Protein Function- Reversible Binding of a Protein to a Ligand: Oxygen-Binding Proteins Flashcards
associação proteína- ligando
Function of many proteins: reversible binding of other molecules= ligands
ligand= any kind of molecule: gás, outra proteína, …
TRANSIENT NATURE of protein ligand interactions is critical to life–> allows rapid + reversible response to changes (environment, metabolism) –> permite funções de sinalização, transporte, regulação
Binding: Quantitative Description
association rate constant ka/ dissociation rate constant kd –> with time: equilibrium of association and dissociation rates –> equilibrium association constant Ka (or eq.dis.const. Kd)
ka [P] [L] = kd [PL]
Ka = [PL]/ [P][L] = 1/ Kd
In practice, we can often determine the fraction of occupied binding sites (θ)
θ= [L]/ ([L]+ Kd)
Ka^= 50% dos sítios ocupados!!
–> medida da afinidade: quanto maior for afinidade, menor é a concentração necessária para atingir 50% saturação, menor é Ka
—- When a ligand is a gas, binding is expressed in terms of partial pressures
θ= [L]/ (Kd +[L])
–> θ= pO2/ (p50 + pO2)
Globins- Função, Necessidade?
Oxygen- Binding Proteins
Biological problems:
• Protein side chains lack affinity for O2
+ H202–> peróxido, superóxido, hidróxido… espécies parcialmente reduzidas de O2 que causam modificações químicas nos AA
• Some transition metals bind O2 well but would
generate free radicals if free in solution
• Organometallic compounds such as heme are more suitable, but Fe2+ in free heme could be oxidized to Fe3+ (very reactive!)
–> Biological solution:
• Capture O2 with heme that is protein bound! –> Myoglobin, hemoglobin
Proteina: ligar Heme + regular função por interações…
Structures of Porphyrin and Heme
• Heme: complex organic ring structure (protoporphyrin IX), (anel tetrapirrólico) with a bound iron atom in its ferrous (Fe2+) state!
[porfirina+ferro= heme]
• The iron atom of heme has 6 coordination bonds: 4 in the plane of (and bonded to) the flat porphyrin ring system and 2 perpendicular to it.
Há vários tipos de heme conforme os grupos R –> associam à proteína de forma lig. diferente
Oxygen binding: structural effect
Oxygen binding (pode-se ligar por baixo ou cima) changes the position of the iron ion!
[deoxyhemoglobin]
iron ion: slightly outside the plane of the porphyrin in deoxymyoglobin heme
[oxyhemoglobin]
- -> moves into the plane of the heme on oxygenation
- -> alteração conformacional na proteína! O grupo inferior (?) (His) tem de se ajustar
Myoglobin
(Mr 16,700 ~150AA; abbreviated Mb): relatively simple oxygen-binding protein (–>single binding site for O2), found in almost all mammals, primarily in muscle tissue.
[fun fact: a baleia tem muito mais mioglobina que os humanos–> usada para armazenar O2 em períodos de apneia]
• made up of eight a-helical segments connected by bends
• topologia alpha (~78% AA)
Myoglobin- Structure
proximal His residue - plane of porphyrin ring system - O2
His distal (His E7, His64) (his=> anel imidazol, é protonável): não faz parte da coordenação direta ao Fe liga ao O2 por ponte de H –> estabilidade, seletividade
Arranjo com his distal evita libertação do ião superóxido! (interação ferro- O2 é uma combinação de híbridos de ressonância: fe2+ e o2 vs. fe3+ e o2 -)
Myoglobin- Oxygen binding and conformational breathing
binding of O2 to the heme in myoglobin also depends on molecular motions/ “breathing” (protein)
heme: deeply buried in the folded polypeptide! Num binding pocket! (No direct path for O2 from the surrounding solution to the ligand binding site)
=> Rapid molecular flexing of the AA side chains –> transient cavities in the protein structure –> paths for O2
(If protein were rigid, O2 could not enter/ leave the heme pocket at a measurable rate)
CO vs. O2 binding to heme
- CO has similar size and shape to O2: it can fit to the same binding site.
