Biochemistry Test 2 End of Ch. 3 and Ch. 4
Chaotropic agents
Allows denaturing without cleaving disulfide bonds
fibrous
Arranged in strands or sheets and relatively simple
fully folded
Chromatography identification technique that is commonly used for preparative separation in which the protein is often able to remain______________
collagen fibrils
Collagen superstructures are formed by cross-linking (covalent bonds between Lys or HyLys, or His amino acid residues. ) of collagen triple-helices to form___________________
Intrinsically Disordered Proteins
Contain protein segments that lack definable structure, Composed of amino acids (lys, arg, glu, pro) whose higher concentration forces less-defined structure
they run alongside each other with C=O and NH is line with a C=O and NH resepctively. the h bonds are not strong because they are at an angle
Describe the orientation of a parallel Beta sheet and strength of h-bonds
The run alongside each other so that the NH is next to a C=O. The hbonds are straight so they are very strong
Describe the orientation of an anti- parallel Beta sheet and strength of h-bonds
glycine and proline rich left handed helix, three collagen chains intertwine into a right handed superhelical triple helix, very high tensile strength, many triple helices assemble into a collagen fibril
Describe the structure of collagen
4-hydroxyproline
Forces the proline ring into a favorable pucker which helps Offers more hydrogen bonds between the three strands of collagen
motifs (folds)
Globular proteins are composed of different ------ folded together
Beta Sheets
Held together by hydrogen bonds between the backbone amides (N-carboxyls) in different strands
most
If a protein has a specific function (e.g., binding insulin), the fraction that binds insulin best after each purification step will contain the _______ of the protein of interest
B.
If a protein is not folded correctly or becomes partially unfolded, what could NOT be a consequence? a. The protein may form an inactive aggregate that leads to disease. b. All of these consequences are possible. c. The protein may be degraded by the proteasome. d. The protein may be remodeled by a chaperone. e. The protein may be refolded.
H-bond
In an α helix, the amino acids get oriented in such a manner that the carbonyl, C=O, group of the nth amino acid can form a __________ with the amido, N-H, group of the (n+4)th amino acid
random coil
Irregular arrangement of the polypeptide chain is called the
Mass spectrometry
MALDI MS and ESI MS can precisely identify the mass of a peptide, and thus the amino acid sequence; can be used to determine posttranslational modifications
electrospray ionization, separation (MS-1), breakage via collision cell, MS-2, Detector
MALDI steps (5)
positive
Negatively charged residues often occur near the __________ end of the helix dipole
the electrophoresis is run perserving the molecules higher order structure
PAGE is native meaning that
trans
Peptide bonds not involving proline are ______
protomer, multimers
Protein with multiple peptides (also known as ____) are __________
3D conformation
Proteins adopt a specific
heat or cold pH extremes organic solvents chaotropic agents: urea and guanidinium hydrochloride
Proteins can be denatured by what four things
common secondary structure elements, reveals regions with unusual backbone structure
Ramachandran plots display two very useful things:
molecular weight
SDS PAGE separates via
a chemical denaturant may be added to remove this structure and turn the molecule into an unstructured molecule whose mobility depends only on its length and mass-to-charge ratio
SDS PAGE- non native meaning that
charge size affinity for a ligand solubility hydrophobicity thermal stability
Separation relies on differences in physical and chemical properties (6 things)
bc the alpha carbon is sp3 hybridized and all are L amino acids so one side chain goes up and the next points down
Side chains protrude from the beta sheet, alternating in an up-and-down direction. Why?
they lower sterics and they contribute to the hydrophobic nature of the helix
Small hydrophobic residues such as Ala and Leu are strong helix formers. Why?
motifs (folds)
Specific arrangement of several secondary structure elements, can be found as recurring structures in numerous proteins
globular
Spherical in shape multiple secondary structures
False
T or F. It is mathematically possible for proteins to fold randomly.
dipole moment
The alpha helix has a large macroscopic _____________
activity
The function of the protein is called the
native fold
This structure is able to fulfill a specific biological function and is called
Hydrogen bonding of amide and carbonyl groups of the peptide bond from opposite strands.
What's holding the strands in the beta sheets together?
e.
