Biochem 2 Flashcards
1
Q
protein structure
A
- arrangement of atoms in 3D
- protein shape
- structure determines function
2
Q
structure can be described on many different levels
A
- primary- amino acid sequence in a linear polypeptide chain, backbone
- secondary- helix, beta pleated sheet; local interactions; defined by interactions between the backbone atoms of the polypeptide chain
- tertiary- one complete protein chain; network of interactions folded; final arrangement of atoms in 3D
- quaternary- separate tertiary chains assembled into oligomeric protein
- proteins have a single shape
3
Q
all proteins
A
go up to tertiary structure
-not all have quaternary
4
Q
planar peptide bond limits possible conformations
A
- peptide bond has 40% double bond character (resonance structure) -> restricts the structure so that it is planar (same plane) -> this means we can only form a few types of secondary bonds that are stable
- two types of peptide bonds:
- trans conformation- account for the majority of peptide bonds -> more stable
- limits conformations for secondary
- cis conformation- 8 kJ/mol less stable than trans conformation (a lot)
- exception- proline- restricts motion of peptide bond -> cis formation is only a little bit less stable than trans (1 in 20 proline are cis)
- 1 in every 1000 are cis normally
5
Q
backbone conformation
A
- can be described by torsion angles
- bc the peptide bonds are planar there angles cannot really be changed
- there are two torsion angles found in the peptide:
- Phi- between the amide carbon and alpha carbon
- Psi- between alpha carbon and carbonyl carbon
- define angles between -180 and +180
6
Q
Ramachandran plot
A
-measure phi and psi for each residue
-plotted on x-axis angles of phi from -180 to 180
-plotted on y-axis angle of psi from -180 to 180
-he found that there are certain angles that were not found - disallowed regions -> and certain angles that were favored- favored region
-tells us about secondary structure - common angle for certain structures like beta sheets are helix
-exceptions: proline- severely restricted in the possible conformations it can form, glycine- flexible, can adopt many more angles of phi and psi, expanded ramachandran plot, many for angles allowed
-gives info on hydrophilic interior and hydrophobic exterior
-
7
Q
disallowed region of ramachandran plot
A
- angles that were not found in residues
- white (Not plotted)
- most likely situation is the protein structure was determined by X-ray crystallography at a low resolution
8
Q
favored regions
A
- dark blue
- commonly observed angles of psi and phi
9
Q
allowed combinations
A
- light blue regions
- sometimes observed angles of psi and phi
- areas around the favored regions
10
Q
secondary structure elements
A
- goal: we want the protein to be stable to perform function
- creates area of local stability so the protein doesnt fall apart -> preserve function
- alpha helix
- beta sheet
- must use phi and psi values that are favored to form secondary structures
- must form a network of interactions that form local stability -> stable structure
- use H bonds
11
Q
alpha helix
A
- takes a polypeptide chain and wraps it around a central point
- forms a right hand helix
- all carbonyl atoms are pointed down (acceptor) -> directly beneath are amide N that is pointed up (donor) -> forms a H bond
- the residue with the carbonyl O is the nth residue and interacts with the amide N of the n+4 residue beneath it
- forms a network of h bonds
- *3.6 residues per turn -> help keep structure so that stretching and squishing wont affect
- *helical pitch- distance between equivalent sites on the helix 5.4 Angstroms
- predicted the structure before we even saw it
- unlikely that an alpha helix would have successive branched amino acids in succession
12
Q
beta sheet
A
- stable local structure
- stabilized by a network of H bonds
- antiparallel- neighboring beta strands run in opposite directions; loops around; strands line up and form straight, strong H bonds
