LECTURE 1: PRINCIPLES OF BIOMOLECULAR STRUCTURES

Question 1

Structure Dictates Function + Most Drugs Target Proteins:

1) What are 4 roles proteins play
2) what confers the role of the protein

Answer 1

1) recognition of other molecules, enzymatic catalysis, signal transduction switch, structural proteins

2) roles are conferred by the structure of the protein

Question 2

Levels of Biomolecular Structure:

1) how many AA and nucleotides are there

2) what are the varying levels of proteins/nucleotides

Answer 2

1) 20 AA or 4 nucleotides

2)

1º: sequence of amino acids/nucleotides

2º: conformation adopted by backbone/main chain of polymer
* polypeptides: form -helices or -chains
* nucleic acids: adopt different types of helical conformation (A- or B- forms)

3º: 3D configuration or fold of the 2º structure elements + linkers between them

4º association of >1 folded chains (3º structures)

Question 3

Structure of AA:
1) what are the 3 groups found in an AA/what groups does an AA consist of

2) what does the variable group/side chain do

3) what is the molecular geometry and stereoisomer of AA

Answer 3

1)
o Amino group: NH2
o Acid group: COOH
o R-variable-group

2) Side-chain and defines the AA type
* AA are classified according to the R-group chemistry
* Some AA can be found in 2 different groups

3) All groups are attached to the C with tetrahedral geometry
o Most natural AA are in L-stereoisomer

Question 4

Aliphatic AA:
1) list all aliphatic AA

Answer 4

1)
o Glycine
o Alanine
o Proline
o Valine
o Leucine

Question 5

Aliphatic AA:
1) where are pro and val often found in the structure of a protein and why?

2) What 2 aliphatic AA are mildly hydrophobic, so where are they found

Answer 5

1) Gly + Pro are often found in loops because of their unique:
o Backbone flexibility glycine
o Backbone rigidity proline

2) Val + Leu are mildly hydrophobic (.˙. found in the core)

Question 6

Hydrophobic AA:
1) list
2) what AA can also be aliphatic
3) where are these AA mostly found

Answer 6

1)
o Isoleucine
o Methionine
o Tryptophan
o Phenylalanine

2) isoleucine

3) hydrophobic core

Question 7

Polar AA:
1) list
2) what do these AA consist of in their side chain
3) what is the function of these types of side chains
4) which AA have electronegative atoms

Answer 7

1)
o Serine
o Threonine
o Tyrosine
o Asparagine
o Glutamine
o Cysteine

2) amides (electronegative) + nucleophillic

3)
- Side chains with electronegative atoms act as e- donors for H-bonds

- Nucleophilic side-chains can be implicated in enzymatic catalysis

4) serine, threonine, tyrosine, cysteine

Question 8

Charged AA
1) list basic
2) list acidic
3) what are the charges of acidic vs basic AA @ pH 7

Answer 8

1)
o Lysine
o Arginine
o Histidine

2)
o Aspartate
o Glutamate

3) basic: +ve @ pH 7
acidic : -ve @ pH

Question 9

Ionizable Groups in Proteins
1) Some side chains have what property that allows them to do what type of RXN

2) What affects the reactivity of these side chains @ pH 7 and what is its general rule

3) give an example of how different reactivity @ pH 7 allows for preferential reaction with N-terminus of a protein at varying pH

Answer 9

1) Exchangeable H+ –> acid-base RXN

2) pKa, increase pKa = weaker acid

3) @ physiological pH 7.4:
o Terminal -amino group will be partically uncharged (NH2)
♣ Therefore can react with a free e- pair
o Side-chain lysine epsilon-amino group (terminal) will be charged (NH3+)

Question 10

Peptide Bond:
1) How is an peptide bond formed and what is lost in the RXN
2) how many resonance structures are there and whats the minor structure
3) what does the resonance structure do
4) draw the RXN

Answer 10

1) through condensation RXN where H2O is lost

2) two resonance forms
♣ Minor/less favored has C=N+ and C-O-

3) Makes all atoms involved to be in the same plane

4)

Question 11

Cis or Trans Peptide Bond:

1) How do these positions impact configuration

2) Cis: X-Z
a) what does this mean
b) whats the issue
c) how often is it observed in protein structures

3) Trans: X-Z
a) what does this mean
b) whats the issue

4) Cis + Trans: X-Pro
a) what does this mean
b) what is the issue and its implication
c) what is this important and what catalyzes the exchange between the 2 forms

Answer 11

1) both cause planar configuration for peptide bond

2) a) The 2 (C alpha + R-group) are on the same side
b) causes them to clash/steric hinderance
c) rarely observed

