Exam 4 Flashcards
Amino acids contain a central _________ carbon
tetrahedral
Is water lost or gain in peptide bind formation?
loose water
What kind of bond is a peptide bond?
covalent
What enzyme catalyzes peptide bond formation?
Peptidyl transferase
What does a peptide bond join?
amino acids
What are the 4 types of amino acids?
- nonpolar/hydrophobic
- polar/hydrophilic
- acidic
- basic
Selenocysteine (SEC, U) is a rare amino acid, what does it look like?
cysteine with selenium replacing the sulfur
What are proteins that depend on Selenocysteines catalytic activity?
selenoenzymes
Pyrrolysine (Pyl, O) is a rare amino acid, what synthesized it?
synthesized from 2 L-lysine
Is the rare amino acid, Pyrrolysine (Pyl, O), positive or negative?
positive
The rare amino acids, Hydroxylysine (Hyl) and Hydroxyproline (Hyp) are post-translationally modified and are found in _____________
connective tissue (collagen)
The rare amino acid, Carboxyglutamic acid, is post-transcriptionally modified how?
glutamic acid undergoes carboxylation
The rare amino acid, Carboxyglutamic acid, is important for _____________
clotting factors
The rare amino acid, Pyroglutamic acid (PCA), is a cyclic form of __________
glutamic acid (glutamine)
Amino acids that do not occur in proteins are know to be responsible for __________ and ____________
hormones and neurotransmitters
What are essential amino acids?
cannot be synthesized from scratch by humans
What are conditionally essential amino acids?
obtained from diet
What are dispensable amino acids?
can be synthesized by humans
How many possible triplets (codons) are possible?
64
(63 without stop codon)
Why are there 64 possible triplets but only 20 amino acids?
Give 2 reasons
- If you increased the number of amino acids beyond 20 then it would be difficult to create suitable tRNA molecules that could be distinguished between one another by the aminoacyl-tRNA synthetase so that the correct amino acid could be added
- these 20 amino acids are fairly stable
What is known as the 21st amino acid and why is it not present anymore in organisms?
it was apart of the anaerobic world before there was oxygen present, but now there is lots of oxygen so its not useful
SELENOCYSTEINE
What is the cationic form of amino acids?
NH3+
COOH
What is the anionic form of amino acids?
NH2
COO-
What is the Zwitterion form of amino acids?
NH3+
COO-
amino acids are weakly _________ acids
polyprotic
What is a polyprotic acid?
has more than one acidic H+ so it can donate for than one H+
The degree of dissociation of amino acids depends on the ______ of the medium
pH
For amino acids that don’t have a dissociable side chain, the first thing to dissociate is _______________ and the second thing is _________
alpha carbonyl group
alpha amino group
What is the pKa of alpha carboxyl group?
2 (acidic)
What is the pKa of alpha amino group?
9 (basic)
What are optically active molecules?
can rotate the plane of polarized light
Optically active molecules are __________
chiral
What amino acid does not have a chiral carbon?
glycine
Chiral centers of amino acids allow for _____________(mirror images but not superimposable)
stereoisomerism (mirror images but not superimposable)
What are the mirror images of chiral amino acids called?
enantiomers
____-amino acids are the most common
L-amino acids
All amino acids absorb __________ wavelengths
infrared
What 3 amino acids absorb UV?
Phe
Tyr
Trp
_______ spectra are characteristic for each amino acid residue in proteins
NMR
What can NMR spectra tell you about a protein?
its 3D structure
Proteins are ______ polymers of amino acids
unbranched
amino acids join head-to-tail through formation of ____________ bonds (peptide)
covalent
The peptide backbone of a protein consists of the repeated sequence ___________
–N–C(alpha)–C(o)
In the peptide backbone sequence, –N–C(alpha)–C(o), what does the N stand for?
amide nitrogen (NH3)
In the peptide backbone sequence, –N–C(alpha)–C(o), what does the C(alpha) stand for?
alpha carbon
In the peptide backbone sequence, –N–C(alpha)–C(o), what does the C(o) stand for?
carbonyl carbon (COOH)
What conformation is the peptide bond usually found in?
trans
What is positive and negative in the peptide bond?
