Biochem Test 2

Enzymatic Cleavage

• Trypsin → cleaves after K and R

• V8 protease → cleaves after E and D

• Chymotrypsin → cleaves after WYFL

• Pepsin → cleaves in between or after WYFL, preserves disulfide bridges

• Thermolysin → cleaves before hydrophobic aa

Proline Rule

the enzymes won’t cut bonds if Proline is after the target aa

Cyanogen Bromide

• CNBr

• cuts after M

• since proteins dont have a lot of M → the protein is cut into larger pieces

Hydroxlamine

• NH2OH

• Cuts between G and N

BNPS- Skatole & O-iodobenzoic acid

• cuts after W

Edman Degradation (8)

• Edman reagent reacts with the N-terminus of the polypeptide

• product → PTH aa that absorbs at 254 nm

• The C-terminus is held in place by a matrix while the protein is washed

• Cycle 1 → cuts off the first aa and identifies it

• Cycle 2 → cuts off the second aa and identifies it

• isn’t efficient for larger proteins

• small protein (100-200 aa) → ED is reliable for 50 cycles

• large protein (2000 aa) → ED is reliable for 10-20 cycles

Carboxypeptidases (5)

• cut off aa from the C-terminus end and give you the sequences backwards

• determines sequence by looking at the order that aa appear in the solution

• can be difficult to tell sequence since some aa can appear in the solution at almost the same time

• Carboxypetidase A → prefers aromatic and branched aa

• Carboxypeptidase B → prefers basic aa (HKR)

ESI (2)

• adds lots of protons onto a protein

• sample is sprayed as fine droplets through a high voltage needle

MALDI

• adds one proton to protein

ESI MS (2)

• as you go to the left → m/z decreases, Z increases

• each peak differs by one proton

Isotopic Distrubution

spacing = 1/z

homologs

proteins with similar sequences

orthologs

homologous proteins found in different species

paralogs

homologous proteins found in the same species

BLAST Substitution Matrix

• determines the probability of an aa being substituted by another aa

• positive score → can be replaced

• negative score → can’t be replaced

Features of a peptide bond (8)

• rigid due to resonance

• partial double bond → longer than SB, shorter than DB

• coplanar

• can’t freely spin or rotate

• Partial positive on NH

• Partial negative on CO

• NH is a H bond donor

• CO is a H bond acceptor

Which conformation are peptide bonds in

trans conformation so that R groups are far from each other

Where do rare cis conformations occur in peptide bonds

right before proline

Phi

angle between alpha C and N

Psi

angle between alpha C and carbonyl C

Ramachandran Plot (4)

• x axis → phi

• y axis → psi

• darker shaded region → more stable

• white region → less stable

Where is each helix found on a Ramachandran plot

• beta sheet → (-phi, +psi)

• right handed alpha helix → (-phi, -psi)

• left handed alpha helix → (+phi, +psi)

Ramachandran plot exceptions (3)

• glycine → large range of accepted phi and psi angles since side chain is just

• Proline → phi is fixed at 60 degrees since side chain connects back into the backbone

• branched side chains → small range of accepted phi and psi angles since beta carbon restricts rotation

Which proteins are the most stable

the ones with the most weak intercations

Weak interactions and their locations (4)

• Van der waals → everywhere

• ionic interactions → surface of protein

• hydrophobic interactions → core, main driving force for folding

• hydrogen bonds → wherever possible (between polar and charged groups)

What are secondary structures and what are the types

• secondary structures are the local folding patterns formed by the protein backbone

• helices

• beta sheet

• beta turn

• coils (intrinscially disordered proteins)

What are super secondary structures and what are the types (4)

• super secondary structures are extensions of secondary structures

• beta hairpins

• beta-alpha-beta

• coiled coils

d=p/n

d → rise per residue

p → pitch

n → residues per turn (rarely a whole number)

Helices are either

right handed (+) or left handed (-)

Alpha helix (4)

• 3.6 resides per turn

• 5.4 pitch

• 1.5 rise per residue

• has 12 residues with a length of 18

handedness of alpha helix

• alpha helices are almost always right handed so side chains are far apart

• left handed alpha helices are rare and tend to contain glycine

alpha helices form hydrogen bonds

within the same chain

Hydrogen bonding in alpha helix (4)

• the carbonyl O and NH that it is bonded to has 3 carbonyls between them

• all hydrogen bonds point in the same direction (creates partial positive on O and partial neg on NH)

• max hydrogen bonds → n-4

• the first 4 amides and last 4 carbonyls don’t have hydrogen bonding

Every loop closed by a hydrogen bond in an alpha helix has

13 atoms

Calculating # of turns

turns = # of residues/n

calculating length

length = # of residues x d

determining if the sequence is long enough

• do length = # of residues x d

• compare that to the given bilayer value

calculating number of amino acids from bilayer thickness

• number of residues = bilayer thickness/d

• if it gives the thickness in nm → do given nm x 10

• round up if you’re answer is a decimal

• if theres a part b and it asks you to calculate the # of amino acids needed for a different type of strand → use the same formula

calculating number of amino acids from bilayer thickness for 2 strands of beta hair pin

• do number of residues = bilayer thickness/d

• multiply your answer by 2

310 helix

• 3 residues per turn

• 6 pitch

Beta structures form H bonds

between different sections or strands

what are beta sheets and what are the two types

• beta sheets are strands from different regions of the polypeptide chain that are linked together by H bonding

