Protein Folding in the cell (L3-6) Flashcards
Which are the main functions of proteins?
- Enzymes catalyze cell chemical reactions
- Membrane proteins form communication channels
- transport of cargo and mechanical forces
Which molecules carries the main functional components in the cell?
Proteins
What is the importance of protein folding?
- Gives its function to the protein
- Provides physical stability
- Provides functional surfaces for interaction with other molecules
*Sequence of amino acid and interactions between them determine structure, function, localization
Name 3 different polymers
- DNA
- RNA
- Peptide chains / Proteins
What is the standard structure for an amino acid?
H2N - alpha C - COOH
often in ionized form at pH 7
(+) H3N - alpha C - COO (-)
What are different characteristics of side chains?
- Hydrophobic, polar or charged (acidic/basic)
- Small or large
- Covalently linked into polypeptides (links are not between side chains, but between the core of AA)
Which are the polar amino acids?
*Not Quite Your Show Time
- Asparagine (NH2) (N)
- Glutamine (NH2) (Q)
- Serine (OH) (S)
- Threonine (OH) (T)
- Tyrosine (ring + OH) (Y)
*H2O = hydrogen bond donor (H linked)
NH3 = hydrogen bond acceptor (N linked)
Which are the acidic amino acids?
- Aspartate
- Glutamate
*Have COO (-)
Which are the basic amino acids?
- Lysine
- Arginine
- Histidine
*Have NH3+ or HN2+
Which are the hydrophobic nonpolar amino acids?
- GAVe LIFe With My Partner in Crime
- Glycine (G)
- Cysteine (C)
- Methionine (M)
- Alanine (A)
- Phenylalanine (F)
- Valine (V)
- Leucine (L)
- Isoleucine (I)
- Proline (P)
- Tryptophan (W)
Explain the peptide bond formation reaction (in the polypeptide backbone)?
- Condensation (H2O released, joining of 2 molecules)
- N - alpha C - C - BOND - N - alpha C
H2O release = OH from C-terminal and H from N-terminal
What part of polypeptides determine the charge and hydrophobicity?
Side chains
What are characteristics of the peptide bond?
- Planar, can’t rotate (bc of hybrid/partial double bond from C=O)
- Peptide bond is uncharged, but polar
- Can form non-covalent contact with other AA
- Only L-amino acids
- Peptide bond always in trans configuration except P (proline), can be trans or cis bc of NH2 loop
Why do we say the polypeptide backbone has limited freedom?
- Peptide bond is planar and can’t rotate
- Rotation around alpha C (both sides)
*Some rotation angles between amino acid residues in a polypeptides a prefered for better interaction
What interactions between residues of a polypeptide act to stabilize structure?
- Hydrogen bonds
- Van der Waals interaction (ALL)
- Ionic bonds
- Hydrophobic interactions (hydrophobic residues assemble ∆S < 0)
- Covalent interactions between cysteines (Disulfide Bonds)
Where can we find disulfide bonds?
Secretory proteins:
- In extracellular proteins
- Inside secretory organelles
NOT in cytosolic proteins (cytosol, nucleus, mitochondria)
Can be intrachain or interchain
What are the main characteristics of tertiary structure?
- Secondary elements organized between each other
- Hydrophobic contacts between 2ndary elements
- Long-rang contacts between residues that are far in primary sequence
- Confers its function to the polypeptide (or part of it)
- Domains are tertiary structure
What are intrinsically disorderd regions in proteins and why are they important?
- Not involved in 2ndary structures
- Provide flexibility to the protein
- Receive post-translation modification (bc more exposed)
What level of structures are dimers, trimer, tetramer, 5-mer, 6-mer, …, oligomers?
Quaternary structures
What are the 4 ways to visualize proteins?
- Polypeptide backbone → only backbone, no 2ndary structure
- Ribbon diagram → backbone + 2ndary structure (not side chains)
- Stick diagram → backbone + side chains
- Space-filling model → filled with volume of atoms
What is a domain? Give an example.
A domain is an independently folded unit within a protein
Different domains = different functions
ex: Hsp70 polypeptide
What are modular domains?
Conserved domains found in many different proteins
They form reversible, specific non-covalent contacts with other molecules
ex: contact with DNA, carbohydrates, lipids, other cofactors, etc.
What is the normal/average length of a domain and the one of a polypeptide?
Most human polypeptides = 100-800 AA long = 12 kDa - 90 kDa MW
Domain = 50 - 200 AA long
What are 4 characteristics of protein interactions
- Non-covalent
- Specific (molecular surface) → “lock and key”
- Often transient → bc thermal motion → molecules constantly moving, colliding
- Binding equilibrium → depends on concentrations of all proteins involved in interaction
What is the difference between identical, similar and divergent amino acid sequences?
Identical = same AA
Similar = same properties, same group, similar structures, charges, both have a ring, etc.
Divergent = very different
*Similarity in sequences suggests evolutionnary conservation even if not identical, bc similar sequences have similar functions
What are protein families?
A set of proteins or domains which have homologous sequences and structures
- related functions
- Can be found in different organisms
Important family = Hsp70
Which polar amino gets involved in hydrophobic interactions?
Tyrosine
Which hydrophobic nonpolar amino acid gets involved in hydrogen bonds?
Tryptophan (bc of N)
When/Why does protein misfolding occur?
