Bio 301 Quiz 2

Peptides

Small condensation products of amino acids

How big are peptides in comparison to proteins

They are smaller (molecular weight < 10 kDa)

• Less than 50 amino acids

What reaction joins two amino acids together

Condensation reaction that results in a loss of water

Do peptides have secondary structure

Yes, some do have secondary structure

Peptide Bond

Bond between 2 amino acids

• Strong and stable - can last up to 7 years in ideal conditions

  • non-protein hydrolysis of proteins doesn’t happen often

    • Need a protein to break the bond because it’s so strong

Residues

Individual amino acids

What are the two ends of a peptide

The N-terminal (amino-terminal) and the C-terminal (carboxyl-terminal)

On what end does the numbering and naming start for a peptide

The N-terminal

How are the R groups usually shown in a peptide

They are usually sticking up and out and the rest is the backbone

True or False: Most proteins are made of multiple polypeptides

FALSE. Most are only made up of one polypeptide

What differentiates polypeptides

Their size, number and modifications

Average molecular weight of a polypeptide

110 g/mol - per amino acid

Prosthetic Groups

Can be attached to proteins to give them different properties

• eg. lipids, carbohydrates, phosphate groups, etc.

Examples of protein classes based off prosthetic groups

• Lipoproteins

• Glycoproteins

• Phosphoproteins

• Hemoproteins

• Flavoproteins

• Metalloproteins

What are the four levels of proteins structure

Primary

Secondary

Tertiary

Quaternary

Primary Structure

The linear sequence of amino acids in a polypeptide

Secondary Structure

The spatial arrangement of the main-chain atoms in a segment of a polypeptide chain

• ⍺-helices

• β-strands

Tertiary Structure

Globular arrangement of the secondary structure, side chains, and other prosthetic groups

What gives proteins their function

This is what calling a protein globular is referring to - in its functional state

Quaternary Structure

Arrangement of multiple proteins into complexes (oligomers)

What is the geometry of a peptide bond

It is essentially planar with 6 atoms (C_⍺, C, O, N H, and C_⍺) lying in a single plane

Why is a peptide bond planar

The bond has partial double-bond character because of resonance which prevents rotation around the bond (it is prohibited)

How was it discovered that the peptide bond is planar/has double-bond character

The C-N bond (the peptide bond - the one between two amino acids) was shorter than a single bond should be but longer than a normal double bond

Peptide Group

Atoms that lie in a single plane in a polypeptide

What does the partial double-bond character of the peptide bond cause

A limited range of conformations

Many phi and psi values are prohibited due to steric interference

What sterochem do the R groups have

They are always trans as this is more stable because it avoids steric hinderance that would be caused during protein folding if the groups were cis

⍺ helix

Positioning of the backbone, without regard to positioning of the side chains

What is the most common form of an ⍺ helix and why

Right-handed helix

• This has the R groups pointed out from the helixas there wouldn’t be much space for them on the inside

How many amino acids are involved in 1 turn of an ⍺-helix and how long is one complete turn

3.6 amino acids and the turn is 5.4 Å

What holds the ⍺-helix together

H-bonds between the carbonyl oxygen of one amino acid and the amide nitrogen of another

These occur every 3-4 amino acids

How are the chemical properties of the amino acids that are hydrogen bonded in an ⍺ helix related

They will have compatible properties

• Ex. A.A at 1 with a negative charge and A.A at 4 with a positive charge

True or False: ⍺-helices are only found in some polypeptides

TRUE. Not all polypeptide sequences adopt ⍺-helical structures

Which amino acids are associated with ⍺-helix formation

Alanine and Leucine which are both small, hydrophobic amino aicds

How does proline impact ⍺-helix formation

It acts as a helix breaker because rotation around the N-C bond is impossible

How does glycine impact ⍺-helix formation

It acts as an ⍺-helix breaker because the tiny R group supports too many other conformations - it’s TOO flexible

How can size and shape of amino acids impact the formation of an ⍺-helix

Amino acids that are too bulky will prevent proper formation of the ⍺-helix

What are the net dipoles in a polypeptide

The N-terminal is positively charged (NH3) and the C-terminal is negatively charged (COO)

How do the net dipoles impact the structure of the polypeptide

Negatively charged amino acids are often near the N-terminal (positively charged) and positively charged amino acids are often near the C-terminal (negatively charged)