positive: N
negative: O
The peptide bond is ______ than single bonds but ______ than double bonds
shorter
longer
Due to the double bond character of the peptide bond, the size atoms of the bond define a ________ plane
amide plane
Is the double bond in peptide bonds usually on the O or N?
neither; it moves between both
What is each amino acid unit called?
residue
What is 2 residues called?
dipeptide
What is 3 residues called?
tripeptide
What is 12-20 residues called?
oligopeptide
What is 20+ (many) residues called?
polypeptide
What is one polypeptide chain called?
monomeric protein
What is more than one polypeptide chain called?
multimeric protein
What is a protein with one kind of chain called?
homomultimeric
What is a protein with two or more kinds of chains called?
heteromultimer
What is an example of a heteromultimer protein?
beta globin has 2 alpha nd 2 beta chains
Is the sequence if amino acids in a protein considered genetic information?
yes
What direction is the amino acid sequence read in?
amino to carboxyl terminus
What 2 groups interact to form a peptide bond?
alpha COOH and alpha NH3+
Peptide formation is the creation of an _________ bond between the carboxyl and amino group
amide bond
Peptide bonds are ___-planar
co-planar
What is the 2 planes (angles) on either side of the alpha carbon formed when a peptide bond occurs?
phi and psi
What are the 2 shapes of proteins?
fibrous
globular
What is the primary structure of proteins?
sequence
What are 3 example of secondary structures of proteins?
helix
sheet
loop/turns
What is the tertiary structure of proteins?
3D shapes (domains)
Are fibrous proteins water soluble or insoluble?
insoluble
What bridges are formed in primary structures of proteins?
disulfide bridges
Disulfide bonds occur in the primary structure of proteins between what amino acid and itself?
Cys
Can multiple secondary structures come together to form a unit?
yes
What is an example of a tetramer (quaternary structure)?
hemoglobin
What is the difference between configuration and conformation?
configuration: breaking bonds
conformation: no bond breaking
Proteins tend to be least soluble at thier ____________ point
isoelectric point
During protein purification, what happens first salting in or out?
salting in
Does salting in or our increase solubility?
salting in
What is the protein purification, Ion exchange?
separates proteins based on their net charge
What is the protein purification, Size exclusion?
separates proteins based on their size
During protein purification, what dramatically increases as the protein is purified?
specific activity
What is specific activity of a protein?
activity of the enzyme per mg of protein
What are 3 methods estimating protein concentrations?
Bradford assay
Lowry assay
BCA (purple complex)
Several methods of estimating protein concentration rely on the reduction of ______ to ______
Cu2+
Cu+
Determination of protein concentration by UV absorption depends on the presence of ____________
aromatic amino acids in proteins
__________ and ________ absorb UV light at 280nm (the very end of spectrum)
tyrosine
tryptophan
What 2 ways can proteins be sequenced?
- real amino acid sequencing
- sequencing the corresponding DNA in the gene
What are the 2 methods of amino acid sequencing that are used together ?
Sanger Method (one at a time)
Mass Spec (cleaves into fragments)
What are the steps of determining protein sequence?
- separate polypeptide chains
- remove S-S (disulfide bonds)
- identify N and C end residues
- cleave each polypeptide into smaller pieces and sequence (Edman and Mass Spec)
- repeat step 4 with different cleavage procedures to make different overlapping fragments
- using overlapping fragments to reconstruct the protein sequence
What end of the residue is susceptible to cleavage by Trypsin, Chymotrypsin, Clostripaon, Staph, and Cyanogen bromide in protein sequencing?
C
What part of the residue is susceptible to cleavage by pH 2.5?
Asp-Pro bonds
What are some uses of amino acid sequences?
- function of protein
- domain structure
- mosaicism in primary structure
- facilitate predications on higher order of structures
- facilitate construction of proves for genes
- evaluation of relationships between proteins (in a species of between species)
What are some biological function of proteins?
- enzymes
- control metabolism and gene expression
- transport proteins
- storage proteins
- movement
- structure
What are 3 structural methods to characterize proteins at atomic resolution?
