Non-Covalent Interactions

How would a mutation in the amino acid sequence of a protein affect: a. The primary structure of the protein b. the tertiary structure of the protein c. the function of the protein

(a) This would change the primary sequence of the protein, as that is the amino acid sequence. (b) it may or may not change the tertiary structure of the protein, depending on where it is and what kind of mutation it is. Some parts of the protein can accommodate mutations without it having a large affect on the overall structure (hypervariable regions). If the mutation is more conservative (amino acids with similar characteristics) this may not affect protein structure significantly. If the mutation is a change in characteristic this may affect the structure of the protein. (c) it may or may not change the function of a protein. If the mutation changes the structure of the protein, or is of an amino acid that is critical for function (invariant position) it would then likely change the function of the protein. It can either increase or decrease function.

What is needed to form a hydrogen bond?

- (1) Electronegative atom with a lone pair - (2) A hydrogen with a partial positive charge - (3) A hydrogen covalently attached to an oxygen or nitrogen (in order for H to have partial positive)

Protein folding pathway

- (1) unfolded polypeptide being translated by the ribosome - As soon as you have initial stretches of the polypeptide, it can start forming secondary structures: alpha helices and beta sheets. - (2) As protein is being made, you start getting secondary structure. - (3) Once you have your entire polypeptide and you have some of those secondary structures, we have a hydrophobic collapse--> all of the different hydrophobic regions of your polypeptide aggregate together in the core of your protein--> this brings different parts of the polypeptide together that aren't necessarily near each other in folded state - (4) So now you have hydrophobic collapse and you have new polar amino acids that are in the vicinity of each other so there's a little bit more of shifting of your structure to optimize all of those weak interactions before it forms that final stable conformation.

Phospholipids

- 2 fatty acids and an alcohol

DNA helix is held together by...

- 2 strands are held together by H-bonds

Neutral lipid

- 3 different fatty acids

Nucleic Acids

- 4 nucleotides - only linear structures - Number of structures= 4^n - 1 million nucleotides per DNA molecule - heteropolymers

What is the physical basis for one molecule (or part of a molecule) interacting with another?

- An attractive force between opposite full or partial charges.

Order of bond strength

- Covalent--> noncovalent (ionic interaction--> van der waals forces); Hydrogen bonding--> dipole- dipole interaction--> london dispersion forces

What is the change in enthalpy when hydrophobic solvent is added vs. when aggregated?

- Dipole induced dipole interaction--> induced dipole induced dipole and dipole dipole thus overall interactions balances out

Water is a permanent dipole. What is meant by this term and why does water have this property?

- Electronegativity of Oxygen leads to an asymmetric distribution of electrons shared between oxygen and hydrogen giving a stable charge separation.

Denaturation of protein is caused by what?

- Heat (increase) - Change in pH - Urea or Guanidinium

H-bond donors

- Hydrogens attached to EN atom

What are the two non-covalent interactions that play the largest role in forming and stabilizing macromolecular structure?

- Hydrophobic interactions (Induced dipole-Induced-dipole), and Hydrogen-bonds

Why is the hydrophobic effect favorable?

- If two or more non polar molecules are added into water, the non polar molecules will aggregate together. This is favorable because it releases the trapped water molecules and allows them to once more form hydrogen bonds with other water molecules. - The surface that interacts with water is smaller--> fewer water molecules trapped in those cages and that increases the entropy of water relative to the hydrophobic regions all spread out.

Aggregation of non polar molecules in water

- In general, when you put something that is non polar in water, you have these water cages that form surrounding the entire molecule - Hydrophobic molecules are diffusing within water and eventually they'll run into each other--> burying some of those hydrophobic surfaces. - the surface is all polar or charged so it forces up all water molecules from those water cages and increases the entropy of those water molecules that were trapped in those cages--> increase in entropy makes overall process thermodynamically favorable

Physical basis of ID-ID

- Induced asymmetric distribution of the electron clouds produce partial and transient opposite charges that attract each other

This helix is amphipathic. What purpose might this serve in the protein's tertiary structure?

- It bridges the hydrophobic core of the protein with the hydrophilic aqueous environment.

Why is it necessary for any biochemist to know the primary sequence of the protein they are working with?

- It is necessary to determine the 3D structure. It is essential to determine which amino acids are essential for function, and then determine their chemical role in the protein function.

What role does the change in entropy play in protein tertiary structure formation?

