Exam 2 Ch.4: Protein Structure and Function Flashcards
Enzymes
catalyze covalent bond breakage or formation
Structural Proteins
provide mechanical support to cells and tissues
Transport Proteins
carry small molecules or ions and transport them throughout the body
Motor Proteins
generate movement in cells and tissues
Storage Proteins
store amino acids or ions
Signal Proteins
carry extracellular signals from cell to cell
Receptor Proteins
detect signals and transmit them to the cells response machinery
Transcription Regulators
bind to DNA to switch genes on or off
Special-Purpose Proteins
highly variable
Disulfide bond
can form between two cysteine side chains
Primary Level of Protein Structure
Chain of amino acids
What dictates the overall 3D shape of a protein?
side chains
What stabilizes protein conformation?
- the sum of weak forces
-van der Waals, electrostatic attractions, hydrogen bonds,
Backbone to backbone bond
hydrogen bond
What governs specific folding of proteins?
- making the most energetically favorable bonds
- meaning proteins folds into the lowest free energy conformation
- folding is a spontaneous process and will release heat
Example of Spontaneous Protein Folding and Refolding
- Protein is exposed to high concentration of urea
- causes denatured protein
- remove urea
- protein refolds
Chaperone Proteins
- assist with protein folding
- spontaneous folding can be time-consuming so chaperones speed up protein folding rates
- come bind to partly fold chains
Benefit and Drawback of Isolation Chambers
- can help prevent aggregation of multiple proteins
- can isolate a single protein
- takes more energy
How do isolation chambers work?
- one polypeptide chain is sequestered by the chaperone
- chamber cap closes chaperone
- isolated polypeptide chain folds correctly
- correctly folded protein is released when cap dissociates
What stage is a protein done in the chaperone protein?
depends because proteins show incredible structural diversity
What are some basic functions of proteins?
transport, structure, signaling, catalyzing reactions
For a polypeptide chain:
What is the structure of its backbone?
Which end is the 1st amino acid? The last?
- they have an N-C-C backbone
- 1st amino acid is the N-terminus
- last amino acid is the C-terminus
What are the forces that govern a strict and specific folding pattern of a single protein?
- proteins folding into the lowest free energy conformation to make the most energetically favorable bonds
What bonds are responsible for protein folding? Where can these bonds occur?
- weak forces such as electrostatic attractions, hydrogen bonds, and van der Waals are responsible for protein folding
- they can occur backbone to backbone, backbone to side chain, side chain to side chain
Secondary Protein Structure
Alpha helix and beta sheet
What stabilizes alpha helices?
- stabilized by hydrogen bonding along the backbone
Structure of Alpha helices?
- amino and carboxyl groups of every 4th amino acid engage in hydrogen bonding
- r groups pointed outwards
- can be right or left handed
bonds from backbone to backbone
hydrogen bond between atoms of two peptide bonds
backbone to side chain
hydrogen bond between atoms of a peptide bond and an amino acid side chain
side chain to side chain
hydrogen bond between atoms of two amino acid side chains
Tertiary Protein Structure
- 3D arrangement of secondary structures
Why is it good that the R groups in alpha helices point outward?
- Because alpha helices are commonly found in membranes
- Hydrophobic R-groups point outward away from the core of the helix and towards hydrophobic membrane
- backbone is typically hydrophilic
What forms coiled- coils and why are they beneficial?
-2-7 alpha helices can form coiled- coils
- helices wrap around each other to minimizes exposure of hydrophobic amino acids side chains to aqueous environment
What holds together beta sheets?
- held together by hydrogen bonds between the strands (backbone of strand)
Direction of beta sheets and r groups?
R-groups poke out on alternating sides (pleated) (top-bottom-top-bottom…)
-can be parallel or antiparallel
Consequence of misfolding proteins?
- can lead to aberrant protein structures which build up and lead to cell death
- causes diseases like Alzheimers, Parkinson’s, Huntingtons
Example of misfolding causing disease?