- X-ray crystallography
- NMR
- Cryo-electron microscopy
Protein function depends on _________ and _______forces
structure
weak, non-covalent forces
The number of protein folding patterns is large but _________
finite
Structures of globular proteins are marginally __________
stable
marginal stability facilitates ________
motion
__________ enables function
motion
What are 4 examples of non-protein amino acids? (neurotransmitteres and hormones)
GABA
epinepherine
histamine
serotonin
Why do polar and non polar amino acids only have 2 pKa values?
one from the carboxyl and one from the amino group
Why do basic and acidic amino acids have 3 pKa values?
one for COOH
one for NH3
one for side chain
What wavelength do amino acids absorb at?
280
What is the length of peptide bonds?
0.133nm
What are the possible phi and psi angles?
nothing with a 0
180 and 180
-60 and 180
Secondary, tertiary, and quaternary structures of proteins are formed and stabilized by ________ forces
weak forces
____________ bonds are formed whenever possible
H bonds
___________ interactions drive protein folding
hydrophobic
__________ interaction usually occur on the surface of a protein
ionic
__________ interactions are ubiquitous
Van der waals
Phi is the angle between what 2 atoms?
C(alpha) – N
Psi is the angle between what 2 atoms?
C(alpha) – C
What does a Ramachandran Map show?
Sterically favored Phi and Psi angles (shaded)
Alpha helices are stabilized by ________
hydrogen bonds
When defining a helix, what is p?
pitch
When defining a helix, what is the number of repeating units?
n
Right handed helix have positive or negative “n”?
positive
Left handed helix have positive or negative “n”?
negative
In a helix hydrogen bonds all point in one direction causing…
dipole moment
in a helix hydrogen bonds form with near by donors and acceptor groups called …..
helix capping
What is a helical wheel?
when 2 helices interact with each other making a circle shape
What are 3 examples of “other” helices?
3(10) helix
pi-helix
left handed helix
What is the peptide unit for left and right handed helices?
left: 3.3
right: 3.6
What is the pitch of left and right handed helices?
left: 9.8
right: 5.4
Beta pleated sheets are made of…
beta strands
Why are antiparallel beta sheets more stable?
the H bonding is more optimum
What do beta turns allow?
peptide chain to reverse directions
How many residues are required for beta turns?
4
How are the atoms bonded in beta turns?
the carbonyl C on residue 1 is H bonded with the amide proton of residue 3
What amino acid is common in beta turns and why?
proline; restricts movement
What is type I beta turns?
proline in position 3
What is type II beta turns?
proline in position 2 and glycine at position 3
________ proteins create maximum internal bonds and minimize solvent contact
globular
___________ proteins create maximum intermolecular bonds and maximize molecule to molecule contact
fibrous
Helices and sheets are often packed ________
closely
Peptide segments between secondary structures tend to be ________ and direct
short
What are 2 ways stability arises in proteins?
- hydrogen bonding
- reduction of surface area (folding)
T/F?? Proteins are usually a mix between hydrophobic and philic?
true
T/F?? most of the polypeptide chain is organized approximately parallel to a single axis
true
fibrous proteins are usually strong/weak and soluble/insoluble?
strong
insoluble
Fibrous proteins usually play a ___________ role in nature
structural
What are 3 examples of fibrous proteins?
keratin
fibrin
collagen
Two right handed alpha helices intertwine to form a ______________ superhelix
left-handed
Left handed twists in coiled coils reduces what?
reduces the number of residues per turns
What does keratin look like?
2 dimers combine to make a antiparallel tetramer
What fibrous protein is a triple helix?
collagen
What is the most abundant protein in vertebrates?
collagen
Ramachandran and Kartha published the first ________________
triple helix
Three left handed helices intertwine to form a _____________ superhelix
right handed
What protein shape is the most diverse functionally?
globular
Are there more fibrous or globular proteins?
globular
What are the 4 classes of globular proteins?
- alpha proteins
- beta proteins
- alpha/beta
- alpha+beta
What are random coils?
segments of proteins that are not helices or sheets
don’t conform to recurring patterns
Why do secondary structures form whenever possible?
