protein “ Binding site ” that is complementary to ligand in size , shape , charge & hydrophobic or hydrophilic characters .
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Introduction • Functions of many proteins involve the reversible binding of other molecules Ligand. • Ligand binds at a site on protein “Binding site ” that is complementary to ligand in size , shape , charge & hydrophobic or hydrophilic characters . • The binding of protein & ligand is often coupled to structural adaptations “Induced fit” , i.e. conformational change in the protein to make the binding site more complementary to ligand , permitting tighter binding . • Substrates are molecules that acted upon by enzymes at “Catalytic site” or “Active site” .
Formation of Binding Site • The binding site forms when amino acids from within the protein come together in the folding • The remaining sequences may play a role in regulating the protein’s activity
Binding Strength • Can be measured directly • Antibodies and antigens are mixing around in a solution, eventually they will bump into each other in a way that the antigen sticks to the antibody, eventually they will separate due to the motion in the molecules • This process continues until the equilibrium is reached – number sticking is constant and number leaving is constant • This can be determined for any protein and its ligand
Equilibrium Constant • Concentration of antigen, antibody and antigen/antibody complex at equilibrium can be measured – equilibrium constant (K) • Larger the K the tighter the binding or the more non-covalent bonds that hold the 2 together
Adjacent Secondary Elements are Often Local in Sequence The degree to which residues that are close in structure are also close in primary sequence is called contact order. A protein with lots of non-local contacts has high contact order.
Connections Between Elements of Secondary Structure Do Not Cross or Form Knots
Myoglobin • Myoglobin (Mr 16,700) – oxygen binding protein of muscle cells. • Myoglobin was the first protein structure to be solved by x-ray crystallography – in the 1950’s by John Kendrew and colleagues • All except two polar side chains of myoglobin are on the outer surface, and all are hydrated (bound to water molecules). • Most of the hydrophobic side chains are buried within the structure.
Each of four P residues occurs at a bend at the end of an a-helix. The protein is about 75% helix (and no b-sheet). • All peptide bonds are in the planar, trans configuration. • contains single heme group . • Dense hydrophobic core – so compact that there is only room for four water molecules in the interior.
Myoglobin – a well-studied protein, which binds oxygen through a heme prosthetic group • Myoglobin structure dictates its O2-binding properties • Hemoglobin is related to myoglobin but is oligomeric – it has four subunits • The oligomeric nature of hemoglobin allows it to bind oxygen cooperatively
Myoglobin structure • Myoglobin was the first protein structure to be solved by x-ray crystallography. • It’s present in muscle cells, where it stores bound oxygen and enhances its transport into mitochondria. • Globular protein, single polypeptide of 153 aa (Mr = 16,700) and single iron-porphyrin ring structure called heme. • This is an example of a prosthetic group, of which there are many different ones required by many different proteins.
Heme is responsible for the deep red-brown color of myoglobin and hemoglobin, and muscle. • Oxygen (O2) is bound by the heme group. Heme is a complex organic ring, protoporphyrin to which an iron atom in its ferrous state, Fe2+, is bound . • The flat heme group rests in a crevice within the protein. • Iron atom in the center of the heme group has two coordination bonds that are perpendicular to the plane of heme. • One is bound to the side chain of H93 (F8, 8th residue of helix F) and the other binds O2 .
Four pyrrole rings Methene bridges Myoglobin Binds O2 with a Heme Group
The Fe2+ atom is about 0.3 Å out of the the plane of the ring, toward His-F8. • A second histidine, His-E7, is on the other side of the heme, close to the bound oxygen. These two His residues are the only polar amino acids in the interior of the protein. • O2 is bound at an angle to Fe-O bond . • Fe moves into plane of heme upon binding O2 – i.e. heme is not a rigid structure. This flexibility is important for function. • Amino acids that contact heme come from different segments of the primary sequence. Formation of the heme binding site requires global tertiary folding.
We can describe the oxygen binding properties of myoglobin quantitatively For any reversible protein-ligand binding interaction: P + L PL Association constant, (units of M–1) Dissociation constant, (units of M) Fraction of ligand-binding sites occupied by ligand, or fractional saturation = .
rearrange and substitute: [L] here refers to concentration of unbound L. However, for most applications L is present at much higher concentration than P, so that unbound L ≈ total L (abbreviated [L]t. Thus we can plot vs [L]t to get a hyperbola. When [L]t = Kd, = 0.5. Half of the binding sites are filled, 50% saturation is achieved. Thus the Kd is equal to the concentration of ligand required to give 50% saturation of the binding sites. (For 50% saturation to give Kd experimentally, the concentration of protein binding sites must be lower than Kd. Why is this? Hint: it has to do with the distinction between unbound L and total L.)
Ligand binding to a protein [L] = q Kd + [L]
Because the ligand for myoglobin, O2, is a gas, the standard equation above is adapted: ; P50 = pO2 necessary to fill 50% of myoglobin sites
Ligand binding to a protein pO2 = q P50 + pO2
Environment of the Heme Group Facilitates O2 Binding Relative to CO
Environment of the Heme Group Facilitates O2 Binding Relative to CO
Hemoglobin structure • Hemoglobin has four polypeptide chains – an oligomeric protein. • It consists of two chains called a-chains and two b-chains (this has nothing to do with helices and sheets). • Each chain has one heme group. Hemoglobin therefore has four binding sites for O2. • The four chains (subunits) are held together by noncovalent forces (hydrophobic and electrostatic) • Quaternary structure is nearly spherical, with 4 chains packed together in a tetrahedron. • Each a-chain is in contact with both b-chains; there is very little contact between the two a’s or the two b’s
Both of the oxygen-binding proteins myoglobin and hemoglobin have a heme prosthetic group.
The Transportation of Blood Oxygen Lung Artery Muscle Vein Any one subunit receives an oxygen molecule will increase the oxygen-binding affinity of the others (R) relaxed state O2 When environmental [O2] decreases, Hb releases oxygen to Mb When environmental [O2] increases, Hb binds oxygen efficiently (T) tense state Hemoglobin Myoglobin Juang RH (2004) BCbasics
The four O2-binding sites are separated from each other by at least 25 Å. • The backbone structures of myoglobin and the chains of hemoglobin are nearly identical. • However, the amino acid sequences show many differences . • Invariant residues have been found in hemoglobin sequences from more than 20 organisms. • Nine positions have the same amino acid in nearly all. • The invariance suggested that these residues must play critical roles in structure/function.
Two invariant residues are near heme, and several others contact heme residues • Residues that stabilize structure by forming H-binds between adjacent helices • identity of nonpolar residues in interior varies, but the changes are always from one non-polar residue to another – the nonpolar character of the interior is conserved. • the most variable residues are on the surface