What are β-sheets

They are made up of multiple β-strands

What holds together β-sheets

Hydrogen bonding between the amide and carbonyl groups of the peptide bond between opposite strands

What are the two orientations of β-sheets

Parallel and antiparallel

What determines if a β sheet is parallel or antiparallel

The directionality of the strands

Parallel sheets: Strands oriented in the same direction

Antiparallel sheets: Strands oriented in opposite directions

What is the distance between 2 amino acids in a β-sheet

3.5 Å

How far/close are amino acids in β-sheets compared to ⍺-helices

The amino acids in β-sheets are much more extended

Which are more favorable parallel or antiparallel β-sheets and why

Antiparallel sheets are more favorable compared to parallel sheets because parallel sheets require long loops to get the strands to go in the same direction

β-turns

Connect ends of 2 adjacent segments of an antiparallel β-sheet

• 180 degree turn

• Involves 4 residues

• H-bonds between the 1st and 4th amino acid

Which amino acids are usually found in β-turns

Glycine and proline (especially proline)

Ramachandran plots

Visualize all psi and phi angles

Test quality of 3D protein strucutres

Can predict the secondary structure based off the phi and psi angles

What are phi and psi angles

They are the dihedral angles associated with each amino acid in a polypeptide

Why can we use phi and psi angles to predict the secondary structure of a region of a protein

⍺-helices and β-sheets are repeating units so they have limited phi and psi angles

Tertiary Structure

Overall spatial arrangement of atoms in a protein

What stabilizes the tertiary structure of a protein

Numerous weak interactions between amino acid side chains

• Mostly hydrophobic and polar interactions

• Can also be stabilized by disulfide bonds

True or False: Interacting aminod acids need to be next to each other in the primary sequence

FALSE

2 Major Classes of Tertiary Structure

1. Fibrous

2. Globular intrinsically disordered

Structures and characteristics of fibrous proteins

⍺-helix, cross linked by disulfide bonds (coiled) - tough, insoluble, protective structures, of varying hardness and flexibility

β - conformation (stacks of β sheets on top of each other) - soft, flexible filaments

collagen triple helix - high tensile strength, without stretch

Collagen

fibrous protein found in connective tissue

What is the secondary structure of collagen

A left-handed, repeating tripeptide unit Gly-X-Y

X is usually Pro

Y is usually 4-Hydroxypro

• Proline allows for the really tight turn that makes these helices so tight

Higher order structure of collagen

Right-handed twisting of 3 separate polypeptides

• 3 left-handed helices come together to make the very rigid structure of collagen

Globular Proteins structure

Fold back on each other

More compact than fibrous proteins

Tend to be made up of more ⍺-helices than β-sheets as the helices are more compact

Order of compaction for ⍺-helices, β-sheets, and a native globular form (most to least compact)

1. Native globular form

2. ⍺-helix

3. β-sheet

True or False: Globular Proteins are insoluble in water

FALSE. They are water-soluble

Examples of globular proteins

Enzymes, transport proteins, motor proteins, regulatory proteins, immunoglobulins

Folds

Repeating motif (units) of secondary structure that makes up the tertiary structure

Folds can be described by the secondary structure that make them up

Can folds be indicative of protein function?

Yes. For example a β barrel could indicate that the protein is a porin/channel

Domain

Part of a polypeptide chain that is independently stable or could undergo movements as a single entity

• Domains may appear as distinct or be difficult to discern

• Small proteins usually have only one domain

• Also usually have their own functions

What connects domains

Linkers which can be very flexible

Intrinsically disordered proteins

Lack definable structure

Often lack hydrophobic cores (would give them secondary structure)

High densities of charged amino acids (Lys, Arg, Glu) and Pro

Facilitates a protein to interact with multiple binding partners

• Metal binding, inhibitors, signaling pathways

Why can intrinsically disordered proteins have multiple binding partners

On their own they do not have secondary structure but they can gain it when associated with their partners and then lose it again when they disassociate

Quaternary Structure

Formed by the assembly of individual polypeptides into a larger functional cluster

1 functional unit made of multiple polypeptides

Oligomer/Multimer

Multisubunit protein

ex. ⍺β protomer = 2 polypeptides

Protomer

Repeating structural unit

Renaturation

Process by which certain denatured globular proteins regain their native structure and biological activity