Hydrogen bonding
Are two adjacent helices usually parallel or antiparallel?
antiparallel
Up-and-down barrels have similar __________
topology
What are jelly role barrels made of?
beta sheets
How are beta helices made?
stacking of parallel beta sheets
____ strand and _____ strand beta sheets exist
2 and 3
Where is the active site in a/b barrels?
in the hole
Where is the active site in twisted a/b domains?
crevice outside the carboxyl end
non-covalent association is usually for ___________ proteins
globular
covalent association is usually for ____________ proteins
fibrous
Can proteins with one polypeptide chain have a quaternary structure?
no
What is 2 protein subunits called?
dimer
What is 3 protein subunits called?
trimer
What is 4 protein subunits called?
tetramer
What are identical protein subunits called?
homo—
What are non-identical protein subunits called?
hetero—–
What are the 5 advantages of quaternary structure?
- stability
- efficiency
- assembly
- cooperatively
- regulation
How are quaternary structures efficient?
small polymerizing units are synthesized more accurately than a single large protein
Quaternary structures assembles _________ sites
catalytic
What is simplest kind of symmetry?
rotational
How do you tell the fold of symmetry?
360/2 = 180
so the fold is 2
Multimeric proteins are symmetric arrangements of _____________ objects
asymmetric
What is isologous?
the same interface (rotational symmetry)
What is heterologous?
two different interfaces (translational symmetry)
Many proteins form tetramers by means of two sets of ______________ interactions
isologous
What is an example of an isologous interaction?
antibody
What are 3 examples of a polymerizing quaternary structure?
tubulin
Hep E
TMV (virus)
What is an example of a protein than can self assemble?
E. coli flagellar motor
What is the cause of flagella spinning so fas?
ions flowing in and out quickly
What is the rpm of flagella?
22,000
What is the cause of Osteogenesis imperfecta?
defect in collagen caused by malformation of protein structure
What is Creutzfeldt-Jacob disease caused by?
prions
What are prions?
misfolded proteins that are infectious to other proteins
What are 6 examples of diseases of protein folding?
- Alzheimer
- FAP
- Cancer
- Creutzfeldt-Jacob
- HE
- Cystic Fibrosis
What are post translational modifcations?
covalent changes to proteins after their biosynthesis
In PTM, functional groups serve as ___________
nucleophiles
Where do PTM occur?
amino acid side chains
N and C terminus
What are the 4 amino acid side chains that contain PTM?
hydroxyl
amine
thiol
carboxylate
What is the most common PTM?
phosphorylation
What amino acids are affected by phosphorylation?
Ser
Thr
Tyr
What functional group is affected by phosphorylation?
-OH
hydroxyl
What mediates phosphorylation?
protein kinase
Is phosphorylation reversible?
yes
What is the consequence of phosphorylation?
increased negative charge changing protein conformation and affecting positively charged ligands
What mediates dephosphorylation?
phosphatase
membrane bound receptor tyrosine kinases are activated by _________
dimerization
When receptor tyrosine kinases are unbound what do they look like?
monomer
proximity of kinase domains promotes ______________
tran-auto-phosphorylation
What does receptor tyrosine kinases activation cause?
downstream signaling
What is Src?
first discovered protein tyrosine kinases
What are the steps of Scr (protein tyrosine kinase)
- De-phosphorylation of C-terminal tyrosine frees SH2 domain
- SH3 dissociates from internal peptide and binds activating ligand
- kinase activation promotes autophosphorylation of tyrosine
- downstream signaling
What is the inactive state of Scr (protein tyrosine kinase) look like for SH2 and SH3?
SH2 bound to C-terminal phosphotyrosine
SH3 bound to internal peptide
High intracellular calcium activates and binds calineurin (phosphatase), how does this affect transcription?
- dephosphorylation of NFAT
- calineurin blocks nuclear export signal
- protein can enter nucleus =
YES transcription
Low intracellular calcium inactivated and removes calineurin (phosphatase), how does this affect transcription?
- nuclear export signal is exposed
- kinase phosphorylates and blocks nuclear import signal
NO transcription
o
o
What are the 3 types of lipidation?
- Myristoylation
- Palmitoylation
- Prenylation
In Myristoylation (lipidation), a myristol group is added where?
N terminal glycine
In Palmitoylation (lipidation), a palmitoyl group is added where?
cysteine
in Prenylation (lipidation), a farnesyl group is added where?
cysteine near C terminus
What facilitates the lipidation, Myristoylation (lipidation)?
