Protein Structure, Folding, and Localization, Exams of Cell Biology

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BIOLOGY 2B03 - Cell Biology Exam Review all what

you need to know new update (covered from

module 1- module 5) McMaster University

l Bio 2B03 Module 1 Lecture 1 Protein Functions:

• Structure: such as tubules, actins and microtubules
• Sensors: Initiate change inside of the cell by relaying information.
• Enzymes: can metabolize chemical reactions
• Regulation: Proteins can be activators or silencers for genes which allows gene expression to be controlled. They can also modify the function of other proteins and turn them on or off.
• Signaling: Proteins act as signals as well as the receptors for communication between cells. They are also the transduction pathways that interpret those signals. Protein Structures:

1. Primary structure:

• The linear array of amino acids.
• There is an amino end (N-terminus) and a carboxyl end (C-terminus).
• Between each amino acid residue is a peptide bond holding it together into a chain
• The peptide bond is between a C- terminus and an N-terminus, it is a C- N bond Amino Acids:
• Each amino acid has a similar structure around the central Alpha carbon.
• They all consist of:

1. Hydrogen
2. Amino group (H 3 N+)
3. Carboxyl group
4. Variable R-group

• The variable group of each individual amino acid will dictate that amino acid’s properties BUT the accumulated R groups will define the properties of the entire polypeptide. Amino Acid Side Chains:
• The side chains of amino acids will dictate their characteristics and will change how the

1. Size
2. Shape
3. Charge
4. Hydrophobicity
5. Reactivity

• **These properties allow amino acids to be classified based on their SOLUBILITY IN WATER or the POLARITY OF THE SIDE CHAIN. Solubility: Definition- a molecule is soluble in water if it can form hydrogen bonds with water which is thermodynamically favorable. A soluble molecule usually carries hydrophilic amino acids residues on its exterior. Hydrophobic molecules:
• Cannot form hydrogen bonds or can form very little, so they are either insoluble in water or only slightly soluble.
• Amino acids with long and saturated hydrocarbon chains in their R group are usually hydrophobic due to this non-polar side group.
• HYDROPHOBIC amino acids tend to be in the interior of cytosolic proteins and form a hydrophobic core.
• A protein found in a hydrophobic environment (i.e membrane) has hydrophobic molecules on the exterior of the protein thus making it hydrophobic and insoluble.
• Hydrophobic amino acids can be divided into Aromatic amino acids AND Aliphatic amino acids (hydrocarbon chain).
• ***Tyr has an OH group and so it can be a part of both hydrophobic and hydrophilic since the OH can hydrogen bond. Hydrophilic molecules:
• Typically charge polarized and capable of hydrogen bonding.
• Includes: molecules with an – OH at one end (O-) & molecules with an – NH 2 at one end (NH 3 +)

• They tend to be on the exterior of water- soluble proteins
• Therefore, hydrophilic molecules can be both CHARGED and UNCHARGED.
• Ser, Thr, Asn, and Glm are uncharged molecules overall but they have polar groups attached. Special Amino Acids:

1. Cysteine- can form covalent bond with other cysteines and create disulphide bridges.
2. Glycine- it is very SMALL since its R group is a hydrogen, it can allow bends in the chain.
3. Proline- its side chain forms a bond back with the amide group and this forms a ring which forces a kink in the peptide chain.
4. Histidine- has an aminodiethyl side chain that shifts between

• charge and – charge depending on the pH of the environment. Peptide Bonds:

• Made through a CONDENSATION reaction: water is released during the formation of a covalent bond.
• It is between the carboxyl end of one amino acid and the amino end of another amino acid.
• Amino acid chains usually have the N-terminus on the left so new amino acids are added to the C-terminus on the right side. Translation Video:
• RNA goes out into outer part of cell.
• Ribosomal units assemble around RNA and translate the info on the RNA into a string of amino acids that become a protein.
• Transfer molecules which carry amino acids match up with the code o the RNA and the amino acid being carried is added to the amino acid chain.
• The structure of the protein depends on the AMINO ACID SEQUENCE.

1. Ionic Bond

• Attraction between positive cation and negative anion.
• An accumulation of these attractions can maintain a protein’s shape.
  2. Hydrogen Bond
• Interaction between a partially charged Hydrogen atom in a molecular dipole and an unpaired electron from another atom
• Ex: a water molecule has 2 partially positively charged H atoms which can then hydrogen bond with the lone electrons on another atom such as C double bonded to O.
  3. Hydrophobic Effects
• When many hydrophobic molecules aggregate in an aqueous solution in order to minimize their interactions with polar water.
• By aggregating together, the surface area exposed to water is minimized.
• The aqueous cytosol can induce these effects.
• The hydrophobic aggregation is much more energetically favorable due to the higher entropy.
  4. Van Der Waals (London Dispersion)
• It is a weak and non-specific attractive force.
• Results from a transient dipole that is induced when two atoms are very close.
• Results in the association of nonpolar molecules that cannot form hydrogen bonds or ionic bonds.
• Ex: setae structures on the bottom of gecko’s feet that allow them to walk on water and ceilings.

