CH 4 The three-dimensional structure of proteins

native confirmation

three-dimensional shapes of proteins with biological activity

primary structure

• the order in which the amino acids in a protein are linked by peptide bonds

• 1-dimensional

secondary structure

• arrangement in space of the atoms in the peptide backbone (C=O and N-H) using H-bonds

• conformation of the backbone constrains the possible arrangement of the side chains in tertiary structure

• a-helix and beta-pleated sheet

• confirmation of the R group/side chains is not part of secondary structure

tertiary structure

• includes the 3-dimensional arrangement of all the atoms in the protein

• includes R group/side chain

• include prosthetic groups

• include noncovalent interactions (hydrophobic, electrostatic(saltbridge), h-bonding), ionic, disulfide bridges

domains/super-secondary structure

• specific clusters of secondary structural motifs in proteins

• combinations of alpha and beta strands produce these

prosthetic group

• portions of proteins that do not consist of amino acids

• Ex) heme group in myoglobin and hemoglobin

subunits

multiple polypeptide chains that make up a larger molecule/ complete protein

quaternary structure

arrangement of subunits with respect to one another mediated by noncovalent interactions (h- bonding, electrostatic, hydrophobic)

2 to 4 separate chains

sickle cell is caused by a change in 1 amino acid residue, which causes a normal healthy RBC to become sickle shaped and not bind O2 efficiently

give an example how primary structure effects protein function

• bond between a-carbon and carbonyl carbon

• bond between a-carbon and amino nitrogen

what bond rotations in amino acids allow for secondary structures

a-helix

• stabilized by h-bonds parallel to the helix axis

• from N-terminus the C-O group of each AA is h-bonded to the N-H group of the AA sequence 4 AA residues away in the sequence

• 3.6 residues per turn

• pitch (the linear distance between corresponding points on successive turns)= 5.4A or 5.4×10^-8cm

confirmation allowed linear arrangement of atoms involved in h-bonds, straight= maximum bond strength

what makes a-helix stable

• proline

• electrostatic repulsion: due to proximity of charged R groups

• steric repulsion: due to proximity of several bulky side chains(all chains outside helix and crowding can occur if bonded with two atoms other than hydrogen)

name some things that can disrupt an a-helix

• creates a bend in backbone due to cyclic structure

• rotation around bond between a-carbon and N is restricted

• prolines alpha-amino group cannot participate in H bonding

why does proline disrupt an a-helix

beta-pleated sheets

• protein backbone is almost fully extended with h-bonding between adjacent structures (antiparallel orientation allows for maximum hydrogen bonding because the oxygen, nitrogen, and hydrogen are in a straight line)

• h-bonds can form between residues on the same or different chains

• h-bonds are perpendicular to the direction of the protein chain

interchain bonds

h-bonds formed between different chains

intrachain bonds

h-bonds formed between residues on the same chain

parallel pleated sheet

peptide chain runs in same direction (aligned in terms of N and C terminus)

antiparallel pleated sheet

peptide chains run in alternating directions

• irregular structure

  • can break up helix

  • found in short stretches

  • 3 residues per turn and 10 atoms in the ring formed by making the h-bond

what is a 3(10) helix

beta bulge

• common non-repetitive irregularity found in antiparallel beta pleated sheets

• occurs between 2 normal beta structure h-bonds and involves 2 residues on one strand and on on the other

• irregular structure

reverse turn

parts of proteins where the polypeptide folds back on itself

• Glycine (less crowding) and proline (cyclic structure) often seen at these

BetaAlphaBeta unit

• supersecondary structure

• 2 parallel beta sheets are connected by a stretch of alpha helix

AlphaAlpha unit

• supersecondary structure

• 2 antiparallel alpha helices so that energetically favorable contacts exist between side chains of the helices

Beta-meander

• supersecondary structure

• antiparallel sheet formed by a series of tight reverse turns connecting stretches of polypeptide chain

Greek Key

• supersecondary structure

• polypeptide chain doubles back on itself

motif

term for a repetitive supersecondary structure that can tell us about the folding of a protein but not the function

collagen fiber

• 3 polypeptide chains wrapped around each other in a triple matrix

  • Left-handed polypeptide helices are twisted together to form a right-handed superhelical structure

• every 3rd position has a Gly so that every 3rd residue on each chain is inside the helix

• found in connective tissue and bone

  • Well packed, rigid, triple helix structure responsible for characteristic tensile strength

• X-Pro-Gly

• X-Hyp-Gly

  • where X= any amino acid

  • where Hyp=hydroxyproline (proline with -OH on 4th carbon)

what’s the amino acid sequence for a collage fiber?