- CO binds heme over 20,000 times better than O2 (carbon in CO has a filled lone electron pair that can be donated to vacant d-orbitals on the Fe2+)
–> The protein pocket decreases affinity for CO, but it still binds about 250 times better than oxygen!!
=> CO is highly toxic, as it competes with oxygen!
-It blocks the function of myoglobin, hemoglobin, and mitochondrial cytochromes involved in oxidative phosphorylation (Fe deixa de estar disponível p/ transporte de O2)
Hemoglobin
(Mr 64,500; abbreviated Hb) is roughly spherical, diameter of nearly 5.5 nm.
- tetrameric protein with four heme prosthetic groups (1 associated with each polypeptide chain)
Hemoglobin- structure
-tetramer of two subunits (alpha2beta2), each similar to myoglobin, but: AAs only 20% identical! –> redundância dos AA (mesma estutura com sequência diferente)
Could Myoglobin Transport O2?
- pO2 in lungs (~ 13 kPa): it sure binds oxygen well
- pO2 in tissues(~4 kPa): it will not release it! Também imediatamente saturada, mesmo a baixa []!
=> For Effective Transport Affinity Must Vary with pO2! Curva sigmoidal, como na hemoglobina
How Can Affinity to Oxygen Change?
COOPERATIVITY (valor energético de transição vai diminuindo)
–> must be a protein with multiple binding sites!
–> that are able to interact with each other
phenomenon= cooperativity
=> dinâmica conformacional: interação entre haver ligação num sítio e a afinidade doutro!
-negative cooperativity
1st binding event reduces
affinity at remaining sites
-positive cooperativity
1st binding event increases affinity at remaining site! –> sigmoidal binding curves –> hemoglobin!
=> stages:
1: no ligand, low-affinity state- “parts” flexible/ somewhat unstable, few conformations facilitate ligand binding
2: ligand bound to 1 subunit –> stabilises a high-affinity conformation! More of the structure is stable, none unstable; rest of polypeptide- higher-affinity conformation!, stabilized by prot-prot interactions of 1st subunit
3: binding of 2nd ligand to 2nd subunit occurs with higher affinity –> positive cooperativity
Hemoglobin- Oxygen binding
Hemoglobin Undergoes a Structural Change on Binding Oxygen (movimento mecânico):
binding of O2 –> heme assumes a more planar conformation –> shifting the position of the proximal His (logo também deslocação do Fe) and the attached F helix (pq his está inserida numa hélice) –> reorganização de conjunto de interações entre subunidades (–>água, interações, dinâmica…)!
This triggers the T → R transition–> Cooperatively:
1st molecule of O2 that interacts with deoxyhemoglobin binds weakly (binds to a subunit in the T state) --> however: conformational changes that are communicated to adjacent subunits--> easier for additional molecules of O2 to bind => T n R transition occurs more readily in the 2nd subunit (once O2 is bound to the 1st subunit) The last (4th) O2 binds to a heme in a subunit that is already in the R state--> much higher affinity than the 1st molecule
T-state vs. R-state
T = tense state
– more interactions, more
stable
– lower affinity for O2
R = relaxed state
– fewer Interactions, more
flexible
– higher affinity for O2
In hemoglobin: Conformational change(T–>R) involves breaking ion pairs between the α1-beta2 interface –> transição “relativamente dramática”, involve quebra de pontes salinas (–> energia algo maior que pontes de H); tem de haver amplitude mecânica suficiente para que haja disrupção
Subunit Interactions in Hemoglobin
The strongest subunit interactions occur between unlike subunits
When oxygen binds there is a large change at the α1β2 contact, with several ion pairs broken
mapa de interações eletrostáticas (T–>R):
- beta2: Asp- e His+ , tal como COO- com Lys+ de alpha1
- alpha1: também Arg+ com Asp- de alpha2 e ao contrário; ainda COO- com NH3+ de alpha 2 e ao contrário
- alpha2: ainda: Lys+ com COO- de beta1
- beta1: também His+ e Asp-
Some ion pairs stabilise the T state of deoxyhemoglobin: His+ e Asp- nas sub. beta; Lys+ e COO- da His entre alpha e beta
Allosteric Regulation
Allosteric protein
– Binding of a ligand to one site affects the binding properties of a different site on the same protein!
