BICD 110 - Quiz 1

Chemical bonds define

1. the 3D structures of macromolecules

2. interactions between macromolecules

Ranking of chemical bonds (strongest to weakest)

1. covalent bonds

2. hydrogen bonds

3. Van der Waals

Covalent bonds

bonds where electrons are shared between atoms

Stereoisomers

two molecules with identical atom connectivity but different spatial arrangments

Hydrogen bonds

bonds formed by the interaction of a hydrogen atom with a partial positive charge with unpaired electrons from another atom

Van der Waals interactions

occurs when any two atoms approach each other and exert a weak attractive force

How to form covalent bonds

dehydration reactions

How to break covalent bonds

hydrolysis reactions

Additive Property of Weak Interactions

the cumulative strength of multiple weak interactions provides significant stability to complex molecular structures

Molecular complementarity

the structural/chemical fit between two molecules

Induced Fit

binding of one molecule changes the conformation of the other, increasing molecular complementarity

Hydrophobic Effect

water pushes hydrophobic molecules together to minimize the cage surface area

Protein Hierarchical structure

1. primary structure

2. secondary structure

3. tertiary structure

4. quaternary structure

5. supramolecular complex

Primary Structure

linear chain of amino acids

Secondary Structure

localized foldings: alpha helices and beta sheets

Tertiary Structure

3D structure of a single polypeptide

Quaternary Structure

association between multiple polypeptides

Supramolecular Complexes

large complexes composed of tens to hundreds of subunits

“Oil Drop” Model of Protein Folding

hydrophobic resides cluster together in the protein core due to the hydrophobic effect, while charged and uncharged polar chains are on the protein surface

Protein structure is dynamic and content dependent

a protein’s structure can undergo a range of conformational changes that are frequently reversible

Phosphorylation

most abundant post-translational protein modification catalyzed by kinases and phosphatases

• serine, threonine, and tyrosine are the major eukaryotic phosphorylation sites

Intrinsically Disordered Regions (IDRs)

unstructured domains of proteins

Liquid-Liquid Phase Separation (LLPS)

proteins with large disordered regions separate themselves into discrete membraneless biocondensates

What leads to LLPS?

many short-lived interactions between regions of IDRs

Major protein classes

• structural - determine cell shape and assist intracellular trafficking

• scaffold - bring proteins together

• enzymes - catalyze biochemical rxns

• membrane transport - move ions and molecules across cellular membranes

• regulatory - signals, sensors, and switches to control cellular activity

• motor - move proteins, organelles, etc.

X-ray crystallography

gold standard for determining protein structures

Cryo-EM

uses particle averaging of purified proteins to computationally determine high-resolution structures

• can be used to determine the structures of large protein complexes

• can potentially solve the structure of proteins in a range of conformations

Cryo-ET

• captures high-resolution image of rotated in vivo samples

• allows determination of protein structure in situ

Considerations for choosing a model system

• What biological process do you want to study?

• What is the phylogenetic position of the species?

• What experimental approaches do you want to use?

• How tractable is the species?

Fractionation

method for protein separation from a complex sample

Key steps of Fractionation

• Prepare starting material

• Apply centrifugal force to separate cellular components

Sucrose Gradients

can be used to better separate smaller particles - larger particles will travel farther into the gradient than smaller particles

Western Blot

used to visualize protein levels within a sample

Key steps of Western Blot

1. Proteins in a sample are separated by size in a gel

2. Proteins are transferred from the gel to a membrane that can be probed

3. The membrane is probed using antibodies that recognize your proteins of interest

Western Blot Step 1

1. Cells are lysed

2. Proteins are denatured

3. Samples are loaded into a well in the gel and an electric current is applied

4. Proteins are separated by size on the gel

Western Blot Step 2

proteins are transferred to a membrane using an applied current

Western Blot Step 3

1. A primary antibody is applied to the membrane, binding the protein of interest

2. A secondary antibody is applied, which recognizes and binds to the Fc region of the primary antibody

3. Because the secondary antibody is covalently linked to an enzyme or fluorophore that produces light, its presence can be detected

Mass spectrometry

can define the components of complex protein mixtures by breaking up proteins into smaller fragments, which are sent into a detector that measures the mass and charge of the fragment

Brightfield Microscopy

imaging biological samples using white light

• can be used on fixed or living samples

• provides low contrast

Electron Microscopy

used to visualize samples at a nanometer resolution

Magnification vs Resolution

Magnification: the increase in apparent size of an object

Resolution: the ability to distinguish between two objects that are very close together

Transmission Electron Microscopy (TEM)

