Structure Determines Function Flashcards
Describe the central dogma of biology
DNA polymerase replicates DNA
RNA polymerase makes sense RNA from DNA
RNA made into mRNA after PTM
mRNA made into proteins in ribosome
Where are proteins synthesized?
Endoplasmic reticulum
Trafficked to Golgi
What are the two secretory pathways for proteins?
Constitutive secretory pathway
Regulated secretory pathway
What happens in the constitutive secretory pathway?
Aka default pathway = does not require signal for cargo to enter this pathway
Transport soluble proteins or plasma membrane proteins
Uses transport vesicles
What happens in the regulated secretory pathway?
Found mainly in specialized cells that release hormones or NTs
Uses secretory vesicles
What is the difference between transport vesicles and secretory vesicles?
Transport = fuse with plasma membrane and release their contents
Secretory = blocked from fusing with plasma membrane without proper signal
Extracellular ligand binds cellular receptors = causing intracellular signal to release block on secretory vesicle, then it fuses with plasma membrane to release cargo
What is the budding event?
Start of forming vesicle
Explain the curvature of a vesicle
Circular part = positive
Stem = negative
Straight parts = zero
What is the difference in charge of a vesicle?***
Cytoplasmic side is negatively charged compared to exoplasmic side
How is vesicle curvature made by lipid properties?
Positive curvature = convex membrane, lipid with large-head group section and small tails
Negative curvature = concave membrane lipids with small head-group section and large tails
What do membrane proteins do when they capture light?
Convert light energy into electric and chemcial potential across the membrane
Example = synthesizing ATP
What is the role of membrane proteins in signalling?
Send signals across membrane into and out of cell
What do membrane proteins control in terms of transport?
Control traffic of metabolites across membrane
Allow DNA to enter cells
Transport entire proteins across cell membrane
How do membrane proteins protect the cell?
Pump undesired molecules out of cell
Function as vacuum cleaner = remove unwanted “polluting hydrophobic molecules from the bilayer”
Name the two types of membrane proteins
Integral membrane proteins (IMPs)
Peripheral membrane proteins
What two architectures are integral membrane proteins limited to?
Alpha helical bundle = 90%
Beta-barral = 10%
How are peripheral membrane proteins bound to membrane?
Loosely bound or associated to IMPs
Can also be anchored to lipid bilayer = GPI-linked of by palmitoylation
What are the two domains of IMPs?
Extracellular domain
Transmembrane domain
Describe the core and loops in the globular-like domain of IMPs
Core = non-polar, densely packed, conserved and contains few functional polar residues
Loops = involved in ligand binding and signal transduction
Describe alpha-helical IMPs with single TM segment and its function
Called a bitopic transmembrane protein = function as recognition and/or adhesion molecules, receptors of growth factor-like messengers
What is the function of bitopic TM proteins’ cytoplasmic region?
Passes signal into the cell by binding soluble elements or cytosekeltal proteins
Give two example of a-helical IMPs with multiple TMS
G-protein coupled receptors = 7 different segments
K+ channel
Describe a-helical IMPs with multiple TMS and their function
Polytopic proteins = usually function as receptors or transporters
What is usually found at the end of integral membrane proteins?
Signalling peptides
What are the most common TMS amino acids and why?
TMS are overall NON-POLAR
Leucine
Isoleucine
Valine
Phenylalanine
Where are polar residues found in TMS?
Buried in core, masked by other polar groups and/or water molecules
What is the difference between globular proteins and TMS in polarity?
TMS core is more polar than surface
Globular protein core is less polar than surface
In TMS, which residues are found in alpha helices and which are found in beta-sheets?
Small residues in closely packed a-helices = Gly, Ala & Ser
b-branched residues = Leu, Val, Ille, Phe
How is the length of TMS determined?
Matches width of HYDROPHOBIC part of membrane
What is the hydropathy index for?
As long as we know the sequence, can then say which amino acids are most hydrophobic
Hydropathy index for any segment = average of hydrophobicity values for its residues
Used to predict where and how many hydrophobic TMS there are
Where are Gly and Pro preferred locations in TM a-helices?
Near kinks
What is special about Pro?
Helix breaker or hinge of motion
What is the role of aromatic amino acids and where are they found in TMS?
Aromatic ‘belt’ anchors proteins to membrane
These amino acids have large, hydrophobic aromatic side chains that interact with the hydrophobic core of the membrane. The aromatic belt acts as an anchor, stabilizing the protein’s insertion into the membrane. This configuration helps the protein to maintain its position and ensures that the hydrophobic transmembrane segments are properly shielded from the aqueous environment, facilitating the protein’s functional role in the membrane.
