Topic 3 Proteins Student W19 PDF

Preview

Summary

Download Topic 3 Proteins Student W19 PDF

Description

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

Biology 201

Dr. Torah Kachur

Topic III – Proteins Introduction: Proteins are the workhorses of the cell and actually do all the functions that a cell requires. They are polymers of amino acids that fold into specific 3D shapes that allow them to have specific functions. However, their functions must be regulated in both time and space. We will look at how proteins fold, how they are formed and also how they can be localized and regulated.

Objectives: ✓ Define: amino acid, peptide bond, stereoisomer, nonpolar, polar, hydrophilic, hydrophobic,

✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

disulfide bridge, primary structure, alpha helix, beta pleated sheet, hydrophobic interaction, chaperone, transcription, translation, aminoacyl-tRNA-synthetase, free ribosomes, bound ribosomes, proprotein, translocation, signal sequence, vesicle, SRP, Signal peptidase, TIM, TOM, Brownian motion Be able to identify the chemical nature of amino acids based on their structure Be able to predict the severity of an amino acid change on protein folding Describe each stage of protein folding, the forces that drive it and the bonds that stabilize it Outline the logic of the denaturation/renaturation experiment Explain why sickle cell anemia is a good example of the relationship between primary structure and function Explain the statement “primary structure determines the function of a protein” Explain what is meant by the term ‘functional domain’ Describe the function of a chaperone Outline the different types of localization signals Draw or describe the process of ER localization Explain the difference between cotranslational and posttranslational protein import Outline the import strategies of the different organelles in the cell Be able to determine where a protein would be localized given a set of localization signals Explain how proteins are inserted into the plasma membrane and where this insertion occurs Draw or describe the signals required for any type of membrane protein arrangement Explain why mitochondrial proteins must be imported Give one example of a protein that uses each of the described translocation mechanisms Explain the role of the voltage potential for mitochondrial protein import

1

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

Biology 201

Dr. Torah Kachur

Outline 1) Proteins do Everything!

2) Structure of Proteins a. Proteins are polymers of amino acids • Twenty different amino acids are used in proteins 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 (Rgroups). • There are 20 biologically relevant amino acids found in proteins

•

The alpha carbon is asymmetric meaning there are two stereoisomeric forms called D- and Lamino acids

2

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

Biology 201

Dr. Torah Kachur

b. 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 • Also the bond angles in water allows for a polarity

Exercise: Classify the following amino acids based on hydrophobicity

•

•

Some amino acids have unique characteristics o Cysteine can form disulfide bonds (type of covalent bonds) between interacting cysteines

3

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

Biology 201

Dr. Torah Kachur o

Tyrosine can be post-translationally modified by phosphorylation (as can Thr and Ser)

c. Peptide bonds polymerize proteins • Chain of amino acids has an intrinsic directionality with the N terminus translated first

•

In a polymer of L-a.a. the side chains alternate on either side of the protein backbone

4

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

Biology 201

Dr. Torah Kachur

d. Proteins fold into 3D shapes • Primary structure dictates folding • Folding dictates function • Bonds stabilize the different levels of protein structure o Secondary structure – H bonding between a.a. backbones ▪ Determined by primary sequence

o

Tertiary structure – disulfide bridges, H bonds, van der Waals interaction, hydrophobic interactions and ionic interactions

5

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

Biology 201

Dr. Torah Kachur

Example problem: A single base-pair mutation has occurred in a gene coding for an enzyme involved in testosterone synthesis. The base mutation results in the change of a single amino acid from a GLY to a HIS? Do you expect the patient with this mutation to be able to produce testosterone? Explain.

o

Quaternary structure – same as tertiary

6

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

Biology 201

Dr. Torah Kachur

e. Folding and Function Relationship • Folding is driven by hydrophobic associations

•

Primary structure determines folding conformation o Evidence: Ribonuclease Denaturation/Renaturation Experiment Researchers found that the enzymatic activity of the Ribonuclease protein is lost if the structure is denatured (unfolded) o The treatment they used to denature the protein breaks disulphide bonds between cysteine residues

o

7

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

Biology 201

Dr. Torah Kachur o

•

Sickle-cell anemia o Single base pair mutation causes hemoglobin to crystallize in the red blood cells and cause the shape of the cell to sickle o This can impede flow through the capillaries as well as limit oxygen carrying capacity

Folding is often spontaneous o Often as the polypeptide is translated it simultaneously folds

8

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

Biology 201 •

Dr. Torah Kachur

Proteins can contain multiple functional domains o Different regions of secondary structure can form different functional regions, or domains, within the protein

Factors affecting folding pattern and protein function

Practice Question: Antibodies are crucial components of the adaptive immune system and are composed of multiple proteins that interact via disulfide bridges. The following treatments were used to elucidate the structure of the antibody. Lane 1: non-reducing conditions Lane 2: reducing conditions Lane 3: universal size marker

Given the data in the gel, what can you conclude about the antibody structure.