- parallel- neighboring beta strands run in same direction; less stable than antiparallel due to angled H bonds
- 2-8 beta strands in a sheet
- stabilized by H bonds (carbonyl O and amide N)
- antiparallel is more stable and more common
13
Q
keratin
A
- structure is dominated by secondary structure
- coiled-coil of alpha helices
- two alpha keratin polypeptides twist around each together to form a helical coiled-coil
- coiled-coil- first coil comes from alpha helix and the second one comes from how the helices coil around each other
- the second coil wraps around due to the offset of 3.5 residues per turn
- forms a strong dimer (2 monomers)
- fibrous protein- long
- pseudo repeat in the primary structure (a-g) -> h-x-x-h-x-x-x
- typically not proline = x
- in position a and d there is a small hydrophobic residue -> forms a hydrophobic surface on the alpha helix
- hydrophobic surfaces on the helices are driven by hydrophobic effect
- hydrophobic sidechains line up on one side of the helix to form a packing interface
- presence of an imperfect repeating primary structure within each keratin monomer -> forms dimer
14
Q
which of the following changes is unlikely to alter the functional characteristics of alpha keratin
A
- substitution of a charge amino acid for a polar amino acid at position c of the 7-residue pesudorepeat
- h-x-x-h-x-x-x
- position c is an x meaning it doesnt matter what residue is there
15
Q
collagen
A
- triple helix containing nonstandard amino acids (not alpha -> three polypeptide chains)
- forms 3 left hand helix and form a triple
- proline prevents right handed
- fibrous protein
- structure is dominated by secondary structure
- 3 residue repeat in the primary structure -> Gly-X-Y
- x= usually proline
- y= hydroxyproline (hyp) or hydroxylysine (hyl)
- 3 residues per turn
- gly is stacked in the middle of the helix -> bc glycine is so small the helix can form very close interactions
- you would NOT find 3 glycine closely packed at the same height in the center of the triple helix bc staggered
- there are NOT equal # of proline and glycine
- proline prevents formation of alpha helices
- at any given position there is a glycine from one chain a proline from another and hyp or hyl from another -> staggered
- glycine allow for stable interaction
- collagen is formed by the hydroxyproline residue
- proline is modified by prolyl hydroxylase using ascorbic acid (vitamin C- co-factor) -> requires additional enzymes after release of the translated polypeptide chain from the ribosome
- cross-linked by fibrous network between lysine in one chain and hydroxylysine in another -> forms fibrils seen under microscope
- irreversible post-translation modifications are required to produce properly functioning collagen molecules
- cross-links that provide additional stability will form without the action of another protein
16
Q
R groups: secondary structure
A
- does not participate in secondary structure interactions
- however, certain amino acids are commonly found in certain structures
- measure the free energy of the helix and change one of the side chain residues of Alanine to every other amino acid one at a time to compare and identify
- the free energy of the new alpha helix is called ΔΔG
- alanine bc its the most favorable residue in a alpha helix
- ex. changing to glycine -> ΔΔG = 4.6 which is very high compared to alanine ΔΔG= .79 -> rarely see glycine in alpha helix
- proline residues break alpha helices and beta sheets -> ΔΔG= >4 -> proline is missing an amide H bc of the ring -> cant form stable H bonds
- alpha helices are often capped by Asn and Gln residues that can fold back and form h bonds with the helix
- rules are used to predict secondary from primary
17
Q
tertiary structure of myoglobin
A
- binds a molecule called heme -> uses to bind oxygen
- single subunit (no quaternary)
18
Q
determining the atomic resolution structures of proteins
A
- x-ray crystallography
- nuclear magnetic resonance
- cryoelectron microscopy
- must purify first!
