CH 4 (L8 + LE +L9) Flashcards
Peptide Bond
comprised of the atoms involved in the peptide bond (C-N) and their four substituents
Resonance Imaging
indicates the bond C-N bond here is shorter than typical C-N single bonds but longer than the typical C=N double bonds
- -> similar with the carbonyl oxygen: slightly longer than typical C=O double bonds
- typically represented as resonance hybrids
planar peptide bonds
Peptide bonds have a second resonance form. This means that the peptide bond (the C=O and N-H) all reside in a single plane. Thus, there is no rotation around that bond.
Resonance forms of the peptide bond: double bond can be between C and O or C and N
trans vs cis
because of the double bond nature of the peptide bond in the peptide group, two conformations are possible: cis (Same) and trans (Across)
- these refer to the position of the alpha-carbons on the plane
- these positions arise when peptides are being synthesized by the ribosomes
trans: alpha-C across plane
cis: alpha-C same plane
cis
less favorable than trans because of STERIC interference between the side chains
Steric effects are the effects seen in molecules that come from the fact that atoms occupy space. When atoms are put close to each other, this costs energy. The electrons near the atoms want to stay away from each other. This can change the way molecules want to react.
Rotations
the rotation around the N-Cα is called phi
the rotation around the C-Cα bond is psi
Ramachandran Plots
calculated values of phi and psi to determine best steric permissibility in a peptide
- the plot is based on observations from hundreds of proteins with known structure
- the observed angles are also plotted in areas not theoretically sterically permissible
cis vs trans
often switch to cis from trans (cis/trans isomerization) leads to loss of function in proteins
- cis/trans isomerases can do this and thereby activate or deactivate TFs
Linus Pauling
(double Laureate)
- proposed alpha helix structure in 1950 with robert corey
- their model took into account observations made from repeating patterns in alpha-keratin
the alpha helix
pitch of 0.54 nm and a rise of 0.15 nm
- these patters were calculated by x-ray crystallography
right-handed alpha helix
stick out right thumb
follow curvature from the base to the tip of the thumb
now look at the helix
- what conformation do you see
the helix can be right or left-handed but most are right-handed
the alpha-helix
each carbonyl oxygen (residue n) is hydrogen bonded to the amide-hydrogen of the residue 4 residues down towards the C-terminus (n+4)
- the 3 amino groups at one end and 3 carboxyl groups at the other lack these H bonds
- R groups point outward from the cylinder and can affect structure
R groups
Alanine is usually found in alpha helices
- tyrosine and asparagine are less common
- glycine tends to destabilize the alpha helix because rotation around its alpha-carbon is unconstrained so they appear in the beginning or end
- proline disrupts the right-handed helical structure and cannot participate in H bonding
the alpha helix
proteins vary in how many alpha helices they contain
- helices vary from 4-5aa to .40aa in length
- turns out the alpha helical structure is very common in nature
- it was discovered to be present in hemoglobin and many other proteins
- it also exists in DNA
plotting
helix is turning to the right (right-handed)
- hydrophilic are clustered in one side (together)-> at surface of protein to interact with water
helical wheel
many alpha helices are amphipathic, with hydrophilic residues (face towards water) on one side and hydrophobic residues (face away from water)on the other - they can be drawn like a wheel
horse live alcohol dehydrogenase
an example of an amphipathic alpha helix is highlighted
leucine zippers
an example of two amphipathic alpha helices coming together is the leucine zipper
- their hydrophobic faces are in contact, specifically the leucine and other residues that form hydrophobic interactions
the 3 sub 10 helix
unlike alpha helices, the 3 sub 10 helices form H bonds between the carbonyl oxygen and the amide hydrogen of residue (n+3) –> this structure makes it tighter
it is less stable because of steric hindrances and awkward geometry
beta structure
- include beta strands (almost fully extended regions) and beta sheets (beta strands that are aligned)
- also proposed by pauling and corey
- beta sheets are stabilized by H bonds between carbonyl oxygen and amide hydrogen on adjacent strands
parallel beta sheets
