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