3) a) The 2 (C alpha + R-group) are on opposite sides
b) no issue because they don’t clash with each other

4) a) C gamma of the side-chain/R-group forms covalent bond with N of backbone (which is already bound to C-alpha)

b) Therefore, clashing occurs in both configurations (cis + trans) with a 50:50 ratio
- Both have similar energies

c) This is important because:
o Enzymes called proline isomerase catalyze the exchange between these 2 forms to facilitate protein folding

Question 12

Torsion Angles in Polypeptides:
1) what is the polypeptide bond in terms of planarity

2) therefore, what are the only variable torsion angles in a polypeptide

3) why are only certain combinations of these angles possible

Answer 12

1) planar

2)
o Phi angle: N — C-alpha
o Psi angle: Calpha — C=O

3) because clashes between the side-chains + carbonyl oxygen (C=O)

Question 13

ALPHA HELIX:
1) what kind of phi + psi torsion angles
2) alpha-helix formed by L-AA is what position
3) 2 ways the helix is stabilized
4) explain the bond between i and i+4
5) where do side chains of helix stick out to

Answer 13

1) negative phi + psi torsion angle
2) right handed
3)
a) H-bonds between the carbonyl oxygen (C=O) of residue i and the amide group i+4
b) dipole (overall strong with axis of helix)

4) at AA 5, there will be a H-bond between the oxygen of C=O and amide group

5) Side chains stick out of helix and all point outwards in the same direction

Question 14

BETA STRAND:
1) what kind of phi + psi torsion angles

2) what shift do these types of angles give the beta strand

3) can it stand on its own?

4) how is it stabilized

5) properties of beta sheet

6) where do side chains of helix stick out to

Answer 14

1) Negative phi and positive psi torsion angles

2) Gives an extended configuration

3) Cannot stand on its own and instead needs to be stabilized

4) Cross-strand H-bonds in both parallel + anti-parallel -sheets

5)
- sheets are pleated by virtue of the tetrahedral C-alpha
- sheets can be parallel or anti-parallel

6) The side chains stick outwards pointing in alternate directions

Question 15

Proteins Fold:
1) what dictates protein folding
2) what is the tertiary structure

4) what is a key step in protein folding and is it efficient

5) where are hydrophobic + aliphatic + aromatic AA found + whats an exception

6) where are charged + polar AA found

7) Where is proline found and why is it special

8) where is glycine found and why is it special

Answer 15

1) primary sequence

2) 3º structure of a protein is the specific arrangement of 2º structure elements With loops + turns connecting them

4) Packing of side-chains to form hydrophobic core + other stabilizing interaction
♣ Packing inside the hydrophobic core tend to be very efficient, leaving little empty cavities

5) tend to be within the core
- Some exceptions apply where aliphatic are exposed

6) exposed to the environment

7) Can induce sharp kinks in the protein
o Allows it to have specific folds + loops

8) Has large liberty because it has no side-chain
- Has a wider degree of freedom in terms of phi + psi angles

Question 16

Protein Folding Process:
1) is it spontaneous or no
2) what elements are formed early in the process
3) what is the “major transition” and what does it require
4) incomplete folding does what to an enzyme and why is the process important
5) ex of consequences of failed folding

Answer 16

1) spontaneous

2) 2º structure elements form very early in the folding process
♣ Helices, in particular, form first
♣ Then beta-sheets (more anti-parallel than parallel)

3) Hydrophobic region move to a “core”
♣ Requires an E barrier to be overcome

4) makes a protein inactive

5) deletion of phe508 in the cystic fibrosis conductance regulator (CFTR) ion channel causes misfolding + trapping in intermediate form degraded CF

Question 17

Interactions Stabilizing Protein Folds:
1) what are the 5 ways

Answer 17

1)
- Covalent peptide bonds
- H-bonds
- salt-bridge
- VdW forces
- Disulfide bonds

Question 18

Interactions Stabilizing Protein Folds:
Covalent peptide bonds
1) what does it maintain

H-Bonds
1) between what atoms
2) distance between non-H atoms
3) energetically favorable?