Bio 2B03 Module 1 Lecture 2 Secondary Structure: Definition: the local chemical interactions that fold a protein that create a conformation of a portion of the polypeptide.

• The secondary structure is the periodic folding of the polypeptide chain into conserved arrangements.
• These structures are determined by H- bonding between the non-variable side groups of the amino acids (aminos and carboxyl groups). Alpha Helix:
• Neighboring amino acids are connected through peptide bonds BUT hydrogen bonds (green dotted lines) connect other amino acid residues to form the alpha helix.
• ***Every amino acid residue forms a Hydrogen bond with an amino acid residue 4 positions away. ***
• There are 3.6 residues per turn.
• This helix pattern is found exactly the same in all proteins since the variable R groups are not involved in determining the structure of the alpha helix.
• So, what is the purpose of the R groups?
• The R groups determine the characteristics of the Alpha Helix, as the alpha helix can be hydrophilic, hydrophobic, or even amphipathic. Beta-Pleated Sheet:
• Structure is defined by the pattern of hydrogen bonding (dotted lines) between the carboxyl and amino groups of the residues.
• The beta stretches of the polypeptide are aligned LATERALLY in the beta sheet, and the backbones (green arrows) of the polypeptide can either run parallel (both terminuses are at the same side) or anti-parallel (terminuses run in opposite directions).
• The position of the hydrogen bonds are NOT REGULAR like in the helix shape.
• ***A beta-sheet can form in a single polypeptide (intramolecular hydrogen bonding) or between 2 different polypeptides (intermolecular hydrogen bonding). ***
• The variable R groups do not contribute to the shape of the sheets.

1 & red for strand 2) interact with each other in an aqueous environment in the cytosol since it is favored.

• In order for a continuous hydrophobic surface to form along the alpha helix, the hydrophobic amino acid residues must be at position 1 and 4 within a heptad repeat (a repeat of 7 amino acids). Ex: abcdefg is one heptad, HPPHCPC is another.
• A simple version of this heptad repeat is called the Leucine zipper, where it goes Leu-X6-Leu- X6 where X6 just means there are any 6 amino acid residues in between.
• The coiled-coil motif is often found in DNA binding proteins since the structure allows it to fir within the grooves of the double stranded DNA helix.

2. Zinc Finger Motif (alpha helix + 2 beta strands)

• Involves an alpha helix and 2 beta strands (a very small beta sheet).
• The secondary structures are held together by the precise interactions of the amino acids.
• In this ex, 2 histidine residues and 2 cysteine residues interact with a zinc cofactor to maintain the motif structure. (can also be zinc interacting with 4 cysteines or 6 cysteines)
• These motifs are often found within the DNA binding proteins, but they can bind to RNA too.

3. Beta-Barrel Motif

• A large beta sheet that loops on itself.
• It’s a collection of 4-10 anti-parallel beta strands that form a sheet, the first and the last strand also H bond and that is why it forms a barrel shape since it closes on itself.
• This is useful for forming a channel or a pore in membranes for example.
• The structure would have to be amphipathic. The exterior would be made of hydrophobic residues since it is lying on the membrane and the R groups pointing into the interior of the barrel would be hydrophilic to allow hydrophilic molecules to pass through the channel.

4. Helix-Loop-Helix motif (2 small alpha helices)

• Made of two small alpha helices held in a specific orientation by NON-COVALENT interactions between specific amino acid residues and a calcium cofactor.
• The shape of the helix-loop-helix can only occur once the polypeptide interacts with the calcium
• Therefore, protein structure and function depend on the cofactor. Tertiary Structure: Definition: the overall 3D structure of a SINGLE polypeptide.
• Domain: a portion of a protein, it is often a functional unit of the protein but can also be structural.
• Domains can fold independently of the rest of the protein and this allows researchers to study domain functions independently of the whole protein.
• Functional Domain: region of protein that performs a certain activity i.e. DNA binding, enzymatic, protein-protein interaction.
• Structural Domain: region of protein that form compact, largely independent globular domains i.e proline-rich, acidic domain. They have a recognizable shape or characteristic
• This is the Src protein.
• It has 2 functional domains, the small and large kinase domains.
• It has 2 structural domains, the SH3 and SH2 domain.
• The structural domains are defined by their similarity to domains that are present in a diverse array of proteins.
• SH3 strands for Src Homology 3 domain and SH2 is similar.
• The SH2 domain is classified by its collection of alpha helices and beta sheets in between; this domain also has an identified function.
• The SH2 domain recognizes phosphotyrosine residues in other proteins, and this domain will always have the same structure and function in other proteins.