Which factor is LEAST likely to result in protein denaturation? a. disruption of weak interactions by boiling b. altering net charge by changing pH c. mixing with organic solvents such as acetone d. exposure to detergents e. changing the salt concentration
e. circular dichroism
Which method would be BEST to use to monitor protein secondary structure during the titration of a denaturing agent? la. iquid chromatography b. x-ray crystallography c. mass spectrometry d. electron microscopy e. circular dichroism
absorptivity is linked to conjugation and tyrptophan has two rings so its the best
Why are tryptophan, tyrosine and phenylaline such good chromophors, which is the best?
angle (phi and psi), r groups
_________ and ________ help determine secondary structure
Isoelectric Focusing
______________________ Can Be Used to Determine the pI of a Protein
hemoglobin
a functional quaternary protein formed by the coming together of four tertiary structures, called globin proteins
ionic bonds in tertiary structure
a type of chemical bond that involves the electrostatic attraction between oppositely charged ions
yes
can non-peptide bonds be rotated?
no
can peptide bonds be rotated?
connective tissue (ie tendons, cartilage, bones, cornea of the eye)
collagen is an important consituent of what
prosthetic groups
covalently attached cofactors
no b/c amides are not basic
does pH change beta sheets quickly
-The electric field pulls proteins according to their charge. -The gel matrix hinders mobility of proteins according to their size and shape.
electrophoresis two steps descripton (what it does, how gel separates them)
permanently charged groups
electrostatic interactions occur between ________ and occur over the longest distance
collagen
example of a fibrous protein
alpha helices and beta sheets
for H-bonding the interactions of N-H and C double bond O lead to what
slower, faster
for MALDI smaller molecules travel ______ when large molecules travel _____
stronger
for ion exchange the higher the charge the _________ the binding
-Hydrogen Bondings -Electrostatic/Ionic -Hydrophobic -van der Waals
four types of non covalent interactions effecting tertiary structure are
cofactor
functional non-amino acid component required for a proteins biological activity to happen
water soluble, hydrophobic, hydrophilic
globular proteins are ________, the inside consists of ________ amino acids while the outside consists of _____ amino acids
native fold
has a large number of favorable interactions within the protein
Proline (most of these consist of beta turns)
has about 6% isomers that are in the cis configuration
chaperones
help Prevent Misfolding and Aggregation of Unfolded Peptides, found when proteins were heat shocked
bc their native structure is thermodynamically favored
how can proteins fold so fast and correctly?
plot the log of the Mr standards against their relative migration (plug unknowns migration in to find log of weight)
how can you calculate the weight of a protein w/ SDS PAGE
very (inner d 4.5 A outer d is 10-12 A)
how compact is the alpha helix
break the S bonds to form SH bonds (reduce), then curl the hair, then oxidize forming new bonds between the S's
how do perms work?
by h-bonding within sheets, london dispersion interactions between sheets
how is the structure of silk fibroin stabilized
5.4 angstroms
how large is a turn in an alpha helix
4, proline in 2 (type 1), glycine in 3 (Type 2)
how many AA are used to accomplish a Beta turn and which two amino acids are common in beta turns at what positions
3.6
how many aa residues per turn in a right handed alpha helix
may cause certain amino acids to avoid water (so they'll be close to one another) effecting the folding of the protein
how might hydrophobic tendencies effect teriarty structure
small, large
in gel filtration, the _______ molecules tavel through the beads and the _______ molecules travel more quickly around the beads
MALDI
is an ionization technique that uses a laser energy absorbing matrix to create ions from large molecules with minimal fragmentation.
