Polypeptides and proteins: structural hierarchy, sequence. Basis of reactivity and hydrolysis Flashcards
1
Q
How many H-bonds would contribute to the stability of an a-helix consisting of 9 aa?
A
-5,
1 bonds to 5, 2 bonds to 6…
**1 ALWAYS bonds with 5**
2
Q
How does X-ray diffraction measure repreating patters
A
- atoms/molecules have similar dimensions to wavelength of x-rays
- isolate protein to have a pure solution
- make a crystal (all proteins are stocked on top fo eachother)
- shine X-ray on crystal, as X-ray hits atoms, it gets diffracted and created a pattern from the diffraction, allows you to figure out spacing in moleucle
- note: typically use angstrom (1A= 10^-10m)
3
Q
What tertiary strucute will protein containing MOSTLY a-helix aa form?
A
- fold into a-helix bundle
- small clusters of breakers set limits of each helix
- non polar aa’s usually every 3-4 places in helix, make a non polar patch/stripe (look at cyclinder, bc of how tightly wound its all on one side of the helix)
ex: PPNPPNNP which fold inside of the bundle - AA’s that perfer b-sheet are present, but scattered
4
Q
How do amino acids “fit together” in teritary strucutre
A
- side chains interlock to maximize the number of close atom to atom contact
- close contact is attracted by weak van der waals (LDF) forces
- 0.1-1 kJ/mol per contact
- goof fit makes hundreds of close contact per macromolecule, helps to hold structure together
*want to miximize number of close contact*
5
Q
What is unique about peptide bonds
A
- peptide bond has two resonance forms, one with a bouble bond (that has a postive charge on N
- double bonds are rigid, fixed in cis or trans geometry (usualy trans) because behaves more like double bond than single
- normal C-N= 1.49A, peptide C-N= 1.32 A, Normal C=N is 1.27A
- rotation only occurs between C-C
- in normal peptide chain, amino and carboxylate are locked in ridigd planar peptide bonds, only the two C-C can rotate freely
6
Q
What is the backbone of the protien
A
- secondary structure consisting of repetitive patterns such as helix
- the polypeptide chain forms a backbone which appears to be linked by C-C and C-N bonds
- flexible due to bond rotation (can rotate about bond axis)
- bonds typically 109 (tetrahedral) or 120 (trigonal planar)
- bond rotation allows peptide chain to adopt variety of shapes
7
Q
How will a beta sheet with polar aa on one side and non polar on another side look when in polar solvent
A
- wraps around to enclose non polar face inside
- is small sheet (3-5 strands) makes OPEN FOLD
if larger sheet (6-8) wraps around forming ANTI PARALEL BARREL
ex: green fluorescent protein
8
Q
How are B-stand and B- sheet formed?
A
- when amino acid alternate orientation
- sheet formed by hrdrogen bonding
- can have parallel sheets: stands in same direction (less ideal for H-bonding **USUALLY NON POLAR
- can have antiparallel sheet: strands in opposite direction (ideal for H-bonding therefore stronger)
9
Q
How does alpha-helix form
A
- forms when aa all have same orienration and a-C bond turns in same direction
- according to paulings model: 3.6 amino acids per turn of helix
- an amino acid is present every 1.5A (minor periodic repeat of a-keratin)
- 5.4 A between each turn in AA (matching major repeat distance of a-keratin
- (5.4A/3.6A=1.5A)
- structure is held together because NH and C=O will form a hydrogen bond (ex: amino acid 1 has H bond with amino acid 5)
- right handed favoured bc left handed side chains placed to close to c=o (strucuture is over crowded
** BE ABLE TO DO CALCULATION ie: if ___ aa “
10
Q
How do polar interactions help stabilize folding pattern
A
- H-bonds may form between donors and acceptors that line up in folded pattern
- lone pairs have strong electrostatic interaction of neg side chains which pair up with pos side chains that are nearby the folded protein
- polar interactions important for maintaining structure are usually in interior, not on surface
- most polar groups face exterior
- charges AA pair with ions in external solution
- H-bonding aa bond to H2O in surrounding
11
Q
Following protein sequence Val-Ile-Leu-Val-Ala-Ile-Val-Cyc-Val
which family of protein teriary strucutres could this sequence be found in
A
- is a beta sheet (b-b-a-b-a-b-b-b-b (sequence of alpha vs beta))
- could be either beta parrel or alpha beta sheet
- full sheet is non polar (cannot be a barrel because non polar on oth sides)
- structure is alpha-beta barrel
12
Q
What structure will sequences which alternate between helix and sheet form
A
- helical sections connect connect the strands, run all in the same direction
- helix lies above or below the plane of the sheet
- paralel B-sheets are LESS stable (angled H-bonds), so much be hidden from water, usually buried in centre of protein, thus made up of mostly non-polar aa
- surrounding the “barrel” sheet are the helixes, helix have the non-polar regions facing inside
**must form barrel is on one side of sheet
13
Q
How do larger proteins fold up?