- Macromolecular structure formation results in a more ordered structure of the molecule, decreasing entropy. However, this also results in a significant increase in the entropy of the surrounding water molecules, leading to an overall increase in entropy, which is thermodynamically favorable.

When is H-bonding most stable and thermodynamically favorable?

- Most stable when linear - most thermodynamically favorable state is when all 4 H-bonds are being satisfied (2 LP and 2 atoms)--> however usually only 3- 31/2 are satisfied

Are London dispersion forces formed by functional groups being pushed away from water?

- NO force thats pushing hydrophobic regions from water - random diffusion--> when they find that state it is more favorable and it will stay there

Would you expect a protein with no hydrophobic amino acids to fold into a stable tertiary structure under physiological conditions? Briefly explain your reasoning. Discuss the change in enthalpy in your answer.

- No, Without the hydrophobic amino acids: The entropy of water does not change from the unfolded to folded state (not trapped in water cages to begin with), but there would be a decrease in entropy of the protein. So the ΔS would be negative (unfavorable) The same type/strength of bonds would be present in the folded and unfolded state, so the change in enthalpy would be zero. Thus, delta G would have to be positive.

Would you expect this to be entropically favorable? Briefly explain your answer

- No, the polypeptide is gaining order, while the non-polar solvent should remain unchanged.

Hydrophobic core does what?

- Optimizes the non covalent interactions the protein has with itself

List the main interactions that stabilize each level of protein structure. Given that most of these are weak interactions how are they able to form a stable native protein conformation?

- Primary: covalent bonds (peptide bonds) - Secondary: hydrogen bonds between backbone atoms - Tertiary: hydrophobic interactions and polar interactions between side chains - Quaternary: hydrophobic interactions and ionic bonds between side chains Even though each interaction does not contribute a lot of energy to the overall structure there are thousands of interactions, and they all add up.

What plays a major role in the stability of a protein?

- Protein structure is stable as long as those non covalent interactions are all intact and the more noncovalent interactions you have within your protein, the more energy it takes to unfold or destabilize that protein. - Measured by how much energy it takes to unfold the protein and strength of weak interactions

What drives protein folding?

- Proteins try to reach the conformation with lowest free energy - Driven by increased entropy of water (highly ordered to higher disorder) and stabilized by formation of weak interactions. - When the protein is unfolded, the hydrophobic regions are ordering water around those hydrophobic amino acids - Once the protein folds, all of those hydrophobic regions tend to be buried in the core of your protein and water molecules have an increase in entropy.

Briefly discuss how this planarity affects protein 3-D structure. Your answer should include comments describing the usual structural relationship between alpha-carbon atoms of adjacent amino acids, and identify the bonds and constraints to free rotation.

- Resonance results in partial double bond characteristic of the peptide bonds, which doesn't allow rotation around the C-N bond. To avoid steric conflicts the alpha carbons are almost always trans and the R groups are almost always on opposite sides of the plane of the peptide bond. Rotation only around the C-Cα or N-Cα is allowed, but this rotation is also constrained due to constraints imposed by R groups. These constraints greatly limit the 3-D orientation of the atoms and thus the possible 3-D structures.

What is the importance of the hydrophobic regions of a protein in making the folding process thermodynamically favorable?

- The hydrophobic regions aggregate, which releases the water from cages, This increases the entropy of water, which is the major thermodynamically favorable component of protein folding.

What similarities does the process of structure formation share for all macromolecules?

- The native structure formation is driven by the hydrophobic effect (thermodynamically favorable increase in entropy of surrounding water) and determined by the aggregation of hydrophobic regions at the core of the structure. The final structure is then stabilized by non-covalent interactions, such as ionic bonds and van der waals interactions.

If the change in entropy is so important for driving macromolecular structure formation, why do the structures need to have weak non-covalent interactions?

- The non-covalent interactions do not contribute to the thermodynamic favorability of folding (over all ΔH is negligible), but they are required to stabilize the final structure once it has formed.

Physical basis of H-bonding

- The permanent dipoles have partial positive and partial negative atoms that form an attractive force between them

How can positive charges be solvated?

- The positive charges on our macromolecules can be solvated by partial negative charges of oxygen on water.

Comparing 2 different proteins with 2 different melting temperatures tells you what?

- The relative stability of those 2 proteins - The higher the melting temperature, the more energy it takes to unfold and the more weak interactions that have to be broken (more stable protein overall)

Why might it be important that secondary structures can be amphipathic?