N-myristol transferase
What facilitates the lipidation, Palmitoylation (lipidation)?
palmitoyltransferase
What facilitates the lipidation, Prenylation (lipidation)?
farnesyltransferase
Methylation is transfer of a methyl group from ________________ to other proteins
SAM
What 2 side chains are most common for methylation?
arginine
lysine
What mediates methylation?
methytransferase
What reverses methylation?
demethylases
What 2 places can acetylation be done?
lysine residue
N terminus
What mediates acetylation?
acetyltransferase
What reverses acetylation?
deacetylase
What is the most important role of acetylation?
regulating histones
Can methylation and acetylation occur on the same side chain?
no
Can multiple PTMs occur on a histone tail?
yes
Where does N-linked glycosylation start?
ER
What amino acid does N-linked glycosylation happen on?
N terminus of asparagine
What does N-linked glycosylation?
oligosaccharyl transferase
Oligosaccharyl transferase is a type of ___________
glycotransferase
Where does N-linked glycosylation continue?
golgi
Where does O-linked glycosylation occur?
golgi
In the Golgi sugars are added in chunks or one by one?
one by one
What amino acids does O-linked glycosylation happen on?
hydroxyl group of serine and threonine
What mediates O-linked glycosylation?
GalNac transferase
What does monoubiquitination regulate?
histones
What does multiubiquitination regulate?
endocytosis
which ubiquitination is most common?
polyubiquitination
What is the steps of ubiquitination?
- E1 attaches to ubiquitin
- E2 interacts with E1+ubiquitin
- E2 attaches E3 and E1+ubiquitin to target protein
What is E1 in ubiquitination?
activating enyzme
What is E2 in ubiquitination?
conjugating enzyme
What is E3 in ubiquitination?
ligase
What is Proteolysis?
cleaving protein bonds
Is Proteolysis reversible?
no
What are 2 exopeptidases (Proteolysis)?
aminopeptidase
carboxypeptidase
What ends of proteins does aminopeptidase
and carboxypeptidase target (Proteolysis)?
aminopeptidase : N
carboxypeptidase: C
What are 2 endopeptidases (Proteolysis)?
Trypsin
chymotrypsin
Where does the endopeptidase, Trypsin, target (Proteolysis)?
C terminus of Lys and Arg
Where does the endopeptidase, chymotrypsin, target (Proteolysis)?
C terminus of Phe, Tyr, and Trp
What are the 4 proteases that target functional groups?
Cysteine protease
Serine protease
Aspartic acid protease
Zinc metalloprotease
What amino acid does disulfide bonds occur between?
cystine
What does disulfide bonds promote?
protein folding
What is an example of a disulfide bond regulating protein folding?
protein disulfide isomerase
Folding of pro-insulin is stabilized by __________
disulfide bonds
_____________ of connecting peptides makes mature insulin
proteolysis
What are primary antibodies and secondary antibodies?
primary: specific for protein of interest
secondary: same as primary but has a tag that sends a signal
Western blot separates proteins by ________ and __________
size/charge
What is the advantage of immunohistochemistry?
can look cell to cell (different cell types)
Does western blot or immunohistochemistry use tissue samples?
immunohistochemistry
Does western blot or immunohistochemistry use fluorescence?
immunohistochemistry
Western blot can detect cleavage?
yes
What is the advantage of Tandem Mass Spec?
you don’t have to know the protein or the modification
How does Tandem Mass Spec work?
by measuring the changes in mass of the peptide sequences (controlled v. modified) you can tell where the changes are
What are the 2 methods of Tandem Mass Spec?
- Peptide Analysis MS
- Peptide Sequencing MS
How does the method Peptide Analysis MS work in Tandem Mass Spec?
you figure out the mass to charge ratio
How does the method Peptide Sequence MS work in Tandem Mass Spec?
fragment the pieces and look for slight shifts in mass from the control to see where and what modification is present
What is the activating step of Scr kinase?
dephosphorylation of SH2 domain
What PTM are not reversible?
lipidation
proteolysis
What functional group is subject to phosphate addition?
Hydroxyl
A protein undergoing proteolysis undergoes a __________ reaction and will have severed bonds between ___________ and __________ functional groups
hydrolysis
carboxyl and amino group
What amino acid does ubiquitantion occur on?
Lys