How did renaturation prove that all you need is the sequence

Anfinsen denatured them in urea and mercaptoethanol and then put them in another solution without that and found that they refolded in the exact same way

Model of Protein Folding

Local secondary structures fold first (ionic interactions play an important role)

Longer range interactions follow (especially hydrophobic effects)

Process continues until the entire polypeptide folds

Secondary Structure Model

Local, short-range interactions occur first, causing small segments of the polypeptide chain to form stable secondary structure

Molten Globule-hydrophobic interactions model

where hydrophobic side chains, which are typically found in the core of the protein, rapidly collapse inwards to avoid water, leading to a compact intermediate

• hydrophobic reactions are what really matters and what allows for everything else - don’t need really need ⍺ helix or β sheet

Nucleation-condensation model

Formation of secondary structure (local) and tertiary (long-range) occur simultaneously

• don’t need secondary to make tertiary because they happen all at once

Is protein folding random/why is it always the same

The free energy funnel. Proteins fold, unfold, and refold until they are at the lowest energy (thermodynamically favorable) conformation which is their native form

Proteostasis

Continual maintenance of the active set of cellular proteins required under a given set of conditions

How do chaperones work

Grab and hold certain regions and then release them when needed → sequential folding

What does misfolded β amyloid do

Promotes aggregation at newly exposed protein-protein interface

• Hyrdophobic regions are now exposed so they stack on top ot each other in ways they shouldn’t

How does protein misfolding lead to disease

Alzheimer = extracellular amyloid deposition by neurons

Parkinson = misfolded ⍺ synuclein aggregates into spherical filamentous masses called lewy bodies

Huntington - involves intracellular aggregation of huntingtin

How can we have more proteins than genes

Proteins can be spliced differently and then they can have different post-translational modifications

What can we use to isolate a mixture of proteins

Charge, size, affinity for a ligand, solubility, hydrophobicity

Ways to lyse a cell

1. Mechanical - sonication, blending, grinding with abrasives, agitation with glass beads

2. Chemical - enzymes and detergents

How can we use differential centrifugation to separate cellular contents

10 minutes would leave the nuclear fraction in the pellet

20 minutes leaves the mitochondrial fraction in the pellet

1 hour leaves the microsomal fraction in the pellet

Column Chromatography

Larger molecules elute first because they don’t have to go through the beads like the smaller molecule

How can aromatic amino acids be used to determine if an amino acid is present

They absorb UV light so that can be usd to see if they are present

Ion-exchange chromatography

Can use Anion-exchange resin (which is positive) or cation exchange-resin (which is negative). If you know the charge of your protein of interest you would put in the resin with the opposite charge so it traps that protein and everything else goes through

Affinity chromatography

Add something that will bind to the protein of interest or also modify the gene and add something that will bind to that (adding histidine tail that has imidazole which will bind to a metal)

Salting out

Add increasing amount of salt which compete to interact with water against the protein.

Proteins crash out at different concentration so this can be used to separate out a protein

Using dialysis to separate out proteins

Use membranes with different molecule weight cut offs to isolate the protein of interest from the samll molecules which can flow out

Used for purification of proteins

SDS page

Lighter proteins go farther down and has nothing to do with charge because they are all made negative by SDS

Can be used to find the molecular weight by comparing to standards

Downside to using many techniques

These can disrupt protein folding

• ex. transmembrane proteins like to associate with lipids and doing all these techniques could cause them to dissosciate and misfold as a result

2D electrophoresis

Taking whole lysate and seperating it by charge and then separating by size

• Proteins stop at where their net charge would be 0 along the spectrum of pH

• Looking to see if protein is expressed or not given certain conditions

• Look at what’s the difference since you are looking at ALL the proteins in the cell since you took the whole lysate

What can denature proteins

• Heat or cold

• pH extremes

• organic solvents

• chaotropic agents: urea and guanidinium hydrochloride

Percent unfolded

Increasing temperature and seeing when the proteins unfold to see if there’s a difference

What makes proteins more stable and how can this affect stability

They are more stable when the are bound to something so you can see if something is binding to something else by seeing if the Tm changes

Tm

Where the protein is 50% unfolded

Elution profile

Tells you which amino acids there are and how many by seeing at which pH they elute out

doesn’t tell you order

How to determine amino acid order

molecule binds to the beginning amino acid which can then be eluted and then does the same thing for the next one so you get the amino acids in order.

Starts getting inaccurate after like 20