Topic 3: Proteins Flashcards
What are proteins?
Functional unit of the cell
Information for function and structure is encoded
How are proteins polymers of amino acids?
Twenty different amino acids are used in protein synthesis
Each protein contains these amino acids in varying proportions
All amino acids have the same basic structure but are distinguished by their side-chains (R-group)
Different amino acids are classified according to their R groups (non-polar, polar uncharged, polar charged acidic (-), polar charged basic (+))
The alpha carbon is asymmetric meaning there are two stereoisomeric forms called D- and L-amino acids
Only L-isomers exist in proteins (in nature)
What is the process of evaluating hydrophobicity?
Water is a polar molecule because it contains a covalent bond where the electrons are shared unequally resulting in a dipole on the atoms
Water is the solvent of life
Oxygen is highly electronegative (high affinity for electrons)
Oxygen and hydrogen have permanent dipoles
What is cysteine?
Can form disulphide bonds (type of covalent bonds) between interacting cysteines
What is tyrosine?
Can be post-translationally modified by phosphorylation (as can Thr and Ser)
How do peptide bonds polymerize proteins?
Chain of amino acids has an intrinsic directionality with the N terminus translated first
Peptide bonds are formed in translation (covalent linkage between a.a.)
In a polymer of L-amino acids the side chains alternate in either side of the protein backbone
Peptide bonds has partial double bond character, therefore peptide is planar (no relation which restricts number of theoretical shapes)
What are primary structures?
Primary structure dictates folding
Folding dictates function
Bonds stabilize the different levels of protein structure
What are secondary structures?
H bonding between a.a. backbones (determined by primary sequence)
Proteins fold spontaneously into lowest energy conformation
Shape maximizes favourable interactions between R groups
What are tertiary structures?
Disulphide bridges, H bonds, van der Waals interaction, hydrophobic interactions and ionic interactions
Forces that drive folding: hydrophilic interactions of non-polar R groups
Forces that stabilize folding: R group interactions
What is the relationship between folding and function?
Folding is driven by hydrophobic associations
Non polar amino acids do not form stable associations with water, therefore it is more favourable for them to aggregate
Non-polar amino acids aggregate in the middle of a folded protein
What was the Ribonuclease Denaturation/Renaturation Experiment?
Used ribonuclease enzyme and denatured it with urea (disrupts stabilizing interaction) = loss of enzymatic activity
Washed out urea to permit refolding = regained enzymatic activity
Showed: primary structures are sufficient to allow proper folding and function
What is sickle-cell anemia?
Shows the importance of primary sequence to folding
Single base pair mutation causes hemoglobin to crystallize in the red blood cells and cause the shape of the cell to sickle
This can impede flow through the capillaries as well as limit oxygen carrying capacity
Caused by a single amino acid change: Glu (-) —> Val (np) in hemoglobin
How is folding spontaneous?
Most protein folding is spontaneous, cotranslational, and sequential
Most proteins cannot be unfolded easily and refolded
How do proteins contain multiple functional domains?
Different regions of secondary structure can form different functional regions, or domains, within the protein
Domains: modules of tertiary structure, unique folded and functional regions of a single protein
What factors affect folding pattern and protein function?
pH: affects stability of ionic bonds
temperature: breaks all bonds
urea: disrupts ionic associations
beta mercaptoethanol: disrupts signal sequence
organic solvents: disrupt hydrophobic association
How do chaperones help proteins fold?
Assist in allowing a protein to achieve its functional conformation, or prevent misfolding or target misfolded proteins or degradation
Assisted self-assembly
Chaperones often shield hydrophobic stretches until folding can complete
Example: heat shock proteins (Hsp), temperature causes unfolding therefore Hsp helps refold, also used in normal conditions, broader role
What are some functions of chaperones other than help proteins fold?
Assembly of multi-unit proteins: assist orientation of subunits
Transport of unfolded polypeptides to specific organelles, e.g. mitochondrial protein import
Target misfolded proteins for degradation by proteasome
Prevents misfolded proteins from aggregating
What is the processing of proproteins?