- There are a variety of places where part of a protein must sit between a hydrophobic environment (core of the protein or interior of a membrane) and a hydrophilic environment (polar amino acids or an aqueous environment).

Physical basis of ionic

- There is an attractive force between the atoms that have full opposite charges

What role does the change in enthalpy play in protein tertiary structure formation?

- There is usually a minimal change in enthalpy associated with macromolecular structure formation. Most interactions that are formed in the final structure can be satisfied in some way in the unfolded state.

Briefly explain how each structure can arise from the primary sequence

- They both arise from having alternating polar and non-polar amino acids in the primary sequence. For a beta strand they must alternate at each position since amino acids next to each in the primary sequence face opposite sides of the beta-sheet. For alpha helices they must alternate about every 2 amino acids, as there are 3.6 amino acids in each turn of the helix.

A methyl group is non-polar. How can it have a partial charge?

- They can form induced dipoles, which are transient unsymmetrical distributions of electrons around the carbon, giving one side a very small partial positive charge and the other side a very small partial negative charge. This transient induced dipole can then interact with other induced dipoles or permanent dipoles. - The nearby electron clouds induce momentary asymmetric distributions of the electron clouds around the methyl groups giving temporary induced dipoles (generating an attractive force between partial opposite charges).

What role does water play in protein tertiary structure formation?

- Water is forced into static ordered structures surrounding the hydrophobic regions of macromolecules. The formation of the native macromolecular structure aggregates these hydrophobic regions together, and away from water. This releases the water from the ordered structures, and increases the entropy of the system.

Why do hydrophobic molecules tend to aggregate in aqueous solution?

- Water molecules in contact with the surface of a hydrophobic molecule have fewer degrees of freedom (i.e. fewer choices of other polar molecules with which to form interactions). Therefore, such molecules are more ordered than molecules in the absence of hydrophobic molecules. Such ordering of water molecules is entropically unfavorable. By aggregating, hydrophobic molecules minimize their surface area and thus the number of water molecules they affect. This favorably increases the entropy of water

Why are water cages unfavorable?

- When a non polar substance is added to water, the water molecules form a cage around the non polar substance. This is not favorable because it limits the water molecules ability to interact with other H20

What constitutes an amphipathic secondary structure?

- When one side (or face) of an alpha helix or beta sheet is non-polar and the other side is polar (based on the characteristics of the constituent amino acids).

Water molecule around a nonpolar solute

- When you put a non polar solute in water, there are some water molecules that have to be next to that solute and those water molecules do not have all of their H-bonds satisfied--> therefore they are making an interaction with a non polar functional group, but its not as strong as other interactions that they could make (dipole-induced dipole interaction is not as favorable as dipole-dipole H-bonds) - water molecules that are around that hydrophobic region get trapped in water cages--> static ordered structure surrounding these hydrophobic regions (releases some of those water molecules from these water cages - Less entropy--> unfavorable

Consider a protein folding in a non-polar solvent: Would you expect this to be enthalpically favorable? Briefly explain your answer.

- Yes, polar regions that have to face the non-polar solvent in the unfolded state can form stronger dipole-dipole interactions in the folded state.

The following statement regarding protein folding, under physiological conditions is from a biochemistry textbook: "The major source for a [large] negative ΔH is energetically favorable interactions between groups in the folded molecule." Based on what you know about protein folding, is there anything you disagree with in this statement? Briefly explain your reasoning.

- Yes, ΔH is generally small in protein folding. The interactions present before and after folding would have similar strengths (Similar number of interactions is not the same thing and was not awarded points, if we exchanged 10 hydrophobic interactions for 10 hydrogen bonds there would be a significant change in enthalpy) If there was a negative ΔH it would be due to increased H-bonding in water, not the interactions formed in the protein.

Biological molecules are largely what?

- amphiphatic--> hydrophilic and hydrophobic regions

All interactions between chemicals are based on what?

- an attractive force between opposite charges--> difference in strength is the magnitude of that partial charge - permanent dipoles have larger partial charges than induced dipole - The strength of the interaction between charges depends on the interaction of the charge--> smaller partial charges are weaker interactions

Simple construction provides what?

- an immense number of possible structures fully capable of providing the necessary diversity required for life. - Diversity is gained through the order in which we connect structures in chains--> polymerization

Van der waals

- anything that involves a partial charge

Lipids

- at least 2 different backbones - at least 12 different fatty acids - up to 7 different R3 substituents, or saccharides - contains various precursors (such as phospholipids)

Heteropolymer

- at least 2 different residues within one compound

What may happen if strands are mutated?