run parallel to one another in the same direction N –> C terminus
antiparallel beta sheets
- run anti-parallel (arrow indicates N –> C terminus direction)
- they can be connected between regions on the same peptide or between different peptides
- anti-parallel sheet is more stable as the H bonds are not distorted
pleated beta sheet
beta sheet is sometimes called the pleated beta sheet because the planar peptide groups meet each other at angles
- some liken it onto an accordion
- the R-groups alternate above and below the plant of the sheet
- comprised of 2-15 strands
influenza virus A neuraminidase
- example of antiparallel beta sheets from the same peptide
- beta strands can be thought of as the true secondary structure of the protein
- beta sheets form once tertiary or quaternary structure has formed
hydrophobic/ hydrophilic interactions
as in alpha helices, beta sheets can contain alternating side chains that project above or below the sheet plane to create amphipathic sheets
- as with pollen protein, this creates the ability for hydrophobic intramolecular interactions (blue) and hydrophilic interactions outside (orange) with solvent
loops and turns
within a given peptide, loops and turns abound which can connect strands or helices
loops
often composed of hydrophilic residues allowing them to exist freely in solution or are found near the surface of a protein
turns
loops with less than or equal to 5 residues are call turns if they change the direction of the peptide
reverse turns aka Beta turns
most common as they usually connect beta strands
Type I and Type II beta turns
4aa structures stabilized by H bonding
- both produce ~180 turns
- -> Type II contains glycine (Gly) as the 3rd residue (60% of the time)
turns
peptides can and often do have many turns
- they are often outside the theoretically proposed Ramachandran plots for angles
- -> glycine, eg, causes less side-chain steric hindrance than other residues because its R group is just a Hydrogen atom
motifs
- folding of a polypeptide results in a closely packed 3D structure –> amino acids that are far apart can now interact–> these structures are stabilized by non-covalent interactions (with the exception of disulfide bridges)
- -> these structures that form are often recognizable structures called MOTIFS, which are collections of alpha helices, beta strands and loops
common motifs
1) helix-loop-helix (HLH)
2) coiled coil (CC)
3) helix bundle (HB)
4) beta alpha beta unit (βαβ unit)
5) hairpin (H)
6) beta meander (β-m)
7) greek key (GK)
8) beta-sandwich (β-s)
helix-loop-helix (HLH)
common structure in calcium-binding proteins
–> in dna binding proteins, this structure is modified and appears as a helix-turn-helix motif
2) coiled coil (CC)
consists of two amphipathic alpha helices
- interact by leucine zipper
- hydrophobic residue interactions in the interior
- looks like a twisted coil cord
3) helix bundle (HB)
- several alpha helices interacting produces a helix bundle
- tend to contain several different orientations
- bundle of alpha helices
4) beta alpha beta unit (βαβ unit)
- two parallel beta strands linked via an alpha helix
- connected by two loops
- the alpha helix connects the C-terminus of one beta strand to the N-terminus of the other
- structure looks like:
beta strand (up), a loop, an alpha helix, a loop, beta strand (up)
5) hairpin
- two adjacent beta strands (antiparallel) connected by a beta turn
- different between a turn and a loop? turns are loops that are less than or equal to 5 residues long
structure looks like:
beta strand (up), turn, beta strand (down)
6) beta meander (β-m)
- antiparallel beta sheet motif connected by loops and turns
- they follow each other in their peptide sequence
- typically joined by larger loops but can be connected by hairpins
structure looks like: a lot of hairpins joined together --> beta strand (up), turn, beta strand (down), beta strand (up), turn, beta strand (down)
7) greek key (GK)
- named for a design in classic Greek pottery
- beta sheet (antiparallel beta strands) created from 4 beta strands with 1-2 in the middle and 3-4 in the outside
structure looks like:
BS1 (down), BS2 (up), BS3 (down), BS4 (up)
BS1 connected to BS4 by a turn, BS2 connected to BS3, BS3 connected to BS4
8) beta-sandwich (β-s)
formed by beta strands or sheets (antiparallel) stacked on top of one another
can be connected by loops and turns
- can exist as beta strands far apart
structure looks like:
2 antiparallel beta strands (connected by loop/turn) stacked on top of each other