• less stable

• can cause scurvy and bleeding gums etc, asorbic acid (vitamin C) is needed to keep the enzyme active and hydroxylate proline

what happens to collagen without hydroxyproline

fibrous proteins

proteins whose overall shape is that of a long, narrow rod and contain long stretches of regular secondary structure such as coils in keratin and the triple helix of collagen

*not soluble in water

• Ex) collagen, fibroin, keratin

Globular proteins

proteins whose overall shape is more or less spherical, helical and pleated sheet sections can be arranged so as to bridge the ends of the sequence close together

*soluble in water

Ex) myoglobin

• Covalent

  • peptide bond between AAs in backbone

  • Disulfide bonds between cysteine AAs (will restrict folding patterns)

• Non-covalent

  • H-bonding: backbone H bonding and between side chains

  • hydrophobic: nonpolar residues cluster in the interior of a protein molecule

  • electrostatic: attraction between oppositely charges groups on surface of a molecule brings groups closer together

  • complexed: side chains give electrons and support a metal ion or something else (heme group)

name the different forces involved in creating tertiary structure

Lowest

A protein folds into the shape with the ____ possible energy oftentimes stabilized by numerous weak covalent bonds

X-ray crystallography

requires proteins to crystalize, beam exposure to crystal creates a diffraction pattern on a photographic plate or radiation counter

• # of electrons in each atom of the molecule scatter the rays and the # of electrons in each atom determine the intensity of the rays (heavier atoms scatter more effectively)

• taken at a series of angles

• scattered rays lead to constructive and destructive interference giving each protein a unique pattern

• can’t use method if protein won’t crystalize

Nuclear magnetic resonance (NMR)

uses proteins in an aqueous solution (most similar to cell environment) to determine 3D structure

Myoglobin

• needed for oxygen storage in muscle

• globular and composed of 1 polypeptide chain (153 amino acids)

• contains 1 heme group

• 8 a-helical regions and 0 beta-pleated sheets

• polar residues on outside and nonpolar found on the inside (except for two polar His residues in the interior)

• Fe(II)/Fe2+

• protoporyprin IX

• 6 binding sites

What’s in a heme group

• His F8: the N atom of the imidazole side chain occupies 1 binding site of the heme group, perp and opposite form binding site, occupation makes sure that only 1 oxygen can bind

• His E7: sterically hinders molecules, decreasing heme affinity for CO2 and bending the O2 ensures that heme will let go

What do the two His residues do for myoglobin

• 4 Nitrogen from the protoporphyrin

• 1 nitrogen from the imidazole side chain of the His F8 reside

• 1 Oxygen molecule

what occupies each of the 6 heme binding sites

hydrophobic interior holds heme group in place and His F8 prevents heme group from becoming free Heme and oxidizing to Fe(III) which will not bind O2

why is the combination of protein and heme needed for O2 storage

denaturation

the unraveling of the 3D structure of a macromolecule caused by the breakdown of noncovalent interactions

• heat

• extreme pH

• detergents

• urea and guanidine

• beta-Mercaptoethanol

list the ways proteins can be denatured

increase temp, increase molecule vibration, disrupt bond and tertiary structure

how does heat denature proteins

charges on protein missing= loss of electrostatic interactions that would normally stabilize native molecule

how does extreme pH denature proteins

disrupt hydrophobic or electrostatic interactions if detergent is charged (SDS)

how do detergents denature proteins

both form h-bonds with protein that are stronger than native bonds withing the protein itself

how do urea and guanidine denature proteins

reduce disulfide bridges to 2 sulhydryl groups

how does beta-Mercaptoethanol denature proteins

1 subunit =1polypeptide chain, chains can combine to dorm dimers, trimers, and tetramers

What’s a subunit

oligomer

• aggregate of several smaller units(monomers)bonding may be covalent or noncovalent

• Ex) hemoglobin is an oligomer, while the alpha and beta subunits are still polymers but hemoglobin as a whole being only 4 units is considered one with each subunit being considered a monomer

allosteric

property of multisubunit proteins such that a conformational change in one subunit induces a change in another subunit

hemoglobin

• O2 transport to tissues

• tetramer, 4 polypeptide chains: a2b2

  • a: 141 residues

  • b: 146 residues

• 4 heme groups

mean the same AA residues are in the same position

what is meant by homologous in regards to the relationship between hemoglobin a and b chains to myoglobin

positive cooperativity

a cooperative effect whereby binding of the first ligand to an enzyme or protein cause the affinity for the next ligan to be higher

hyperbolic

a characteristic of a curve on a graph such that it rises quickly and then levels out