- can't form H-bonds--> strands will still form a double helix, but it will just be very easy to break it apart. - thus, H-bonds help structure stay together after it is formed.

Homoolymer

- every residue is the same initial precursor

Hydrogen bonding can form between...

- favorable for water to H-bond with itself, alcohols, carbonyls, carboxylic acids, amino groups

Polymerization

- formation of macromolecules from precursors (releasing H20)

What are the different types of biopolymers?

- homopolymer, heteropolymer (linear and branched)

What interactions will hold this helix in the correct position in the protein?

- hydrophobic would hold it towards the interior, and polar interactions would orient that face towards the exterior of the protein.

Macromolecular structure formation is driven by what?

- increase in entropy of water upon aggregation of hydrophobic regions

Dipole- Dipole

- interactions between permanent dipoles

Would you expect this to be thermodynamically favorable? Briefly explain your answer

- it depends on if the favorable change in enthalpy outweighs the unfavorable change in entropy. Depending on your answers to part a or b, you could also justify Yes or No.

What happens when you put salt in water?

- it dissolves--> partial positive charges on H can interact with negative charges on functional groups - shields the negative charge and separates them from each other

H-Bond acceptors

- lone pairs on EN atoms

Melting temperature

- midpoint where 50% of protein is unfolded - how much energy has to be put in order to denature or unfold that protein

Salvation of ions

- nonrandom orientation of water molecules - charges can be solvated and interact favorably with water (relatively favorable)

What are biological macromolecules?

- nucleic acids - proteins - carbohydrates - lipids (even though it doesn't have repeating unit)

Proteins

- only 20 naturally occurring amino acids - only linear structures - heteropolymers - number of structures 20^n

Polysaccharides

- only about 8 sugars - linear and branched molecules - homopolymers and heteropolymers

Properties of water

- polar molecule - has a permanent dipole (electronegativity of oxygen) - Dipole between O and H - Hydrogen has a small partial positive charge while the oxygen has a small partial negative charge

Folding funnels

- relates overall conformations of polypeptide to the overall energy or stability of that protein. - unfolded polypeptide has a lot of entropy--> there's a lot of states that the unfolded polypeptide can be in but its in a relatively high energy state - As each individual polypeptide folds, there's fewer conformations that you'll find that polypeptide in until all of them reach that most stable single native structure that has free energy.

Macromolecular structure formation is stabilized by what?

- stabilized by (held together by) non-covalent interactions - Enthalpy does not largely contribute to structure forming but does help hold together once structure is formed

Hydrogen bonding

- the interaction between the partial positive charge of a hydrogen covalently attached to an electronegative atom and a lone pair on an electronegative atom

If you were to mutate a surface exposed Ser to Ile. How might that affect the folding of the whole protein?

-Hydrophobic ILE could pull that part of protein into hydrophobic core (tertiary structure) OR: - would have an exposed hydrophobic a.a. that might bind to other polypeptides (aggregation?) (Quaternary structure)

Briefly discuss the role of hydrogen bonding in protein 3-D structure. Identify the level of structure being discussed and provide an example of the atoms involved in forming the relevant H-bonds

-In secondary structure, formed between backbone atoms (carbonyl groups to amino groups) -In tertiary (and quaternary) structure, formed between polar side chains, and between polar side chains and water.

H-bond vs, induced dipole induced dipole

-They are both based on the attractive force between opposite charges, as they both involve partial charges -The H-bond is a permanent dipole with a larger charge separation (larger magnitude partial charges) leading to a stronger interaction. While, the Induced-dipole - Induced Dipole interaction is due to shifting electron clouds giving temporary dipoles with smaller partial charges, leading to the weaker interaction.

a) Involves non-covalent interactions b) Has a specific pattern for each protein under physiological conditions. c) Always involves covalent bonds d) Dictates the other order of structure e) Involves more than one polypeptide f) Is random and has no particular pattern in a given protein g) Involves Hydrogen bonds only between atoms in the polypeptide backbone. h) Involves electrostatic and hydrophobic interactions between side chains i) May involve disulfide bonds.

Primary:__b,c,d______________________ Secondary:______a,b,g______________________ Tertiary:___a,b,h,i____________________ Quaternary:_____ a,b,e,h,i_________________________