19
Q
x-ray crystallography
A
- generate highly purified protein
- takes advantage of the fact if we throw an object at something at all different angles -> it will deflect off -> we can measure the angle and infer the shape
- we “throw” x-rays at our proteins to determine shape
- x-rays are small enough that they will interact with the electrons in the protein -> electrons will scatter the x-rays
- higher amplification more organized
- pack the proteins in a uniform arrangements in a protein crystal -> orients all the protein molecules in the exact same orientation -> amplifies the signal (static)
- we then can pass an x-ray beam through the protein crystal -> protein interacts and scatters/diffracts -> forms highly ordered spots that we can detect on an x-ray film
- diffraction pattern is formed we can back-calculate what the protein looks like
- what actually interacts with these x-ray are the electrons -> generates an electron density map -> model are atoms inside this electron density
- depending on the resolution we will have a higher/lower degree of confidence
- gives information on the primary and secondary structure of the protein as well due to high resolution
- carries a negative entropy term
- can determine quaternary
20
Q
resolution
A
-our ability to separate two objects
-high resolution- distance is small that you can separate these two objects
.5A- highest resolution
6A- lower resolution
-as resolution increases so does confidence
21
Q
nuclear magnetic resonance (NMR)
A
- produces ensembles of structures that reveal dynamic areas of tertiary structure
- expand this one dimensional shift spectra into 2 dimensional spectra
- we generate these plots by using the nuclear overhouser effect
- 1D plot is on one line and then we spread it out in 2 ways (separate it in two dimensions)
- were not doing this in a protein crystal -> we are using a protein that is dissolved in water (not static) -> allows us to see dynamic tertiary structure
- you get many structures are overlay them -> you notice that some elements of the structure are constant (stable secondary structure elements) while others are changing confirmations
- give you an idea about how proteins are moving in solution
22
Q
cryoelectron microscopy
A
- protein is highly purified and freezed -> one structure
- we vitrify the protein -> rapid freezing process that happens so fast that the water molecules cant form an ordered opaque structure -> transparent
- pass electrons through our specimen and they interact with our protein molecules -> scatter
- light microscopy with a magnetic lens to focus these electrons on a single image point
- if we do this many of times (million) and superimpose all of those molecules on top of each other -> average those to end up with very high resolution structures of our protein
- determined to <3.0A resolution (resolution is not as high as x-ray crystallography map)
- visualize tertiary structure without having to make protein crystals
- gives information on the secondary structure as well but the resolution is not high enough to infer about the primary structure
23
Q
Which is false
A
- NMR produces a dynamic picture of protein molecule in solution (true, the protein is not static)
- x-ray crystallography can give information on primary and secondary structure of protein (true, if we determine tertiary structure we can infer information about the amino acid residues)
- cryoEM can give information on secondary structure of protein (true, can give information about secondary structure but not high enough resolution to determine primary)
- *you will have greater confidence in crystal structure determine to a 3A then 1A (false, 1A will be a better resolution and more confidence)
24
Q
side chain location varies with polarity
A
- hydrophilic residues on the outside
- hydrophobic side chains aggregate inside the protein
- important for stable structure
25
Q
secondary structures form common motifs
A
- beta strand loops around forms a alpha helix loops around again and forms another beta strand that forms a beta sheet -> beta alpha beta loop
- beta barrel- network of sequential beta strand to form a barrel (green fluorescent protein GFP- acts as a tag)
26
Q
classification by motif
A
- draw topology diagrams to map out where all the secondary structure elements are location and interact
- allows us to note where the secondary structure elements are in the tertiary structure of a protein
- allows us to compare it to other proteins -> superimpose topology maps -> if structure is similar so is function
27
Q
intrinsically disordered proteins
A
- there are parts of proteins that adopt multiple shapes
- ex. p53 can be divided into 3 different parts -> middle- ordered; end terminal residues- disordered; C terminus- disordered
- polypeptide chain is constantly going to be moving
- malleable- can change shape
- C-terminus is constantly changing bc it interacts with multiple different proteins in different ways
- multiple functions multiple structures
- x-ray crystallography will only observe the middle portion
28
Q
large polypeptides form domains
A
- can fold into smaller subunits -> domains
- troponin C- barbell structure
