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!
Concerted cooperativity
modelo “tudo ou nada”, Monod, Wyman, Changeux 1965
-all subunits are postulated to be in the same conformation,
(either all low affinity, circ./ inactive or all high affinity/ active, squ.)=> undergo transition simultaneously
-Depending on the equilibrium, Keq, between circ. and squ. forms, the binding of one or more ligand molecules (L) will pull the equilibrium toward the squa. form
Sequential cooperativity
Daniel Koshland 1966
each individual subunit can be in either the circ. or squ. form! (ligand binding can induce a change of conformation in an individual subunit)
=> many conformations, potential intermediate states possible!
Nem todas são preferenciais/ as mais eficientes
CO binding to hemoglobin
tight binding–> COHb can accumulate over time –>mov. cumulativo
Healthy indiv: <1% of total Hb is complexed as COHb
CoHb ~50%: loss of consciousness –> efeito na afinidade dos restantes grupos heme para O2
=> a hemoglobin tetramer with two bound CO molecules can efficiently bind O2 in the lungs—but it releases very little of it in the tissues!
Effect of BPG on O2 binding to Hg
BPG concentration in normal human blood: ~ 5 mM at sea level, ~ 8 mM at high altitudes
Sea level: Hg nearly saturated with O2 in the lungs, but just over 60% saturated in the tissues=> amount of O2 released in the tissues~ 38% of the maximum that can be carried in the blood!
High altitudes: O2 delivery declines by about 1/4, to 30% of maximum.
Mas: An increase in BPG concentration decreases the affinity of hemoglobin for O2 => ~ 37% of what can be carried is again delivered to the tissues
=> alteração fisiológica (aumento BPG) compensa alteração altitude (também menos O2 disponível…?) –> Fração de O2 transportado prat. igual! (também + BPG em pessoas com hipoxia, menor oxigenação dos tecidos, problemas sistema circulatório…)
Hg fetal
Regulation of O2 binding to Hg by BPG has an important role in fetal development
–> A fetus must extract O2 from its mother’s blood => fetal Hg must have greater affinity than the maternal hemoglobin for O2
(se igual: competição!)
• The fetus synthesizes gamma subunits rather than alphabeta subunits, forming alpha2gamma2 Hg => tetramer with much lower affinity for BPG than normal adult Hg–> correspondingly higher affinity for O2
Dif. subunidades de Hg todas codificadas num gene –> desenvolvimento: vão se alterando Hg! (alguns res. dif–> interação dif –> afinidade, função dif) variação conforme necessidades, vão ficando mais complexas
Globinopatias
-anemia das células falsiformes (1 AA mutado–> altera conform., expõe zonas hidrofóbicas –> promove interações…. => quando HbS é desoxigenada, fica insolúvel –> forma polímeros que agregam em fibras tubulares –> deformação eritrócitos…)
-talassémia
mutações: inactivam subunidade, deficiência funcional se mutação nos 2 alelos–> não tem beta-globina
