Protein folding and stability Flashcards
Protein folding
Protein folding= a physical process by which a polypeptide folds into its characteristic 3-dimensional structure from a random coil.
• Folded proteins correspond to an energetic minimum and are marginally stable
• Folded structure is dictated by primary sequence. (& ditado pela física: exclusivamente interações entre cadeias laterais!)
• Proteins fold through preferential pathways
• Protein folding occurs spontaneously under physiological conditions
–> Proteins fold into a unique and well-defined 3D structure – the native state
One of the largest unsolved puzzles in modern biochemistry: how proteins fold
Redundancy
Conformational preferences of AA
Conformational preferences of AA are not strong!
Many sequences can adopt alternative conformations in different proteins. –> Redundância (cerca 5x?), não há determinismo
(Ex the sequence VDLLKN assumes an α helix in one protein context and a β strand in another)
Favorable Interactions in Proteins
Stabilised by numerous weak interactions between AA side chains: Hydrophobic interactions Hydrogen bonds London dispersion Electrostatic interactions
Interacting AA are not necessarily next to each other in the primary sequence.
Cooperatividade: o estabelecimento de uma interação facilita o da segunda…
–> The protein conformation with the lowest free energy (that is, the most stable conformation) is the one with the maximum number of weak interactions!
The Anfinsen experiment - Ribonuclease Refolding
• Ribonuclease= small protein with 8 cysteines linked via 4 disulfide bonds.
• Urea + 2-mercaptoethanol (/DDT?) fully denatures ribonuclease
• When urea and 2-mercaptoethanol are removed (diálise), the protein
spontaneously refolds, and the correct disulfide bonds are reformed (analisado pela atividade enzimática).
–> The sequence alone determines the native conformation.
(When denatured structure is reoxidised under denaturing condition–> “disulphide scrambling” –> removal of urea, addition of a minute amount of 2-mercaptoethanol: native structure)
• The experiment is quite “simple” but so important it earned Chris Anfinsen the 1972 Chemistry Nobel Prize.
Protein Folding Pathways
Progressive stabilization of (micro)intermediates (a partir de certo momento: “ponto de não retorno”) rather than random search (trial and error)!
Denatured: Local regions with sufficient structural preference tend to adopt their favoured structures initially –> They come together to form a nucleus with a native- like, but still mobile, structure–> it fully condenses to form the native, more rigid structure: Native
Protein Stability
Proteins are marginally stable–> Flexibilidade! Necessita de “conformational breathing”= dinâmica (se mutação –> hiperestabilidade: MAU!)
STABILIZING FORCES:
– internal interactions (deltaH):
High number of weak interactions, predominates as a stabilizing force
– hydrophobic effect(-TdeltaS):
Nonpolar groups are clustered together–> decrease in the extent of the solvation layer (each group no longer presents its entire surface to the solution)–> favorable increase in entropy (da água)
DESTABILIZING FORCE:
– Conformational entropy (-TdeltaS, maior que efeito hidrof.)
–> NET: 20-60kJ/mol
(Weak interactions disrupted by: 4-30kJ/mol! single covalent bond: 200-460 kJ/mol)
–> Modest changes in the protein’s environment can cause structural changes that can affect function!!!!!!!
Protein denaturants
• Temperature
Perturbation of stabilizing interactions through thermal energy –> aumento movs brownianos–> instabilidade, rutura (ex: estrelar ovo, albumina desagrega–> branco)
• Chemical denaturants
chaotropic agents such as urea, guanidinium chloride (GuHCl), guanidinium tiocyanide (GuSCN) –> perturbam a organização das mols. de água à superfície
• pH
Protonation/deprotonation of side chains (Lys, Arg, His, Glu, Asp) that affect electrostatic interactions
• Reducing agents
for S-S containing proteins! beta- mercaptoetanol or Dithiothreitol (DTT) (–>experiência Afindsen)
Quantitative analysis of the protein stability
Through determination of thermodynamic parameters
(study of the reversible protein unfolding reaction)
–> Protein denaturation curve ([denaturant] vs. [protein unfolded], medida por métodos espetrofotométricos)
Forma sigmoidal com transição abrupta!! Decorre da cooperatividade
Chemical denaturants
CHAOTROPIC agents
–> Disrupt the H-bonding network between water molecules: disorder
1–> weakens the hydrophobic effect (bc hydrophobic molecules are more easily solvated)
2–> free water molecules can compete with intraprotein interactions
- -> disruption of the secondary structure
- -> denaturants interact directly with polar residues and the protein peptide backbone
- -> nonnative conformations are stabilized –> reduces the stability of the native state of proteins
Urea / Guanidinium chloride / Guanidinium thiocyanate
Urea → GuHCl (~2x more efficient) → GuSCN (≈3x more efficient)
Protein folding in the cell
-Physical principles of protein self-assembly are valid in vivo;
- occurs in polyribosomes that bring different chains to proximity (Pseudohelical organization along the mRNA: maximizes distance between nascent chains on adjacent ribosomes–> reducing the probability of aggregation-prone intermolecular interactions that limit productive folding)
- folding is tightly coupled to many other processes that control the protein life cycle
- envolve catalizadores
- ocorre num ambiente congestionado macromolecular (cell: Protein/nucleic acids= 80-400 mg/ml)
Proteostasis
Synthesis, Assembly, and Degradation of Proteins in vivo
1- synthesis: [Ribosome]–> nascent polypeptide
2- multiple pathways –> protein folding: folding intermediate
–> [chaperones]: catalyse
-folding –> native protein
(& prevent -remodelling –> misfolded protein +disaggregation–> amorphous aggregates! Ex of chaperons: Hsp40, Hsp70…)
Small proteins (<100 AA) fold spontaneously
- irreversibly unfolded proteins–> sequestration, degradation (additional pathways) [ex ubiquitin-proteasome system–>peptide fragments]
(Partially unfolded proteins (intermediates that escape the quality-control activities of the chaperones/degradative pathways) may aggregate –> amorphous aggregates/ oligomers/ amyloid fibrils)
Chaperonins
GroEL, Hsp60: “shaker molecular”, heptameric ring, with 2 chambers: alternate in the binding and facilitated folding of client proteins.
Folding occurs within the cis chamber (durations: time of hydrolysis of the 7 ATPs bound to the subunits in the ring)
–> GroES & ADP molecules then dissociate –> protein is released.
Misfolded proteins
multiple pathways to disease!
abnormal folding and structural changes due to biochemical or genetic factors! –> Protein misfolding
• Loss of protein function
(ex destabilisation)
• toxic gain of protein function (ex formation of amyloids)
Protein folding diseases and affected proteins
- Amyloid diseases
- Chaperonopaties
- Non-amyloid diseases
Over 50% of metabolic diseases result in protein misfolding!
(ver Quizlet)
Amyloids
highly ordered supramolecular protein assemblies
Protein molecules with regions of β sheet undergo partial folding.
In some molecules, before folding is complete, the β-sheet regions of one polypeptide associate with the same region of another, forming the nucleus of an amyloid! –>interações indevidas, padrão “cross beta”((?), alguma semelhança à fibroina)
Additional protein molecules slowly associate with the amyloid–> amyloid fibril