In Alzheimers and Parkinsons disease, insoluble amyloid fibers are made from misfiled alpha-synuclein, which builds up to a toxic concentration and kills neurons
What are prions?
an infectious form of misfodled proteins
- Ex: mad-cow disease, chronic wasting disease, Creutzfeldt-Jakob disease
How do prions work?
can induce normal proteins of the same type to misfiled in to the same misfolded form
What would the differences in R-groups be between an alpha-helix in a cytoplasmic protein versus an alpha-helix in a transmembrane protein?
cytoplasmic protein: alpha helices in a coiled coil to minimize exposure of hydrophobic amino acid side chains to aqueous environment
transmembrane protein: hydrophobic r-groups point outward away from the core of the helix, backbone is usually hydrophilic
What forces form a coiled-coil?
2-7 Alpha helices that wrap around each other to minimize exposure of hydrophobic amino acids side chains to aqueous environment
What makes a Beta-sheet pleated? Parallel or anti-parallel?
pleated - r-groups poke out of alternating sides
Parallel vs antiparallel: n-terminus/ c-terminus going the same way vs not
Tertiary Structure
3D arrangement of secondary structure
Main types: fibrous and globular
- Globular: enzymes, etc
- Fibrous: structural/ strength
What is a protein domain?
any segment of a polypeptide chain that can fold independently into a compact stable structure
- associated with specific functions (DNA binding domain, catalytic activity, etc.
Effect of mutation in a domain?
mutation in a specific domain can affect the function of that domain
Protein Families
Families have proteins with similar structures, but over time have gained slight differences that give them slightly different functions
Quaternary Structure
- multiple subunits (optional)
Subunit
individual folded polypeptide chain
- dimer/trimer/tetratrimer
- Heterodimer vx homodimer
Homodimer
two proteins with the same amino acid sequence that bind together to form a function
Heterotetramer
- four individual subunit with differing amino acid sequences that work together to perform a function
What are the types of structures that subunits form?
- helices, rings
Actin fibers
helical structures made up of many individual actin proteins
Microtubules
long hollow tubes of the protein tubulin
Protein Cross-Linking
- what it does
- where it’s common
- example
- proteins can be stabilized by covalent cross linking
- very common in extracellular matrix proteins
- a type of cross linking is the formation of disulfide bond between cysteine amino acids
Elastin
an ECM protein that’s stabilized by covalent cross links
How do proteins work?
their properties/functions depend on physical interactions with other molecules
Ligands
a substance that forms a complex with (binds to) a specific protein
How do ligands form a complex?
-forms a complex thanks to the sum of its non-covalent interactions
- these interactions define is specificity, “Hand in glove” or “Lock in Key”
Structure of IgG antibodies?
are made up of 2 heavy chains, and 2 light chains
Antigen
- something that causes an immune response
(each antibody can recognize one specific antigen)
Antigen binding sites
-where antigens bind to an antibody
- antigens are bound at the antigen-binding sites at two ends of an IgG antibody
Antibody specificity
each antibody binds with a distinct antigen
Structure of an antibody?
- 2 heavy chain, 2 light chain
- constant domains: sequences constant from antibody to antibody
- variable domains: vary between antibodies; antigen binding domains vary between antibodies
- structure determines function
How do antibodies defend us against infection?
- antibodies cross-link antigens into aggregates (clump together)
- antibody-antigen aggregates are ingested by phagocytic cells
Uses of Antibodies in research?
1) molecule purification
2) protein visualization
Molecule Purification
- using antibodies to purify molecules
- immunoprecipitation
- immunoaffinity column chromatography
Protein visualization
once antibodies are bound to protein of interest with fluorescent tag, we can shine specific light and see the protein
Steps of molecule purification? Know both types
What are 3 ways that enzymes can catalyze a reactions?
(a) enzyme binds to two substrate molecules and orients them precisely to encourage a reaction to occur between them
(b) binding of substrate to enzyme rearranges electrons in the substrate, creating partial negative and positive charges that favor a reaction
(c) enzyme strains the bound substrate molecule, forcing it towards a transition state that favors a reaction
Vmax
maximum rate of a reaction
KM
the concentration of a substrate where an enzyme works at half its maximum speed
- small KM: strong binding (strong affinity)
- large KM: weak binding
Enzyme reaction rate
determined by substrate concentration and affinity
- measured by Vmax and KM
Therapeutics
- drugs mostly target enzymes
ex: Rifampin and Gleevac (Imatinib)
Coenzymes
small molecules that aid the function of enzymes
- ex: heme groups bound to hemoglobin (heme groups reversibly bind to oxygen gas through its iron atom) (without heme, hemoglobin can bind oxygen)
Why is it important to regulate protein activity?