– positive or negative
– homotropic
• normal ligand of the protein is the allosteric regulator
– or heterotropic
• A different ligand affects binding of the normal ligand
Cooperativity= positive homotropic regulation
Hemoglobin- other functions
Hemoglobin also Transports
H+ and CO2
Hemoglobin carries 2 end products of cellular respiration: H+ and CO2 from the tissues to the lungs and kidneys (where they are excreted)
Inside a red blood cell, CO2 (produzido por células de tecido) reacts with water to form carbonic acid (H2CO3), in a reaction catalyzed by: carbonic anhydrase. Carbonic acid dissociates to form HCO3 - and H+ –> drop in pH inside the red cell= acidificação da célula
(Para pulmões: contrário, CO2 entra nos pulmões)
pH Effect on O2 Binding to Hemoglobin
Actively metabolizing tissues generate H+, lowering the pH of the blood near the tissues relative to the lungs (catalyzed by carbonic anhydrase)
(CO2 + H2O ↔ HCO3− + H+)
The pH difference between lungs and metabolic tissues increases efficiency of the O2 transport! –> effect of pH and CO2 concentration on the binding and release of oxygen by hemoglobin= Bohr effect
Bohr effect- chemical basis
Hb Affinity for oxygen depends on the pH
H+ binds to Hb and stabilizes the T state pq. protonates His146, which then forms a salt bridge with Asp94 –> leads to the release of O2 in the tissues!
Hemoglobin and CO2 Export
- CO2 is produced by metabolism in tissues and must be exported.
- 15–20% of CO2 is exported in the form of a carbamate on the amino terminal residues of each of the polypeptide subunits (COO-) (4 cadeias= 4 N-terminais por Hb, como [] Hb no eritrócito é alta–> grande quantidade de residues terminais disponíveis)
• Notice:
– The formation of a carbamate yields a proton & forms additional salt bridges,
stabilizing the T state = Bohr effect
2,3-Bisphosphoglycerate
Regulates O2 Binding
• Negative heterotropic regulator of Hb function
(different from ligand, diminui afinidade!) –> diminui a afinidade do O2 pela Hg
• Present at mM concentrations in erythrocytes
• produced from an intermediate in glycolysis
- Small negatively charged molecule, binds to the positively charged central cavity of Hb + stabilises the T states –> diminui afinidade de Hg para O2
- oxygenation: The binding pocket for BPG disappears! Transition to the R state follows
Sickle-Cell Anemia= Anemia das células falsiformes
Due to a Mutation in Hemoglobin
• Glu6 → Val in the beta chain of Hb
• The new Valine side chain can bind to a different Hb molecule to form a strand similar to the amyloidgenic proteins! –> This sickles the red blood cells.
(= interação deixa de ser funcional –> hb perde função)
=> alteração da conformação –> exposição de uma superfície hidrofóbica (Phe, Leu… que estavam resguardadas) –>moléculas agregam + cristalizam, formam fibras insolúveis! Muito rígidas e resistentes à degradação (proteases… não têm centros ativos!)
- Untreated homozygous individuals generally die in childhood.
- Sickle cell hemoglobin is called hemoglobin S (HbS)
Cooperativity- quantitative description
=> Hill equation (Archibald Hill, PN 1922)
log (theta/ (1-theta))= nlog[L] -logKd
=> Hill plot (log pO2 vs. log (theta/(1-theta))
declive= coeficiente de Hill, nH –> medida da cooperatividade:
n =1 no cooperativity
n > 1 positive cooperativity
n <1 negative cooperativity
[em teoria: nh no max. = n –> completa cooperatividade, todos sítios de lig. ligados simultaneamente, sem proteínas parcialmente saturadas; mas na pratica sempre menor]
hemoglobina: 1 (estado baixa afinidade, T) –>3–>1 (estado alta afinidade, R)
mioglobina: 1! sempre!
