Biochemistry Test 2 End of Ch. 3 and Ch. 4

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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


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