A
- in different domains
- fold into 10-20 kDa sections (called domains)
- each domain can ahve different folding pattern
- larger proteins often modular in nature
14
Q
Summarize the way a protein folds based on amino acids
A
1) amino acids “elect” secondary strucute (majority rules)
2) “breaker” aas sllow for folding, introduce flexible sections
3) distribution of non polar aa in sequence determines which part folds inwards
4) polar aa interact well with aqueous surroundings
5) pattern of large and small side chains is arranges so that secondary strucutre components pair up with best possible fit (maximize intramolecular forces)
*in tertiary its pattern of distribution, not how mny polar vs non polar
15
Q
What are conformations
A
- represent STATE of molecule that can be interconverted by BOND ROTATION without BREAKING BONDS
- (conformation, not breaking any bonds, just rotating and getting new structure)
note: marcomolecules like proteins are usually concerned with conformations
16
Q
What is quaternanry strucute
A
- only in some proteins
- multiple proteinds come together and interact to form overall protein
- made of multiple subunits
17
Q
What are configurations
A
- can only be interchanged by BREAKING COVALENT BONDS not be bond rotation
- ex: cis and trans forms of molecule about double bond
18
Q
How does the primar structure contain all information for secondary and tertiary structure?
A
- ribonuclease is an enzyme which catalyses hydrolysis f RNA
- 8 Cys make 4 specific disulfide pairs in properly folded protein
- ribonuclease is denatured (catalytic activity lost) by by placing it in 8M urea solution with 2-mercaptoethanol
- urea weakens hydrophoic effect, allows proein to unfold
- 2-mercaptoethanol is a reducing agent that convert disulfides back into original unlinked Cys-SH
- ribonuclease can refold to original strucutre when urea is removed
- first remove urea so ribonuclease refolds
- expose to O2 so disulfide bodns form
- refolded enzyme has normal catalytic activity and original pairs of cys in disulfide bonds
- if exposed to O2 before refolding, disulfides pair up randomly, wrongsly folded (inactive)
19
Q
What is restricted bond rotation
A
- state of C=N peptide bond is trigonal planar (ridgid bc double bond)
- C=N linked to alpha-C atoms with tetrahedral shape, thus creating a 109 bind.
- adopt 2 patterns: helical state bond rotation in same direction
- extended state, bond rotation alterting direction (zig zag structure for peptide chain)
- if non repetitive pattern= random coil
20
Q
What determines which secondary structure is formed?
A
- ala, arg, gln, glu, his, leu, lys, met, (phe) tend to form a-helix (defult formation)
- trp, tyr, (phe), val, iil, thr, cys tend to form b-sheet
- local majority determines which structure forms
21
Q
How does hydrophonic interactions effect protein folding
A
- folding encloses most of non-polar amino acids in the core
- non polar aa group together to minimize contact with H2O (hydrophobic effect)
- polar aa form outer layer bc interact well with surrounding water or with ions in solution
22
Q
What are secondary strucute breakers
A
- gly, pro, asn, asp, ser (GPNDS)
- have side chains which interfere with secondary H-bonds
- if 2 breakers in group of 4 amino acids it interups structure
- forms a turn or flexible loop, allows the polypeptide main chain to change direction drastically (can be 180)
- between breakers they form a or b
23
Q
How do we investigate the strucure of a protein
A
- before you can dientify aa you need to break peptide bonds, release amino acids
- peptide bonds are hydrolysed with help of catalyst to relsease individual amino acids,
- water cannot hydrolyse alone (needs help of catalyst)
24
Q
What is the most common structure for protein
A
- globular strucutre
- folding requires breaks in rigid secondary structure
- clusters of 2-3 secondary strucutre breakers (Gly, Pro, Asn, Asp, Ser, GPNDS) in a run of 4 AA
- this region allows for flexible loops and turns, polypeptide can change irection to allow for folding
25
What is acid hydrolysis
- use 6M HCL at 110 for 24-72 hours
- trp is destropyed
- side chain amine of Asn and Gln are converted to carboxylic acid form releasing amino group as ammonia
26
What is a native state, how does denaturing effect the native state
- native state= unique 3D tertiary strucute required for their function
- denaturation unfolds proteins, unfolded form may be unstructured or aggregated (often irreversible)
- causes loss of function
- can be denatured by eat, disruptive solvents or harsh detergents
27
What is van der waals interaction?