Q9 at the end of Chapter 3
know how to do this problem
Mycoplasma pneumoniae
- understanding the protein composition and functionality of multiunit proteins helps us understand the biochemistry of living organism
- the larger multiunit proteins are sometimes even large enough to be seen in electron micrographs
- M. pneumoniae is a pathogen classified as the causative agent of atypical bacteria pneumonia (fluid in air sacs)
- in kids, it causes tracheobronchitis
- mild disease sometimes “walking pneumonia”
- no PGN, resistant to antibacterials
- outbreaks: schools, dorms, military barracks, nursing homes
- community-acquired
- spread by respiratory droplets from sneezing and coughing
bacterial flagella
- a motor protein composed of multiple units that come together to form a functional motor that propels bacteria through liquid media
- great example of quaternary structure
- world’s most efficient motor”
association vs. dissociation
- some multiunit protein subunits associate so strongly, they rarely dissociate
- some associate very briefly and quickly dissociate
- what brings them together? (what associates them) –> weak interactions: H bonds, charge-charge, van der Waals, hydrophobic interactions
Enolase
enzyme in glycolysis (2PG –> PEP)
Comprised of 2 subunits
- brought together because of complementary H bonding
Association constant (Ka)
- the association of peptides into quaternary structure from their constituents can be expressed as an association constant Ka
- the concentration of the associated quaternary form [P1:P2] divided by the concentration of the constituents [P1][P2] gives us Ka
P1+P2 P1:P2
Formula:
Ka = [P1:P2] / [P1][P2]
Dissociation constant (Kd)
- the dissociation of peptides from quaternary structure into their constituents can be expressed as a dissociation constant Kd
- the concentration of the dissociated constituents divided by the concentration of the associated protein gives us Kd
Formula: Kd = [P1][P2] / [P1:P2]
Association constants
typical association constants range from 10^8 M-1 to 10^14 M-1
- the lower relevant range would be around 10^4 M-1
- to break it down: the higher the [P1] and [P2], the higher the likelihood that these will interact
Associations
- the association of the TFs that bring in RNA Pol to a promoter ranges from 10^5 to 10^7 M-1
Why would these be relevant?
–> the greater the binding, presumably the more it can affect RNA Pol activity
At 1, what state is all the protein in?
represents the concentration at which 1/2 the molecules are free and 1/2 in complex or the reciprocal of K2
The interactome
- determining the interactions of proteins in organism led to the development of interactome maps
- these lead to a better understanding of organism in biochemistry
–> an interactome is the whole set of molecular interactions in a particular cell
denaturation
disruption of the native form (the conformation it normally/naturally goes into)of a protein
–> environmental changed are typically needed to accomplish this, some proteins are more easily denatured than others
Heat
proteins have optimal temp. at which they form and in which they operate
- adding heat (energy) to a system will denature proteins
- ribonuclease A –> was heat denatured (y axis = denatured fraction)
Protein Melting Temperature (Tm)
- most proteins have a characteristic Tm
- this represents the temperature wherein the protein is halfway between transitioning from native state to denatured (midpoiint of the graph)
chemical denaturation
- chaotropic agents disrupt H bonding in water
- common chemicals used to denature are urea and guanidium chloride are thought to allow water molecules to solvate nonpolar groups in the interior of proteins
- SDS is also thought to denature by penetrating a protein’s interior
Disulfide Bridges
- proximity of cysteine residues in 3D space allows the possibility of disulfide bridges to be formed in a protein
- these are covalent bonds that can give a lot of structural stability to a folded protein
- cysteine residues don’t have to be close - on the peptide, they are found on amino acids 26 and 84 –> they are not close together in sequence, but are close together in 3D space
- reaction requires oxidation of thiol groups, thought to be performed by oxidized glutathione
denaturation/ renaturation
- bovine ribonuclease A has been studied as a model for denaturation and renaturation
- in the presence of urea and 2-mercaptoethanol, the protein is denatured
- different conditions can produce an inactive form rather than renatured form