Answer 18

1) Maintains the polypeptide chain

SPACE

1) Between a donor X-H atom pair and Y
i. X = electronegative (N or O)
ii. Y = atom with free e- pair

2) < 3.5 Å between non-H atoms

3) Energetically favorable

Question 19

Interactions Stabilizing Protein Folds:
Salt Bridges:
1) what kind of bond and between what + example of AA that participates
2) more stable than H-bond? if so whats a unique feature

Answer 19

1) H-bond between 2 groups of full opposite charge (.˙. electrostatic interaction)
i. AA glutamic + aspartic acid

2) More stable than just H-bond .˙. can withstand higher temperatures

Question 20

Interactions Stabilizing Protein Folds:
VdW Interactions

1) when does it become relevant
2) what does it maintain

Answer 20

1) Becomes relevant at short range and in large quantities
2) Helps maintain tight packing

Question 21

Interactions Stabilizing Protein Folds:
Disulfide Bonds

1) between what atoms

2) done under what conditions and what kind of RXN (therefore, what is lost)

3) What kind of protein is this bond found in (give EX)

Answer 21

1) Between 2 cysteine side chains

2) Under oxidizing conditions, condensation RXN loss of H2O

3) Found in extracellular proteins (not found in proteins in cytosol)
EX: Light + heavy chains of antibodies and linked by these bonds

Question 22

Hydration Shell:
1) what is it?

2) how important are the H2O molecules

3) how are the H2O molecules stabilized and can they be seen (and when)

4) properties of the h2O molecules

Answer 22

1) layer of bound H2O molecules on the surface of a proteins

2) These H2O molecules are integral part of structure

3) Stabilized by H-bonds to polar groups on surface of protein
♣ Some can be seen in crystal structures

4) typically ordered however
- Some H2O molecules in the hydration shell are mobile + exchange rapidly with the bulk liquid water pool

Question 23

Entropy:
1) gibbs free energy equation

2) - Delta G means what

3) how can that be done

Answer 23

1) ∆G = ∆H - T∆S

2) Favorable reactions have -∆G (.˙. spontaneous)

3) - delta G can be done by having +∆S
♣ +∆S = more disorder
♣ -∆S = less disorder

Question 24

Hydrophobic Force:
1) what can decrease delta S

2) what happens to the hydration shell when proteins fold and side chains pack together

3) what does this result in and provide and what does this lead to

Answer 24

1) Ordered H2O molecules decorating proteins decrease ∆S

2) When proteins fold and side-chains pack together, they “liberate” H2O molecule –> +∆S

3) H2O molecules at the interface are now free so more disorder amongst them
* Increasing ∆S and decreasing ∆G
* This is the kinetic drive for the combination of hydrophobic cores
* Increase entropy of water (increase disorder) drives the formation of the hydrophobic core

Question 25

Hydrophobic Force:
1) how is the loss of delta S in ordered H2O molecules (H-bonds between water and the protein) lost

2) how is this loss compensated?

Answer 25

1) loss of ∆S in ordered H2O molecules happens through H-bonding with the protein is lost through the hydrophobic force and the coming together of proteins

2) compensated by H-bonds forming within the protein

Question 26

Protein Stability:
1) what determines the overall stability of a protein

2) What occurs at higher Temp and why

Answer 26

1) The sum of the stabilizing interactions

2) The protein denatures because the entropy-driven hydrophobic effect does not hold the protein fol

Question 27

Temp-Sensitive (TS) Mutations:
1) what does it do to the protein
2) give an example
3) useful in what study

Answer 27

1) Makes the protein more sensitive to changes in temperature

2) yeast strain combine with drugs that alter the protein and see which rescues the growth at a specific temperature (37)

3) Useful to study the function of proteins in cells + discovering stabilizing molecules

Question 28

Chaotropic Agents:
1) Examples

2) what do they do

3) why does this happen

4) what consequence does this have on the protein

5) does the same thing occur in the opposite direction

Answer 28

1) urea + guanidium hydrochloride

2) decreases the entropy of water

3) this organic molecule is non-tetrahedral therefore will change the intermolecular bonding between H2O molecules

4) increase entropy or disorder of protein that H2O surrounds –> protein is denatured + cannot fold properly

5) YES… when increasing entropy of water where delta G decreases –> very spontaneous RXN where now proteins can crash due to very strong hydrophobic effect where proteins liberate more water —> protein crystallizes

Question 29

Chaperones:
1) what do they do

2) how do they do so (2 ways)

3) if they were not present, what would occur/be risked

Answer 29

1) Chaperones assist/catalyze the folding of a protein (they are proteins that accompany other proteins)

2) a) allowing the protein to escape its trapped intermediate that wouldn’t lead to its native state

b) OR shuffling conformation + allowing the protein to “search” + find the lowest energy state without interacting with other proteins

3) Exposing hydrophobic side-chains from unfolded proteins WHICH risks forming aggregations

Question 30

PTM:
1) after what process can a protein undergo PTM

2) what can proteins as a whole undergo to activate

3) examples of AA modified w/ new chemical groups

Answer 30

1) After ribosomal translation, a polypeptide can undergo many PTM

2) Proteins can be cleaved by proteases –> activating them + structurally changing

3)

o Phosphrylation
♣ Ser, Thr + Tyr
♣ Activate or inactivate proteins
♣ Induce new interactions
♣ Cause re-localization or secretion
o Ubiquitination
♣ Lys
♣ Signal degradation or re-localization
o Glycosylation
♣ Ser + Asn
♣ Induce secretion or re-folding
o Methylation + hydroxylation
♣ Lys
o N-terminal acetylation
o Lipidation
o Etc.