- each globular side has its own tertiary structure and function -> its own domain
- autonomous
- polypeptides over 200 *residues fold into 2 or more domains
- small molecule binding sites are often located between domains
- not formed by the assembly of multiple protomers into an oligomer -> does not define its oligomeric state
- provide specific enzyme functions to multi-domain proteins
29
Q
structure is conserved more than sequence
A
-if we look at two different domains that carryout the same function and are found in two diff organisms -> their primary sequence will be very different but the structure will be similar
-domains are the fundamental unit of protein evolution:
they do this by providing a few different properties…
1. stable protein patterns
2. tolerate primary structure changes -> doesnt affect structure/function
3. tuned to essential biological functions -> we can take the same domain and duplicate it and if one loses its function the other domain can compensate; changing a residue can lose one function but adopt another
ex. cytochrome C needs to bind a molecule called heme
-if we looked at the sequence of residues in all different organisms they are all changed but function remains the same bc the structure is the same
-residues that are critical for structure and function did not change in any of the animals
30
Q
multiple subunits have quaternary structure
A
- subunit=protomer
- increasing the number of subunits is more efficient than increasing the length of the polypeptide chain
- multisubunit proteins form oligomers of many protomers
- oligomerization packs subunits together and forms interfaces allow for communication between protomers
- communicate through changes in their structure
- allow it to tune its function
- ex. if one subunit binds oxygen it will communicate and tell the other subunits to bind as well through structure changes
31
Q
subunits are symmetrically arranged
A
- DNA sliding clamp PCNA- 3 protomers
- bacteriophage MS2 Capsid- 180 protomers
32
Q
DNA sliding clamp PCNA
A
- symmetrically arranged
- 3 protomers
- binds and slides along DNA and unwinds it
- 3 fold rotational symmetry
- if we rotate molecules by 180 degrees all the subunits would superimpose on each other
33
Q
bacteriophage MS2 capsid
A
- symmetrically arranged
- 180 protomers
- often see in large oligomeric form
- capsid forms spontaneously on its own
34
Q
protein folding
A
-high energy state to low
35
Q
hydrophobic effect on protein stability
A
- hydrophobic effect has greatest effect on protein stability
- desire of hydrophobic sidechains to stay away from water and hide inside the protein
- folded protein is only 0.4kJ/mol more stable than unfolded proteins (proteins are not that stable)
- use properties of side chains to predict which sidechains will be buried inside protein (more hydrophobic deeper inside)
- do this by using a hydropathy plot
36
Q
hydropathy plot
A
- scores each amino acid based on how hydrophobic it is
- ex. Phe will have a positive score, lys will have a negative score
- we can then plot them on the primary sequence of the protein
- look for long stretches of hydrophobic residues
- predict the stretches will be inside the protein core
- important for membrane proteins (dont exist in aqueous state) -> predict of the protein may have transmembrane helices and which dont
37
Q
electrostatic interactions that contribute weakly to protein stability
A
- van der waals interactions are important in the interior of proteins (dipole induce dipole interactions (mainly see in buried nonpolar residues)
- hydrogen bonds make only minor contribution to stability but are important for defining folding pathway
- salt bridges are found on the exterior of proteins but are rarely conserved
38
Q
hydrogen bonds
A
-make only minor contribution to stability but are important for *defining folding pathway (not really involved in folding)
-help dictate what secondary structure is formed and at what rate -> dictates how the protein folds
-HOWEVER not directly involved in the folding
-
39
Q
salt bridges
A
- ionic interactions
- pos charge lys and neg charge glu -> if they are in close proximity -> salt bridge
- rarely conserved
- usually on the surface of proteins
- found in extremophiles (extreme conditions) -> help stabilize in these conditions
40
Q
denaturation
A
- bc proteins are not that stable they can be denatured
- loses native state
- increasing temperature
- exposing hydrophobic pores-> aggregating it
- unfolding proteins
- chemical denaturation
- detergents denature- SDS
- pH denatures- disrupts H bonds pattern -> lose tertiary structure -> unfold
41
Q
native state
A
-final folded state of the protein where it carries out its function
42
Q
denaturation by temperature
A
- S shaped curve -> process is cooperative (little by little and then all the sudden it unfolds rapidly)
- at the midpoint (50% folded) -> melting temperature (Tm) -> quantifies how stable our protein is
- each protein has its own Tm
- exposing hydrophobic pores-> aggregating it
- unfolding proteins
- changing an Ala residue thats buried in hydrophobic core of cytochrome C to Arg will affect the melting temperature of cytochrome C