- a fully renatured form can result
How to fold
- that ribonuclease A could be denatured and would renature by itself when removed from denaturing conditions points to the concept that:
- -> Peptide folding instructions are contained within the amino acid sequence
- in vivo when improper disulfide bridges form, enzymes like protein disulfide isomerase (PDI) can catalyze reduction of the incorrect bonds
Energy well
as nascent peptides are being born from ribosomes, the prevailing thought is that they are slowly going into their native conformation and into a low-energy state thereby making it more stable
- most believe they can fold spontaneously to form these structures
- one change induces another and so forth
forces keeping things together
aside from cysteine disulfide bridges, weak forces are thought to keep peptides together:
- h bonds, charge-charge interactions, van der waals forces, hydrophobic interactions
molten globules
weak interactions are independently weak but en masse are thought to stabilize proteins
- because they are weak, they can give a protein flexibility and resilience to undergo small conformational changes in response to environmental changes
- multidomain proteins are thought to fold domains independently, more than 300 aa and folding would go too slowly
- the intermediates forming as it folds are called molten globules
Folding
models would support that as proteins fold, they form partial secondary structures and rapidly change conformation until they reach their native state
Hydrophobic effect
proteins are more stable in water when their hydrophobic residues are hidden in the core
- this is thought to make the protein collapse into a more compact molten globule
- this effect is also thought to drive polar groups into the interior that neutralize their charge by H bonding to one another
Hydrogen bonds
- help with folding and stabilizing structures
- if formed in the interior of the protein, they are more stable than those that form on the surface because they don’t compete with water molecules
van der waals and charge-charge interactions
- the contribution of these forces are hard to calculate and test in a given polypeptide though they are thought to contribute some stability
- most ionic side chains are found at the surface of proteins, though if in the interior, they’d presumably be stronger than those on the outside because they would not compete with water
chaperoning the fold
folding is NOT considered a random process
- tertiary structure depends on primary structure
- incorrect folding can slow down proper folding or lead to a state that falls out of solution
- cells contain molecular chaperones that enhance protein folding. they bind both newly folded peptides and unassembled protein subunits or those that aggregate
Heat Shock Proteins (HSPs)
- class of chaperone that is activated in response to changes in temperature
- they aid the cell in helping denatured proteins refold. this is important for survival
- the major HSP is Hsp70 found in all species except some archaebacteria called DnaK in bacteria
example: E.coli chaperonin GroE - has a space in the middle where unassembled protein goes
Chaperone Proteins
chaperonin assists proteins into tertiary form
Process as follows:
Unfolded polypeptide goes inside chaperone (nATP - energy required) –> energy released (nADP + nPi) –> chaperone releases now folded polypeptide
Human Type-III Collagen
left-handed, triple helical structure with diverse functions from skin to tendons
- 3aa/turn, 0.94 nm pitch
- extended structure stabilized by interchain hydrogen bonds
- rich in G-X-Y repeats
- -> X is often proline
- -> Y is often modified proline
5-Hydroxylysine
- collagen contains a modified lysine that are covalently bonded to carbohydrate residues making collagen a GLYCOPROTEIN
- both hydroxylysine and hydroxypoline are hydroxylated after incorporation into polypeptide, the enzymatic pathway requires ascorbic acid (vitamin C)
Schiff Base
cross-linking between the helices also occurs when some of the side chains of lysine and hydroxylysine are converted to ALDEHYDE (-COH) groups to make ALLYSINE and HYDROXYALLYSINE
- these will react with neighboring lysine residues to form Schiff bases that form between carbonyl groups and amines
The end result of this reaction is a compound in which the C=O double bond is replaced by a C=N double bond. This type of compound is known as an imine, or Schiff base.
Cross-linking
two allysine residues can also condense to form intramolecular cross-links