Question 31

Membrane Proteins:
1) What are the membrane thicknesses of the polar + non-polar regions

2) what can membrane proteins be in terms of location

3) what do integral membrane proteins have that peripheral don’t

Answer 31

1)
o Non-polar is 30 Å thick
o Polar is 5-10 Å thick

2) Integral + Peripheral

3) Integral proteins have hydrophobic region as helix bundles or beta-barrels
* Where hydrophobic AA are found interacting with non-polar region of lipid chain

Question 32

Protein Domain:
1) what are they and where are they found

2) are they autonomous and how long are they

3) are they modular

4) what does this allow for

5) ortholog definition + ex

6) paralog definition + ex

7) whats special about multiple domain proteins

Answer 32

1) found in globular proteins, they are conserved segment of a polypeptide chain that forms a rigid tertiary structure

2) Autonomous folding units w/ hydrophobic core , 25-900 AA long

3) yes

4) Allows for insertions + deletions

5) Homologs evolved from common gene with speciation
* EX: Pyruvate kinase in human + E.coli

6) Evolved by gene duplication within the same genome
* EX: human Src + Abi kinases

7) Multi-domain proteins can acquire functions conferred by the individual activity of each domain

Question 33

Quaternary Structure:
1) Anemia

a) what mutation and where in RBC

b) this mutation in RBC results in what novel structure/interaction

c) this novelty leads to what

d) why is this advantageous for parasitic activity

Answer 33

a) Mutation Gln Val in beta2 subunit of Hb

b) Creates novel interface between beta1 + beta2

c) This interaction propagates + creates a fiber of Hb tetramer –> distorting RBC (problems between + within tetramer

d) advantageous for parasitic survival (RBC leaks nutrients needed for parasite to survive)

Question 34

Quaternary Structure:
2) Axenfeld-Rieger syndrome

a) caused by what mutation and whats the result

b) is this mutation dominant or recessive

c)what is required for proper activity and what does this mutation cause

d) is id dominant negative, what does this mean

Answer 34

a) Caused by mutations in PITX2 transcription
* Truncation or missense mutation in DNA binding domain

b) Mutations are dominantly inherited

c) Fully functional dimer is required for activity
* Prevents translation (because its dominant, even if one of the dimer is messed up or half one then it is non-functional)

d) YES, it is Dominant negative:
o Mutant product competes with standard for DNA binding

Question 35

Protein-Protein Interactions:
1) different to intra protein interactions?

2) have more of what kind of bond than protein core

3) what is unique about obligate proteins and what is a property because of this

4) where are non-obligate proteins usually found and what an you measure

Answer 35

1) No different than interactions found within protein domains
o Involve same forces

2) Protein-protein interactions may have more H2O molecules than protein cored

3) Subunits of obligate proteins are not stable + soluble on their own
o Therefore, if one is without its partner, it will be unfolded and degraded
- Therefore, have high affinities

4) Non-obligate usually found in signaling where there is transitional state between bound and unbound and therefore can measure an affinity constant

Question 36

SA
1) what is accessible SA
2) what is buried
3) what does larger buried SA mean

4) what is the most important determinant of protein-protein interaction/ protein-drug interaction and which protein (obligate vs non) exhibits this the best

Answer 36

1) VdW surface contact + reentrant surface generated with molecular probe (like water)

2) Difference between the sum of the individual subunits surface area and that of the final complex

3) Larger correlates with stronger interaction

4) Shape complementarity
o Obligate protein interactions show better complementarity then non-obligate interactions

Question 37

Dynamics:
1) the larger a protein motion is the…

2) whats the time duration of small scale motions

3) whats the time duration of larger scale motions

4) what can larger motions be induced by

5) within complexes, what are the types of interactions + their kinetics

6) what machine + unit described the amplitude of internal motion and what is the range of the value (whats does it mean)

Answer 37

1) the slower they are + the more energy they require

2) (atomic vibrations) are very fast

3) (conformational changes) occurs in msec

4) catalyzed or induced by PTM

5)
o Tight interactions have slower kinetics
o Weak interactions are faster

7) NMR Spec
o shows the S2 parameter
♣ Describes the amplitude of internal motion + ranged from 0-1
* 1 indicates absolute rigidity