9

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

Biology 201

Dr. Torah Kachur

Practice Problem The image to the right shows the association of the nucleosome (middle) with DNA (outer rings). The graph depicts an experiment that used increasing salt concentrations (X-axis) on the association of DNA and histones (Y axis) and they looked at two variables 1) Effect of increasing salt concentration on histone-DNA association 2) Effect of acetylation of Lysine residues on histone H3.

What can you determine about the bonds that hold DNA and histones together. Why?

What can you determine about the effect of acetylation of histone H3 on DNA-histone association?

Lysine 9 is acetylated in Histone H3 and a mutation was found that changes Lysine 9 into a Glycine. What would you expect to be the consequence of this mutation?

10

How is this the because?

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

Biology 201 f.

Dr. Torah Kachur

Chaperones help proteins fold • Assist in allowing a protein to achieve its functional conformation, or prevent misfolding or target misfolded proteins for degradation • Assisted self-assembly

• •

Several different chaperones can be used to assist in protein folding. Many serve to prevent polypeptides from premature (or incorrect) folding.

Other functions of Chaperones;

11

Biology 201

Dr. Torah Kachur

3) Protein Synthesis – Translation a) Introduction to Genetic Expression • Genetic information flows from DNA via mRNA to protein where the DNA dictates the primary sequence of amino acids through the genetic code. • Transcription occurs in the nucleus and will not be covered in this course • All translation starts on free ribosomes in the cytoplasm

12

Biology 201

Dr. Torah Kachur

4) Protein Modifications and Regulation a. Processing of proproteins • Some proteins are produced in an inactive form that require cleavage for activation

b. Chemical modifications • Covalent attachment of chemical groups can regulate protein function • Can be –

c. Complex formation i. Many proteins act in a complex and must associate with other members of the polypeptide

13

Biology 201

Dr. Torah Kachur

5) Protein localization and import a) Proteins must be sorted • There are specific amino acid sequences that target proteins for different destinations within the cell •

14

Biology 201

Dr. Torah Kachur

Different types of signal exist: • All translation begins in the cytoplasm on free ribosomes • A continuous stretch of amino acid sequence, typically 15-60 amino acids long

b) There are 3 mechanisms for moving proteins amongst compartments 1. Nuclear import

2. Protein translocators

3. Transport vesicles

15

Biology 201

Dr. Torah Kachur

Your drawing: 3 mechanisms of import

16

Biology 201

Dr. Torah Kachur

c) Cotranslational Import •

• •

Some proteins are moved into their destination during translation, in particular, any protein destined for within the endomembrane system will be translated at the ER on bound ribosomes How do you distinguish a protein destined for the ER vs. cytosol? Polypeptides destined for the ER encode an N-terminal ER signal sequence.

•

The Signal Recognition Particle (SRP)

•

SRP docking

•

Translocation

17

Biology 201

Dr. Torah Kachur

18

Biology 201

Dr. Torah Kachur

d) 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 α-helical transmembrane segment (20-30 hydrophobic amino acids). proteins can cross the membrane once or many times.

•

Draw a transmembrane protein

• • •

19

Biology 201

•

•

Draw stop transfer insertion

•

Draw start transfer insertion

Dr. Torah Kachur

Multipass proteins o Some integral membrane proteins span the bilayer multiple times o This is accomplished by alternating start and stop transfer sequences

20

Biology 201

Dr. Torah Kachur

Example question: Draw or describe how a protein would be inserted into the membrane with 5 transmembrane segments and the N-terminus on the cytosolic face. Draw or describe the relevant sequences in the protein that would facilitate this.

Example Question The following image depicts a wildtype (normal) cell in A and a cell infected with the polio virus in B. A) Looking specifically at the endoplasmic reticulum in these cells (as depicted by the brackets) what can you reasonably conclude about the direct effect of polio virus infection?

B) What type of microscopy is this?

21

Biology 201

Dr. Torah Kachur

e) Post-translational Import • Some proteins are only localized to their destination after translation has completed • This occurs for proteins destined for: mitochondria, peroxisome, chloroplast and the nucleus • We have already seen the post-translational process of the nucleus, we will now focus on the mitochondria • Once completed the proteins will be transported to specific organelles due to the presence of special targeting signals. Mitochondrial Protein Import •

Mitochondria can synthesize ~5% of their own proteins but the remaining proteins are derived from nuclear DNA and need to get into this organelle

22

Biology 201

Dr. Torah Kachur

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 two translocation complexes – the TOM and TIM complexes

Chaperones are crucial to ensuring transport o Nascent polypeptides with a transit sequence are bound by Hsp70 in the cytoplasm

23

Biology 201

Dr. Torah Kachur

Energy for Transport • •

As part of cellular respiration there is a voltage difference across the MIM This membrane potential is used to drive protein translocation along with ATP hydrolysis

24...