Some proteins are produced in an inactive form that require cleavage for action (e.g. caspases)
Many proteins are synthesized in an inactive form to allow rapid response to a stimulus
What are chemical modifications?
Covalent attachment of chemical groups can regulate protein function
Can be: phosphorylation (most common), acetylation, methylation, glycosylation, etc.
What is complex formation?
Many proteins act in a complex and must associate with other members of the polypeptide
E.g. CDK/cyclin complex of cell cycle, when cyclin is present the complex can be functional, all steps in protein formation and function are highly regulated
How are proteins sorted in localization and import?
There are specific amino acid sequences that target proteins for different destinations within the cell
What makes an organelle unique is the type of protein, each organelle has a unique collection of proteins
Therefore proteins must contain sorting signal and mechanisms to deliver proteins to destinations
How do different types of signals exist in protein localization and import?
All translation behind in the cytoplasm on free ribosomes
A continuous stretch of amino acid sequence, typically 15-60 amino acids long
What are the three mechanisms for moving proteins amongst compartments?
Nuclear Import: soluble, folded proteins are made in the cytoplasm and transported across NPC —> fully folded
Protein Translocators at ER and mitochondria: transport of unfolded proteins across membrane through narrow translocators
Transport Vesicles: proteins made at RER are moved by organelles of endomembrane system using vesicles
What is cotranslational import?
At RER
Some proteins are moved to their destination during translation, in particular, any protein destined for which the endomembrane system will be translated at the ER on bound ribosomes
How do you distinguish between proteins destined for ER vs. cytosol?
Signal sequence (SS) ~15-30 (+) charged amino acids followed by a hydrophobic stretch
SS directs nascent protein/protein complex in ER
What is the signal recognition particle (SRP)?
SS is read by SRP
Recognizes the SS and directs the nascent polypeptide to the ER where there is an SRP
SRP in cytoplasm binds to SS and pauses translation
SRP-SS-ribosome complex moves to RER to dock and complete translation
What is translocation?
Signal sequence cleaved in ER lumen by signal peptidase
ATP dependent movement of peptide into lumen of ER
Translation completes into the lumen of the ER
What is integral membrane protein insertion?
Similar process of protein synthesis as generation of soluble ER proteins
The completed protein remains embedded in the ER membrane
Anchoring into the ER membrane is aided by at least one alpha-helical transmembrane segment (20-30 hydrophobic amino acids)
Proteins can cross the membrane once or many times
Two mechanisms proposed for anchoring proteins into the ER membrane
What are two mechanisms of cotranslational import?
Proteins with a SS and a STOP transfer sequence
Proteins with an internal START transfer sequence
What is stop transfer insertion?
Stop transfer sequences causes TMD to be related into membrane
Translation completes in C term in cytoplasm
What is start transfer insertion?
Translation begins in cytoplasm, continues until START sequence directs it to RER
What are multipass proteins?
Some integral membrane proteins span the bilateral multiple times
This is accomplished by alternating START and STOP transfer sequences
What is mitochondrial protein import?
Mitochondria can synthesize ~5% of their own proteins but the remaining proteins are derived from nuclear DNA and need to get this organelle
Nuclear encoded mitochondrial proteins must be translated in cytoplasm, imported into mitochondria
What is the localization signal in the mitochondrial protein import?
Transmit sequence
Alpha helix with alternating hydrophobic and hydrophilic amino acids at N terminus
What is import into the matrix of the mitochondrion?
To get into the matrix of the mitochondrion, the protein must pass through both the inner and outer mitochondrial membranes
This requires the two translocation complexes: TOM and TIM complexes
Protein is unfolded to cross TOM and TIM complex, refolds in matrix, refolding is facilitated by matrix chaperones
How are chaperones crucial to ensuring mitochondrial protein import?
Nascent polypeptides with a transit sequence are bound by Hsp70 in the cytoplasm (ushers protein into mitochondria to cytoplasm)
Where does the energy for transport come from?
As part of cellular respiration there is a voltage difference across the MIM
Established by electron transport chain
This membrane potential is used to drive protein translocation along with ATP hydrolysis
Membrane potential provides energy for import