• Modifications after translation alter the variable R groups such as through the linkage of a chemical group to the variable R groups.
• The modifications alter the chemical properties of the R groups and change the protein structure and function.
• It is because of these modifications that we see more than 20 distinct amino acids (up to 100) in a post-modified protein.
  1. Acetylation: addition of an acetyl group (reversible)
• Protects against intracellular protease degradation (80% of proteins in cytosol have at least one acetylated amino acid residue). Ex: adding an acetyl to lysine makes it called acetyl lysine.
• Acetylation can also modify the activity of a protein, for example, histone proteins associate with DNA molecules in order to form chromatin, but when a histone is acetylated, this alters the association of histone and DNA and thus the gene expression is regulated.
  2. Methylation: addition of methyl group (reversible)
• Histidine molecules are often modified to form 3 -methyl histidine.
• Methylation of histone proteins at histidine residues alters gene expression by weakening the association between the histone protein and the DNA.
  3. Phosphorylation: transfer of phosphate group from ATP to the – OH group of serine, tyrosine, or threonine by kinases.
• Only ser, try, and thr, can be modified this way due to their hydroxyl group.
• Phosphorylation is catalyzed by kinases.
• Phosphatase: enzymes which mediate removal of the phosphate (dephosphorylation).
• Both phosphorylation and dephosphorylation can activate and deactivate proteins by changing its shape or ability to interact with a substrate.
  4. Hydroxylation: adding of an OH group.
• Proline can be converted to 3-hydroxy proline which is important in changing the structure of the protein.
• Ex: formation of a triple helical coiled-coil of collagen required 3 alpha helices to join together through the hydroxylation of certain residues in order to form the functional collagen. Collagen can exist with one single helix, but it is only functional with 3 helices.
• The enzyme which catalyzes the hydroxylation in the cell requires vitamin C.
  5. Carboxylation: addition of carboxyl group (COO-)
• Changes properties of amino acids by adding a NEGATIVE CHARGE.
• Ex: carboxylation of glutamate changes it to gamma-carboxyglutamate which now has 2 negative charges instead of 1 and this may facilitate ion bond formation or allow a positively charged cofactor to bind.
  6. Glycosylation: addition of carbohydrate
• Sugar molecules are added to specific amino acid R groups.
• Glycosylation occurs in the Golgi apparatus.
• Many cell surfaces and secreted proteins are glycosylated since it protects proteins from proteolysis and allows for proper protein folding.
  7. Lipidation: addition of lipid molecule
• Important for anchoring proteins to hydrophobic bio-membranes.

Native Conformation:

• The most thermodynamically stable conformation.
• It depends on the primary amino acid sequence.
• Protein folding is:
  1. Spontaneous
  2. Reversible
  3. Unique
• These 3 points were proposed by Christian Anfinsen in 1973 based on studies of the ribonuclease A protein.
• Anfinsen added a high concentration of urea to break down the hydrogen bonds and disrupt the hydrophobic interactions of the protein.
• He also added bet mercaptoethanol which breaks disulphide bridges which were present due to the cysteines in the protein.
• These tactics caused the protein to denature and unfold. (Extra: high salt concentrations and temperature can also denature)
• Using dialysis, the denaturants were removed, and the protein refolded in exactly the same way since its primary structure was intact.
• This shows that protein folding is spontaneous since it refolded by itself. It is reversible since it was renatured through dialysis. And it is unique since it folded into exactly the same shape which is unique to its primary structure. Villin Protein Folding:
• Protein folding is spontaneous and based on trial and error, a protein will fold and refold until it reaches the most thermodynamically favored state.
• A small 36 residue alpha helical protein.
• Folding stabilized the hydrophobic interaction when in the aqueous cytosol.
• The protein is not static, but it is spending most of its time in its stable conformation. Sickle cell anemia: misfolded hemoglobin:
• Hemoglobin is a tetramer (4 polypeptides) and it has 2 native conformations based on whether oxygen is present.
• This tetramer is a soluble, globular protein complex.