Occur before they are completely folded, in the folding process
- Immediatly after protein synthesis
- If a required ligand is not available
- Genetic mutation in AA sequence (ex: cystic fibrosis)
- harmeful environmental conditions (ex: heat)
- Aging → ledd efficient protein quality control mechanism
How can aging be responsible for disease at a protein folding level?
Aging → Deacreased efficiency of protein quality control mechanism → lose of protein homeostasis → aggregates of misfolded proteins (ex: amyloid) → Neurodegeneration (Alzheimer, parkinson, ALS, dementia)
Why can some mutations be non-pathogenic?
If an AA is substituted for a similar amino acid, it will interact in a similar manner with the rest of the sequence → similar folding → similar native state → similar function
ex: Asp → Asn
What does protein homeostasis / proteostasis refer to?
Extensive NETWORK of components that act to maintain proteins in correct:
- Concentration
- Conformation
- Subcellular location
To cooperatively achieve stability and functional features of proteome
*Chaperones are at the center of the protein quality control network
Are PTM permanent?
No, they act as On-Off switches for cells
In what conditions are HSP upregulated?
In stress conditions
*Levels of HSP are tightly regulated in order to match the levels of unfolded/misfolded proteins depending on stress
What are the 2 main categories of Heat Shock Proteins?
- Heat Shock Response (HSR)
- Cytosolic and nuclear proteins
- Protects against cell death - Unfolded Protein Response (UPR)
- ER proteins
- Can promote cell death if stress is too severe
Which amino acids get involved in van der Waals interactions?
ALL OF THEM
What are different types of modification proteins can undergo post-translation? (not reaction names)
- Cleaved into smaller proteins by peptidases (specifically transmembrane proteins get their cytoplasmic part cut to go and send a signal)
- Covalent modification of N-terminus (co-translational)
- Covalent modifications of side chains → introduce functional groups to proteins
What are different roles of side chain PTM?
- Can change surface of conformation of protein
- Can create or block binding site for other proteins
- Useful as switched bc fast
*many modifications are regulated and reversible
What molecules mediate ALL PTM?
Enzymes
What are the main types of PTM?
- Phosphorylation
- Methylation
- Acetylation
- Glycosylation, Sumoylation, Ubiquitination
What if the role/effect of phosphorylation? Which AA can be phosphorylated?
- Major regulatory mechanism
- Phosphorylation can be required for specialized binding of specific domains
- Adding phosphoryl group changes the charge (neutral to negative) and size
Need hydroxyl groups → S, T, Y only
Which enzymes are involved in phosphorylation?
Kinases tranfer phosphate from ATP
Phosphatases remove phosphate
What are the different Kinase and Phosphatase families?
Kinase:
- Ser/Thr kinases
- Tyr kinases
- dual specificity (Ser/Thr and Tyr)
*Same for Phosphatase families
What molecules are used to study the role of phosphorylation in protein function?
Phosphomimic (S by D) If you want your molecule to be permanently in the phosphorylated state
Change phospho-serine (PO4 2-) for Aspartic acid (COO-)
De-phosphorylated (S by A) If you want to study the effect of your molecule being never phosphorylated
Serine → Alanine (same structure but no OH on Alanine)
Give an example of specialized binding to phosphorylated AA.
WD40 domain of Cdc4 interacts with Sic 1 CPD peptide which has been phosphorylated
p-Thr fits perfectly in pocket → negative p-Thr interacts with positive arginines (electrostatic intractions)
Which amino acid can be acetylated?
How does it occur?
Lysine (K)
Lysine acetyltransferases (KATs) and deacetylases (KDACs) recognize specific sequences, also Histone acetyltransferases
NH3+ at bottom of the chain → H-N-Acetyl group (Given by Acetyl CoA)
Acetyl = −C(=O)−CH 3
What is the effect of Acetylation of Lysine?
- Increase in size of the side chain
- Change in the charge (positive → neutral)
Regulates transcription:
Lys is positive → interacts with negative DNA → compact DNA → Heterochromatin (no transcription)
Acetylated Lysine → neutral → less interaction with DNA → Euchromatin
*ON-OFF activation and repression of gene expression
Which amino acids can be methylated?
Lysine (K) (can’t be methylated and acetylated at the same time, they are competing reactions) → mono, di, trimethylation
Arginine (R) → mono, Asymmetric di-, Symmetric dimethylation
*NO change in charges (keep their positive charge compared to acetylation)
Which enzymes are responsible for methylation?
What is the reaction?
Lysine methyltransferase (KMT) and Lysine demethylases (KDM) or RMT and RDM
Reaction changes the N-H into N-CH3
How do PTM (specifically methylation an d acetylation) change the binding capacities of a protein?
Provides new binding sites
methylation and acetylation → Important role in DNA repair mechanisms and Transcription
When in the process of protein synthesis can post-translational modification occur?
After the protein has been fully folded
What is the native state of a protein?
What is it determined by?
- Complete folded conformation
- State of minimal energy of that AA sequence, Thermodynamically favoured
Linear conformaiton → Native State → ∆G < 0 (Spontaneous) - Stabilized by hydrophobic contacts (exclusion of water)
- Some domains require ligand factor to be stable
Determined by primary sequence of AA
What is a folding intermediate?
- Has some 2ndary structure, but tertiary structure is incomplete
- Some hydrophobic side chains exposed instead of buried
- More of the polypeptide is flexible and disordered
Which side chain interactions are the strongest/more abundants?
Strength:
Disulfide > Ionic > Hydrophobic > Hydrogen > Van der Waals
Quantity:
Van der Waals / hydrogen / hydrophobic > ionic / disulfide