Quaternary Structure
is formed by the assembly of individual polypeptides into a larger functional cluster
electrophoresis
is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field
CD
measures the molar absorption difference of left- and right-circularly polarized light as chromophores in the chiral environment produce characteristic signals (peptide bonds depend on the chain conformation and therefore produce signals)
affinity
method (general not specific name) of separating biochemical mixtures based on a highly specific interaction between antigen and antibody, enzyme and substrate, or receptor and ligand
heme in myoglobin
name an example of a prosthetic group
hydrophobic effect, van der waals, salt bridges, h-bonds, electrostatic interactions
name five favorable interactions in proteins
primary: covalent secondary: h-bonding tert and quat: Ionic bonds, disulfide bonds, hydrophobic interactions, hydrogen bonding
name the interactions that stabilize each protein structure (primary, secondardy, tertiary quatenary)
Glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp)
name the nine hydrophobic amino acids
Coenzymes
organic cofactors or helper molecules
domains
proteins often have regions that can fold and function as an independent entity from the whole protein. These regions are
protein levels are maintained
proteostasis is the cellular process by which:
posttranslational modifications
refers to the covalent and generally enzymatic modification of proteins during or after protein biosynthesis, can occur on amino acid side chains or at the termini by modififyin an existing functional group
Ramachandran plot
shows the distribution f and y dihedral angles that are found in a protein
bind components, affinity wash off all components except the target protein (remains bound to ligand), elute the target protein and salts, run through size exclusion resin
steps for affinity sep. (4)
Edman Degradation
successive rounds of N-terminal modification, cleavage, and identification, can be used to identify protein with known sequence
fibroin, antiparallel beta sheet structure with small side chains to allow close packing (ala, gly)
the main protein in silk from moths and spiders, name it and describe its structure
tertiary structure
the overall spatial arrangement of atoms in a protein
carbohydrates, immunoglobulin G
the prosthetic group and example for glycoprotein
heme (iron porphydrin), hemoglobin
the prosthetic group and example for hemoproteins
phosphate groups, casein of milk
the prosthetic group and example for phosphoproteins
lipids
the prosthetic group lipoproteins
secondary structure
the spatial arrangement of the polypeptide backbone
aromatic AAs (tryptophan and tyrosine)
the strongest chromophores are:
entropy
there is an ________ cost to folding the protein into one specific native fold
alpha helices and beta sheets
two regular arrangements of the poly peptide backbone are?
identify
we can combine Isoelectric Focusing and SDS page to help __________ our protein
phi, psi
what angle represents the alpha C-amide N bond, what angle represents the alpha-C carbonyl C bond?
pH
what are electrostatic interactions dependent on
pro: No need to crystallize the protein; Can see many hydrogens/carbons/nitrogens cons: Difficult for insoluble proteins; Works best with small proteins
what are the pros and cons to NMR
pros: no size limit, well established cons: difficult for membrane proteins and binding proteins, can't see H's
what are the pros and cons to xray crystallography (2 each)
electrostatic interactions
what are the strongest interactions between proteins
parallel and anti parallel
what are the two major orientations of beta sheets
Edman degradation and Mass spectrometry
what are the two ways of determining protein sequence
fibrous and globular proteins
what are two major classes of tertiary structure
polyacrylamide gel electrophoresis
what is PAGE
functional non-amino acid component, metal ions or organic molecules
what is a cofactor, name some examples
NAD+ in lactate dehydrogenase
what is an example of a coenzyme
two alpha helices
what makes up alpha keratine
amino acids
what makes up the primary structure of a protein
is formed by the assembly of individual polypeptides into a larger functional cluster
what makes up the quatenary structure of a protein
alpha helices and beta sheets
what makes up the secondary structure of a protein
fibrous and globular arrangements stabilized by weak interactions in amino side chains
what makes up the tertiary structure of a protein
hydrogen bonds between adjacent segments that may not be nearby
what stabilizes a beta sheet
hydrogen bonds between nearby residues
what stabilizes an alpha helix
numerous weak interactions between amino acid side chains (hydrophobic and polar interactions, disulfide bonds)
what stabilizes tertiary structure
amino is positive, carboxyl is negative
what terminus of the helix dipole is positive and which is negative
least reactive carbonyl, rigid and nearly planar, dipole moment favors trans configuration
what three things does resonance define about the peptide bond?
phosphorylation
whats the most common example of posttranslational modification
when strands in beta sheets change directions
when do beta turns occur
out
where do the side chains point in the right handed alpha helix
right, right
which are more common, right handed helices or left handed helices and which are longer
MALDI
which is more gentle MALDI or Edman
they cause steric crowding, can give a chance to form favorable h-bonding interactions along the backbone
why are some phi and psi combinations disfavored while others are favored
They have a second resonance form where the lone pair on the N will donate
why can't peptide bonds be rotated?
some bacteria make d-amino acids or glycine may be involved
why do left handed helices show up in nature
its tiny R-group makes it too flexible, supporting more than just the helix conformation
why does glycine act as a helix breaker
due to its amine group existing within a ring, it has different angles which make rotation from the N-alpha C (phi) angle impossible and therefore hard to fit into the coil
why does proline act as a helix breaker?
when examining new projects over the psi angle at -45 for right and 45 for left there is a strong steric hindrance for the left
why is the right handed favored