- prevents waste
- some reactions are only beneficial at certain times and in certain cellular locations
Proteasome Parts
- regulatory cap (blocks core to stop proteasome from destroying stuff)
- catalytic core
- regulatory cap
1st Strategy to regulate protein activity
- balance proteins production (transcription/translation) and degradation
2nd Strategy to regulate protein activity
Control the location of the protein
3rd Strategy to regulate protein activity
Feedback inhibition
4th Strategy to regulate protein activity
Phosphorylation of the protein
Process of Feedback Inhibition
- the end product of a downstream reaction binds and inhibits an enzyme earlier in the pathway of the downstream reaction
- binds to a regulatory site on the enzyme
- this is a form of negative regulation (positive regulation also exist)
How does Feedback Inhibition work?
- works through an allosteric site
- enzymes have at least 2 sites: active site, allosteric site
- binding of inhibitor to allosteric site induces conformational change
- lowers affinity of active site for substrate
What are the advantages of feedback inhibition?
- Ex. synthesis of amino acids form aspartate
- very specific
- nearly instantaneous
- can be reversed as soon as concentrations of product decrease
Phosphorylation
- serine, threonine, and tyrosine amino acids side chains can become phosphorylated
- Phosphorylation can activate or inactivate a protein
- causes conformational change
Kinases vs Phosphatases
- phosphorylation is reversible
- Kinsases: phosphorylation
- Phosphatases: removes phosphate
- phosphate is usually transferred from ATP
How do protein modifications impact protein function?
Things impacted: activity, stability, binding partners and cellular location
- many proteins have multiple/many sites of covalent modification
Phosphorylation
on/off switch; also creates docking site for binding partners
homogenization
gently mechanical procedures where the plasma membrane of cells can be ruptured so that the cell contents are released
Types:
- break apart cells with high-frequency sound
- use a mild detergent to make holes in plasma membrane
- force cells through small hole using pressure
- shear cells between a close-fitting rotating plunger and the thick walls of a glass vessel
homogenate or extract
thick soup, contains large and small molecules form the cysts, such as enzymes, ribosomes, and metabolites, as well as all of the membrane-enclosed organelle
Differential Centrifugation
- repeated centrifugation at progressively higher speeds with fractionate cell homogenates into their components
- centrifugation separates cell competes on the basis of size and density. the larger and denser components experience the greatest centrifugal forces and move most rapidly. they sediment to form a pellet at the bottom of the tube, while smaller, less dense components remaining suspension above, a portion called the supernatant
Gel electrophoresis
when an electric field is applied to a solution containing protein molecules, the proteins will migrate in a direction and at a speed that reflects their size and net charge.
SDS polyacrylamide-gel electrophoresis (SDS-PAGE)
individual polypeptide chains forma complex with negatively charged molecules of sodium dodecyl sulfate (SDS) and therefore migrate as
Gel Electrophoresis - Western Blotting
- proteins from gel electrophoresis are transferred onto a membrane
- Antibodies are used to detect a specific protein of interest
Immunoprecipitation
- mixture of antibodies
- add specific anti-A antibodies
- collect aggregate of A molecules and anti-A antibodies by centrifugation
Immunoaffinity Column Chromatography
- bead coated with anti-A antibodies
- column packed with these beads
- mixture of molecules flows through column
- flow through is discarded
- elute antigen A from beads
- collect pure antigen a
Acetylation
on lysine amino acid
- impacts protein: DNA interaction of histones
Ubiquitination
targets proteins for degradation
How can antibodies be used in research?
- Molecule Purification
->Immunoprecipitation
->Immunoaffinity column chromatography - Protein Visualization
What is Molecule Purification and what types are there?
- uses antibodies to purify molecules
- Immunoprecipitation
- Protein Visualization
What is Immunoprecipitation
1) mixture of molecules,
2) add specific anti-A antibodies
3) collect aggregate of A molecules
and anti-A molecules by centrifugation
What is immunoaffinity column chromatography?
1) mixture of molecule
2) goes into column packed with beads coated with anti-A antibodies
3) discard flow through
4) elute antigen A from beads
5) collect pure antigen A