1. Preventing incorrect folding
2. Preventing incorrect associations with other proteins

• These mechanisms are seen in all organisms and are not specific to certain types of proteins. Molecular Chaperones:
• These are monomeric proteins.
• They bind to hydrophobic amino acid residues on a polypeptide and prevent the protein from forming incorrect folds due to hydrophobic interactions within an aqueous environment within a protein or with other proteins.
• An example of a chaperone proteins are Heat-Shock Proteins (HSP) which are expressed in high levels when under conditions of stress such as high temperature. This is because high temperatures cause proteins to denature and unfold so these chaperones are expressed in high levels to refold various proteins in the cell.
• Hsp70 is an example and it’s found in cytosol and mitochondria. Other examples: BiP in ER and DnaK in bacteria. Hsp70:
• Contains 2 domains: The Nucleotide Binding Domain (blue) and the Substrate-binding Domain (orange).
• **The substrate can either be unfolded proteins or a nascent (still being translated) protein.
• The unfolded protein will have a hydrophobic patch which will be exposed to the aqueous cytosol, this will interact and rapidly bind with the hydrophobic region on Hsp70.
• ATP hydrolysis to ADP is stimulated by the Co-Chaperone named DNAJ or Hsp40. And this hydrolysis to ADP changes the conformation of the Hsp70 chaperone in order to change the relative shape of the unfolded target protein.
• ADP is released from Hsp70 by the Nucleotide Exchange Factor GrpE or BAG1.
• A new ATP comes into the nucleotide binding domain, the folded protein is released and the Hsp70 is free to bind to another unfolded protein. Chaperonins (“isolation”):
• Large cylindrical macromolecular complexes containing many different proteins.
• The collection of proteins in the complex form a chamber or a barrel and this is the region where the unfolded protein can move into and fold in isolation.
• Example: TCiP in cytosol, GroEL in bacteria or chloroplast and Hsp60 in mitochondria.

• The example on the right is a chaperonin from bacteria.
• It consists of 2 large subunits (GroES = small and GroEL = large)
• Multiple proteins make up the walls of the 2 GroEL or large subunits that are attached to one another at their bases and capped off by a GroES or small subunit or left uncapped.
• The GroEL is a hollow chamber that forms an isolation chamber or folding chamber.
• Either GroEL can be used as a folding chamber as they are independent of each other. Process:
• The 2 GroEL chambers are ALTERNATELY used.
• In step 1, the bottom chamber is releasing its GroES cap and ADP since it has completed its task.
• The top chamber is binding to a new ATP and an unfolded protein and is about to begin the process. A new GroES cap binds to the top chamber and isolated the unfolded protein.
• **The chamber changes conformation and enlarges its chamber in order to allow more room for folding.
• The chamber remains closed for several seconds as the protein folds without any assistance from the chaperonin, the chaperonin is only serving as an isolation chamber.
• ATP hydrolysis to ADP allows the GroES cap to come off and the protein diffuse out. If it has not folded properly then it will repeat the cycle but it will use the bottom chamber instead. GroEL Conformations:
• There are different conformations of the GroEL depending on whether it is bound to GroES or not.
• GroEL + GroES = GroEL “relaxed” conformation. This conformation causes the GroEL to be larger on the inside in order to allow the protein in the isolation chamber to have ample space for fold.
• GroEL on its own = GroEL “tight” conformation. This conformation is when there is no protein inside the GroEL chamber and therefore it does not need to be large on the inside.

• Unfolded proteins are a risk since they lack appropriate functions, but also because they can aggregate and form complexes that are harmful to the cell.
• The proteins that will be degraded are selectively tagged and sent to the proteasome for degradation.
• Degradation is a 2 step process.
  1. Tag the protein with an Ubiquitin Tag. It is attached by covalent attachment to a specific amino acid residue on the protein. Ubiquitin itself is a folded protein with a unique sequence.
  2. The Ubiquitin Tag is recognized by proteolytic machinery of the cell and the target protein is cleaved into short peptide sequences, effectively eliminating protein function. Ubiquitinylation:
• Covalent attachment of Ubiquitin to the target proteins for degradation requires 3 enzymes.
• E1: is the Ubiquitin Activating Enzyme. It recognizes the free Ubiquitin in the cytosol and picks it up.
• E2: is Ubiquitin Conjugating Enzyme, it facilitates the attachment of Ubiquitin to the target protein.
• E3: is Ubiquitin Ligase. It is the E3 Ligase that is going to recognize the specific target for degradation and attach Ubiquitin to it.

• ATP hydrolysis allows for Ub to attach to E1.
• Activated Ub is transferred to Cysteine on E2.
• Target protein was previously recognized and captured by E3.
• E3 and E2 interact and Ub is transferred from E2 to target protein.
• Polyubiquitinylation is needed since one Ub is not enough, E1 can bind with many E2s, which can bind with hundreds of E3s. It is the E3 which provides specificity of degradation. The Proteasome:
• The Proteasome is where degradation occurs. It is a large protein complex with similar structure to chaperonins.
• A wall is created by many identical subunits to create a hollow interior or barrel, it has caps at both ends which enclose the contents.