1. Sampling

2. Samples are chemically crosslinked (fixed) and stained with heavy metals to provide contrast

3. Ultrathin sections of the sample are cut using a diamond knife

4. An electron beam is focused onto the sample and electrons that pass through the sample onto a detector show up as light regions on the image

Fluorescence microscopy

used to visualize specific features within biological samples

Fluorochromes

chemical compounds that fluoresce = absorb light of a specific wavelength and emit light at a longer wavelength

Immunofluorescence

using antibodies to visualize proteins

Steps of Immunofluorescence

1. Chemically fix and permeabilize the sample

2. Incubate the sample with a primary antibody

3. Incubate the sample with a fluorochrome-conjugated secondary antibody

4. Image sample on a fluorescence microscope

Fluorescence microscopes

are engineered to collect specific wavelengths

• excitation light is reflected by the dichroic mirror and emitted light passes through the mirror and is collected on the detector

Multiple proteins can be visualized at the same time by co-staining

• primary antibodies must be from different animal species

• secondary antibodies must be conjugated to fluorochromes with different excitation/emission spectra

Fluorophores

can be used to monitor dynamic protein behavior by absorbing light of a given wavelength and emitting light of a longer wavelength

GFP

first protein to have fluorescent activity entirely on its own

• can be genetically encoded

Lipid bilayers are essential permeability barriers

essential to compartmentalize the cellular components and separate the cell from its environment

Rank molecule types according to the ability to freely diffuse across a plasma membrane

1. hydrophobic molecules

2. small polar molecules

3. large polar molecules

4. ions

Endosymbiont hypothesis

mitochondria and chloroplasts arose from engulfed prokaryotes

Phospholipids

structural subunit of phospholipid bilayers

Phospholipid structure

• head group is composed of the phosphate and polar group and is hydrophilic

• fatty acid chains are attached to a carboxyl group and are hydrophobic

Phospholipids self assemble into one of three specific structures

• bilayers

• micelles

• liposomes

Lipid composition impacts

• bilayer thickness

• membrane curvature

• layer fluidity

Phosphoglycerides

the most abundant class of phospholipids in most membranes

Bond saturation is an important determinant of membrane properties

cis C=C bonds introduce a rigid kink that prevents tight packing in a membrane layer

Temperature alters the dynamics of lipid bilayers

increases in temperature increase the disorder of the tails, leading to less packing and increased fluidity

Sterols impact membrane fluidity

@ low temperature: sterols increase fluidity by preventing tight packing

@ high temperature: sterols decrease fluidity (due to rigid structure)

What drives membrane self-assembly

hydrophobic effect and Van der Waals interactions between fatty acyl chains

Features that decrease transition temperature of lipids

• unsaturated bonds

• tail kinks or packing defects

• shorter acyl chains

Features that increase transition temperature of lipids

• saturated bonds (straight tails = tighter packing)

• longer acyl chains (more interactions between tails)

Fluid Mosaic Model

states that lipid bilayers behave similar to two-dimensional fluids as phospholipids can move rapidly within a membrane face

Fluorescence recovery after photobleaching (FRAP)

widely used technique that can measure diffusion of lipids or proteins within a membrane

Steps of FRAP

1. Label protein/phospholipid of interest (FP, covalently linked tags)

2. photobleach a small region of the signal

3. mesure the fluorescence recovery after photobleaching

4. quantitative analysis to determine rates of recovery

Transmembrane alpha-helices

typically 20-25aa long and composed of hydrophobic amino acids that are stabilized by Van der Waals interactions with lipid tails

beta-barrel-containing transmembrane proteins

modified beta-sheet that has hydrophobic resides on the exterior and hydrophilic ones on the interior

Monotypic membrane proteins

have one alpha helix buried in only one face of the membrane

Acylation

the covalent attachment of a fatty acyl group to an N-terminal glycine residue

Prenylation

the covalent attachment of a 15-carbon farnesyl or 20 carbon geranylgeranyl group to a C-terminal cysteine residue

GPI anchors

the covalent attachment of phosphatidylinositol to several sugar resides and the C-terminus of the proteins

The orientation of a protein in a lipid bilayer is very important

as membranes bud and fuse, the membrane faces are conserved

Functions of rER

• protein insertion into membranes

• protein folding

• protein quality control

Pulse-Chase Experiments (strategy)

label newly synthesized proteins and track how they move through the cell

Steps of Pulse-Chase Experiment

1. Pulse - briefly expose cells to a radioactive amino acid

2. Wash - remove unincorporated radioactive aas

3. Chase - wait a certain amount of time and then fix the cells

4. Image - determine localization of radioactive proteins via TEM

Pulse-Chase Experiment (Results)

identified rER as the start of the secretory pathway

Pulse-chase experiments with purified ER membranes (goals)

to test whether secretory proteins are imported into the ER lumen

Pulse-chase experiments with purified ER membranes (steps)