What do Arg and Lys do in TMS?
They are positively charged, so interact with negatively charged lipid head groups of cytoplasmic memrbane
Why are some TMS distorted and how are they compensated?
Distorted because they exceed the thickness of bilayer
Compensated by their tertiary structure
What happens to medium and long TM helices?
Medium = tilted at angle
Long = helix-distorting kink (Proline)
Name the two functional roles of distortion?
Allow greater proximity between helices in the membrane
Create binding sites
What two numbers characterize beta-sheet integral membrane proteins?***
Strand number = antiparallel beta sheets
Shear number = total shift in strand registry between first and last strand
This determines hydrophobic packing arrangement and diameter of the barrel
Example of beta-sheet integral membrane protein
Bacterial porins
3 properties of bacterial porins
Non-selective channel in outer membrane
Some are toxins that can form pores in host’s cell memebrane
Some are attachment sites for phage and bacterial toxins
How are TMS adapted for better packing?
Have ridges and grooves = created by different amino acids/MOTIFS
Then tilted at 20 degrees
What do PTMs do and where do they occur?
PTMs mainly occur in Golgi
Increase functional diversity of proteins by covalent additional of functional groups etc
What PTM changes physical-chemical properties?
Glycosylation = increases stability and water solubility
What PTM regulates activity of protein?
Hormone-induced phosphorylation = turns activity of many enzymes on and off
What PTM affects protein transport?
Acylation serves as membrane anchor
What PTM regulates half-life of protein?
Ubiquitination tags proteins for proteolysis
Describe the steps in insulin PTM
- Signal peptide removal
- Disulphide bond formation
- C-peptide cleavage
Which amino acids do N-linked and O-linked glycosylation occur on?
N-linked glycans attached to Asparagine or Arginine side chains
O-linked glycans mainly attached to Ser or Thr side chains
Where does N-linked glycosylation take place and what is its function?
In the ER = N-glycosylation directs INITIAL steps of protein folding and its quality control
All newly synthesized glycoprotein to interact with lectin-based chaperone system in ER
Where does O-linked glycosylation take place, and what is its function?
Primarily takes place in Golgi
Accelerates proteins degradation and decreases protein stability
Regulating activities such as aggregation and phase separation
How is glycosylation important in the immune response?
Effective immune response depends on successful activation and maturation of dendritic cells
Abnormally glycosylated protein antigens impair the function of dendritic cells = allowing cells to evade the host’s immune response
Role of phosphorylation and enzymes involved
Kinase and phosphatase
Activate, inactivate or change binding affinities = phosphorylation-induced conformational change
Role of ubiquitylation and enzymes involved
Ubiquitin ligase and deubiquitinases
Covalent bond between C-terminal of ubiquitin (proteint) and lysine residue
Marked for degradation
What is the role of protein metalation?
Metal cation addition = bound directly or via prosthetic group
Many roles = stabilization, ligand binding, catalysis, electron transport
How are metals or metal clusters inserted into polypeptides?
Either transferred from a pool of free metal ions
OR
Via delivery by metallocharperones
At what stage are metals added to polypeptides, and what can removal of metal results in?
Metal can be inserted at unfolded state, intermediate state or native folded state
Removal from protein’s native state can cause unfolding/misfolding = leading to disease
Or can result in a foled APO conformation with vacant metal binding site
Apo state = metal free state
Name 3 methods of detecting PTMs
Western Blotting
Mass spectrometry
Immunofluorescence
How does western blotting detect PTMs?
Different proteins separated from cell according to MW in the gel
Transferred to membrane for blotting with 1 & 2 Ab for the selected PTMs
Advantages and disadvantages of western blotting
Able to investigate endogenous alterations of PTMs
No specific tools required
Specific Ab against target protein required
No site specificity
False negatives possible = due to PTM modifications may block Ab binding site of target protein
How does mass spectrometry detect PTMs?
Digest protein lysate of interest, by specific protease (trypsin)
Enrich the specific PTM and analyse
After collecting data = comupatational algorithms are employed to identify peptides and proteins
Used to detect PTMs substrates and map PTM sites
Advantages and disadvantages of mass spectrometry
Can detect large numbers of modified proteins with high sensitivity and specificity
Time-consuming and highly PTM specific
How does immunofluorescence detect PTMs?
Useful to investigate global and spatial changes in PTM profiles in tissues or cells
Advantages and disadvantages of immunofluorescence
Used to examine localization of proteins throughout the cell
Can determine localization of specific forms of proteins in response to different cell conditions
Not available to identify a target specific PTM