• Parallel sheets → chains run in the same direction, causes bent H bonds that are less stable

• antiparallel sheets → chains run in opposite directions, causes straight H bonds that are more stable

parallel beta sheet n and d value

• n = 2

• d = 3.2

antiparallel n and d value

• n=2

• d=3.4

how many strands do beta sheets contain

2-22 strands

parallel beta sheets have

hydrophobic side chains on both sides

antiparallel beta sheets have

hydrohobic side chains on one side

True or false: beta sheets can be a mic of parallel and antiparallel

true

Handedness of beta sheets

right handed twist

Beta turns allow a protein to

reverse it’s direction

H bonding in Beta turns occurs

between carbonyl O of 1st amino acid and NH that is 4 carbons away from it

Beta turns are found

on the outer surface of proteins

Type 1

common orentation

Type 2

180 flip of middle peptide bond

which aa are found in Beta turns

proline and glycine

Beta turns help form

antiparallel Beta sheets by connecting adjacent strands

Random coils are

loop regions of varying lengths and shapes that connect alpha helices and beta sheets

Random coils are not

random or coiled → they just don’t have a recurring bonding pattern

random coils are located

on the surface of proteins

Hydrogen bonding of random coils

• random coils form H bonds with surrounding water molecules

Random coils contain what type of amino acid?

lots of charged and polar aa

conservation and flexibility of random coils

• not conserved → their sequence can vary

• flexible

Proteins function in what state

proteins function in a partially unfolded state and have high flexibility

Intrinsically unstructured proteins (2)

• unstructured by themselves but adopt a well defined structure when they bind to target proteins or ligands

• use a large surface area when interacting with other molecules

Intrinsically unstructured proteins contain what type of aa

lots of polar aa

Intrinsically unstructured proteins lack

hydrophobic aa which keeps them form folding up tightly like normal proteins

amino acid preferences (4)

• aa have the tendency to form certain secondary structures but there are no set rules

• alpha helix → glutamine (Q) and Methionine

• beta sheet → aa with longer or branched chains

• beta turn → NSPG

Coiled coils

• made up of 2 or more helices that wind around each other to form a supercoil/super helix

handedness of coiled coils

left handed twist

coiled coil n (2)

n=3.5

side chains repeat positions every 7 aa

stability of coiled coils (2)

• hydrophobic aa are buried inside where 2 helices touch

• further stability comes from ionic interactions

beta hairpin

• contains 2 adjacent antiparallel strands connected by a short loop

• seen in trypsin inhibitors

Beta-alpha-beta motif (3)

• 2 adjacent parallel beta strands

• alpha helix connects two strands

• 1st loop is conserved → aa sequence remains the same

Handedness of proteins (4)

• proteins tend to twist slightly in a right handed direction

• beta hairpin loops back easily → common

• parallel strands with right handed loop → common since connecting loop is right handed

• parallel strands with left handed loop → rare

a protein domain is

a part of a protein that can fold into a stable tertiary structure in an aqueous solution

a domain is a single continuous chunk of amino acids

protein domains (4)

• 250 aa in length

• have their own specific independent function

• can combine to make multi domain proteins

• 90% of domains reoccur in different proteins

large proteins contain

more domains, but not larger domains

proteins with multiple domains evolved through

gene fusion

when is entropy lost?

when protein pieces join together, they lose random movement

when is entropy gained?

when protein peieces join together, their hydrophobic parts get buried inside their core

multimeric assumbies

proteins made of different parts are symmetric arrangements built out of asymetric parts

what are the most common protein structures

• dimers (2 subunits)

• tetramers (4 subunits)

cells code for

small subunits and repeat the emall subunits to build large proteins

when proteins bind together, and are created at the _

catalytic sites and regulatory sites are created where the proteins touch

when a molecule binds to a subunit ___ occurs and ___

a conformational change occurs in the subunit, and that subunit communicates with the next subunits making then more or less ready to bind to their own molecules

even though residues are paked tightly together

small avities (tiny empty spaces) are present

____ % of a proteins volume is ___

25% of a proteins volume is empty space

cavities provide ___

flexability that helps conformation changes and movement

is proetin folding driven by H or S

it can be driven by both

• H → main contributor to protein folding

• S → stabilizes folded proteins

are proteins stable or unstable

proteins are slightly unstable which allows rfor movement and assembly of structure

denaturation is

extrenal stress such as heat or chemical treatmemt that causes a protein to lose its 3D shape and function

once a protein hits a specefic temp

it starts rapidly unfolding and loses its function

which structures form first and what is that driven by

secondary structures form first and are driven by hydrogen bonding

the folding process includes

one or more intermideate states, including a transition state called molten gobules (paritally packed protein strucutres)

chaperons are ___ and are reffered to as

found in all cells and are reffered to as HSP (heat shock proteins)

3 primary chaperons

HSP 60, HSP 70, HSP 90

HSP 60 and HSP 70

help new proteins to fold properly

HSP 90

is the chaperone for single transduction proteins in eukaryotes

what drives protein folding

the binding and hydrolysis of ATP by the 3 chaperones

Chaperons keep proteins from

forming clumps by blocking unproductive interactions between hydrophobic aa untile productive interactions can occur