- weak electrostatic attraction between atoms that are close, but not covalently bonded to each other (LDF)
- random fluctuations in distribution of nucleus and electrons create short-lived dipoles inducing dipoles in close neighbours
**Free energy of interaction:**
* atoms too close together [A] *repel strongly* (pos free energy)
* Atoms at ideal distance [B], close to each other
* atoms further away are attracted [C]
* force fades away when atoms are more than 2-3 diameters apart [D]
28
What is tertiary structure
overall pattern of folding of whole polypeptide chain
- A-keratin is totally a-helix and fibroin (b-keratin) is antiparallel to B sheet
- collegen has unique triple helix strucute: strucute requies repeating units of gly-pro-X
29
What is a nucleophile?
An atom with a lone pair of electrons available to share
- seek out other groups that are electron deficient (electrophiles)
Ex: O, N and S often have lone pairs and are nucleophiles
note: O is weak nucleophile, but when O- it is a strong nucleotphile
30
How do covalent/non covalent bonds influence protein structure
- covalent: links aa in specific sequence
- non covalent interactions dictate folding pattern & stability (hydrophoib effect and van der waals are most important non-covalent interactions)
- hydrophobic effect locates non-polar aa in core of folded protein, contrbuted to ~50% of total energy stabilizing native state
- 5kJ/mol per CH, CH2, or CH3 moved out of contact with H2O
- polar aa face exterior (interact well with water)
31
what is chemical reactivity
arises from an unbalanced distribution of valence electrons
32
How many clusters of secondary sturcute breakers are found in this peptide
MVCLWTTSKPGEIMQLAKHGPKNFITWQVQV
- SKPG (HAS 3 BREAKERS)
- GPKN (HAS 3)
33
What are disulfide bonds
- form when pairs of Cys-SH groups react with O2 releasing H2O
- makes a strong covalent bond to help hold the folded protein together
- few proteins have disulfide bonds: mostly proteins designed to function outside cells since O2 needed
34
What is the simplest possible tertiary structure
a continuous secondary structure
35
What is secondary structure
- regular repetitive patterns, such as helical sections in myoglobin, that run along short sections of peptide chain
- alpha helix
36
What is primary structre
linear sequence of thee amino acids
37
What is nucleophilic substitution/displacement
- incoming nucleophile X attacks target atom C to displace leaving group Y
38
What other fomr can helices on both side of sheet give
- sandwhich structure
- "filling" is the non polar sheet between two layers of A helix,
- b-sheet is often twisted for better packing
- ex: part of lactate dehydrogenase
39
What strucute will MOSTLY B- sheet aa form
- fold into antiparallel B-sheet
- anti-paralel sheet is more stable because H-bonds are arranged in straight line
- side chains project out of the sheet: odd on one side and even on the other
- sheet can then be non polar on one side and polar on the other
40
What are the dimensions for B- sheet
- match B-keratin patterns
- R-groups come "out of the page"
- 7.0 A betwen R-groups( R1 and R3), 3.5 between central carbons
- amino acids with larger structures perfer forming Beta strucute
- trp, tyr, phe: big
- Val, Ile, Thr: have branch on Beta-carbon
- Cys has large S on B-C
41
How do atoms use thier lone pairs
- as a hydrogen bond acceptor
- if it is accepting an H, it is considered a bse
- as a nulceophile when it shared the lone apir with another electron- deficient atom to make a new bond
42
What is tertiary strucute
- overall pattern of 3D folding of the while polypeptide
- polypeptide chain
43
What is the major and minor pattern of alpha keratin
major: 5.4
minor: 1.5
44
What is the major and minor pattern of B-keratin
major: 7.0
minor: 3.5