1. incubate the sample for a brief time with radiolabeled amino acids

2. homogenize cells, forming microsomes

3. isolate microsomes with bound ribosomes (by centrifugation)

4. treat purified microsomes with protease

   1. (+) detergent: dissolves ER membrane, proteins not protected from digestion

   2. (-) detergent: proteins in ER membrane are protected from digestion

Pulse-chase experiments with purified ER membranes (conclusions)

newly synthesized proteins are contained inside microsomes

Signal Sequence Hypothesis

proteins encode a signal sequence that targets them to the rER

Three components for rER targeting

• signal sequence

• SRP

• SRP receptor

Translation and translocation occur simultaneously (experiment)

1. if microsomes are added after protein synthesis, protein is not internalized in microsomes and digested by added protease

2. if microsomes are added before protein synthesis, protein internalized in microsomes, protected from digestion and the signal sequence is removed

Cotranslational Translocation (Steps)

1. N-terminal SS emerges first from the ribosome during nascent protein synthesis

2. SRP binds both the SS and ribosome, arresting protein synthesis

3. SRP-nascent polypeptide chain-ribosome complex binds to the SRP receptor in the ER membrane and SRP and SRP receptor each bind one GTP

4. Transfer of the polypeptide-ribosome to the translocon (Sec61)

   1. SS transferred to a hydrophobic binding site next to the central pore

   2. SRP and SRP receptor hydrolyze their bound GTP

   3. Protein synthesis resumes

5. The polypeptide elongates, providing the force to move the polypeptide through the translocon and the SS is cleaved by signal peptidase

6. Growing peptide chain contines

7. Translation comples at mRNA stop codon - ribosome is released

8. The rest of the nascent protein is drawn into the ER and folds into its native conformation, as the translocon closes

Type I TM protein

single-spanning membrane with a cleavable signal sequence

Inserting Type I

1. SRP-mediated targeting to the SRP receptor

2. Transfer of the signal sequence to Sec61

3. Translation resumes and SS cleaved

4. A stop-transfer anchor sequence (STAS) is recognized by Sec61, which releases the alpha-helix into the membrane

Type II TM protein

single-pass membrane protein (internal SA sequence, N-terminus in cytosol)

Inserting Type II

1. SRP binds the internal SA sequence and interacts with the SRP receptor

2. Positively charged amino acids on N-terminal side of SA sequence orient nascent polypeptide chain in the translocon with N-terminus in the cytosol

3. Chain elongation extrudes remainder of nascent protein into the ER lumen

4. Internal SA sequence moves laterally through a hydrophobic cleft between translocon subunits to anchor protein and protein synthesis completes

Type III TM protein

single-pass membrane protein (internal SA sequence, C-terminus in cytosol)

Inserting Type III

Insertion is similar to that of Type II except the positively charged residues on C-terminal side of the SA sequence, so elongation is completed in the cytosol

Type IV TM Protein

multipass proteins

Inserting Type IV

• The first topogenic segment utilizes the SRP/SRP-dependent pathways (like Type II/III proteins)

• The remaining transmembrane segments do not engage with SRP

• Can be oriented with N-terminus in either cytosol or lumen

Hydropathy profiles

can be used to predict transmembrane regions

Tail-anchored proteins

• no N-terminal signal sequence

• hydrophobic C-terminus (not available for membrane insertion until protein synthesis is complete)

Inserting Tail-Anchored Proteins

1. Sgt2, Get4, and Get5 - sequester nascent protein tail anchor sequence before transferring it to dimeric Get3-ATP

2. Get3-ATP-nascent protein complex docks onto the ER membrane via interactions with dimeric Get1/Get2 receptor

3. Get3 ATP hydrolysis triggers release of the tail anchored protein and associated release into the ER membrane, facilitated by Get1/Get2

4. Get3 releases ADP, releasing it from Get1/Get2

GPI-anchored proteins

a variation on Type I TM proteins

Inserting GPI-anchored proteins

1. Protein is targeted and inserted like a Type I TM protein

2. GPI transamidase cleaves the precursor protein near the stop-transfer anchor sequence and covalently link the C-terminus to the GPI anchor

Post-translational modifications in the ER lumen

• specific proteolytic cleavages

• disulfide bond formation

• glycosylation - covalent attachment of carbohydrates

• protein folding and multimeric assembly

Disulfide bonds

stabilize the tertiary and quaternary structures of proteins