• Ex) myoglobin

sigmoidal

referring to an S-shaped curve on a graph, characteristic of cooperative interactions

• Ex) hemoglobin

Hemoglobin’s curve being lower that myoglobin’s means at any pressure myoglobin will have a higher saturation percentage than hemoglobin (will always have a higher affinity for O2)

What’s the relationship between Hemo and Myo on binding curve

• in lungs O2 pressure=100 torr= 100% oxygen saturation

• in capillaries/active muscles= 26 torr= <50% oxygen saturation

  • positive cooperativity in reverse: if hemo loses 1 O2 it gets easier to lose the rest

How does Hemo’s transport and release of O2 work

binding of O2 and other molecules like CO2 and H+ change the confirmation of the protein which could change function

(O2 brings the b chains closer together)

(H+ and CO2 affect hemo affinity for O2 by decreasing it)

explain the allosteric changes hemo has with O2, CO2, and H+

• Cell metabolizes releasing CO2 and H2O

• carbonic anhydrase turns it into H2CO3

• pKa of H2CO3 is 6.35 while blood is 7.4 so it immediately drops its H becoming H+ +HCO3-

• H+ makes the environment more acidic

• H+ protonates key amino acids on hemoglobin such as N-terminals of the alpha- chains and His146 of the beta -chains

• protonated His is attracted to, and stabilized by, a salt bridge to Asp94. This favors the deoxygenated form of hemoglobin.

• hemo releases O2 to the cell or wherever its needed

• The oxygenated form of hemoglobin is a stronger acid (has a lower pKa) meaning it wants to give up H more than deoxygenated form

  • In other words, deoxygenated hemoglobin has a higher affinity for H+ than does the oxygenated form.

explain how H+ affects decreases hemoglobin affinity for O2

• cell metabolizes giving off H2O and CO2

• carbonic anhydrase combines into H2CO3

• pKa of H2CO3 is 6.35 while blood is 7.4 so it immediately drops its H becoming H+ +HCO3-

  • 90% of dissolved CO2 is in this form

• mainly transported to the lungs by being dissolved in blood

• Hemo binds to O2 at lungs and releases H+

• H+ and HCO3- recombine to H2CO3

• H2CO3 dissociates to CO2 and H2O

  • lungs have low CO2 so moves down concentration gradient

• CO2 is breathed out

• **at high CO2 concentrations hemo binds CO2 as carbamate

  • alpha amino terminals into anions, which can then interact with the -chain Arg141, also stabilizing the deoxygenated form.

explain how CO2 is transported to the lungs

• lowers hemoglobin’s affinity for oxygen, meaning hemoglobin needs a higher pO₂ (more torr) to reach the same saturation.

  • With BPG: partial pressure at which 50% saturation is at 26 torr

  • Without BPG: 50% saturation is at 1 torr

• No BPG means little O2 would be released from capillaries

how does 2,3-bisphosphylate (BPG) affect oxygen affinity

• fetal hemo is a2y2 where y2 doesn’t bind BPG as strongly

  • one less bond/contact due to Ser instead of His at the salt bridge, neutral instead of + charge)

• = fetal hemo has a higher affinity for oxygen

  • needs to steal from mom so it has to be stronger than mom’s affinity if they were equal strength O2 wouldn’t go anywhere or at least not as effectively

explain how fetal hemo allows the transfer of oxygen from mom to baby

bioinformatics

application of computer methods to processing large amounts of information in biochem

homology

finding known sequences in polymers, finding a similar known sequence in a database and comparing it to the knew protein but must be >25-30% similarity

fold recognition

comparison with well-known folding motifs common to many secondary structures

de novo prediction

bases on 1st principles from chem, biology, and physics confirmed by X-ray crystallography

phospholipids

polar head and nonpolar tail creating a lipid bilayer

liposome

spherical aggregates of lipids arranged so that polar heads face outside and in contact w/ water while nonpolar tails are inside sequestered from water its a spontaneous process (deltaSuniverse>0)

• H2O surrounding nonpolar molecules can bind with each other but have fewer possibilities than if they were surrounded by other water molecules which prevents dispersion of energy

• each droplet of nonpolar material disrups H2Os ability to h-bond to more H2O

• Fewer H2O molecules are inconvenienced by just one big droplet (layers) than a lot of smaller ones (mixed)

why is liposome formation and layer separation a spontaneous process

can form aggregates due to exposed hydrophobic regions that end up on the surface instead of inside which can interact with other hydrophobic regions on other proteins

what happens if proteins don’t fold correctly

prevent misfolding and speed the folding process which prevents aggregates and sometimes require ATP

• Ex) Reduce (SH-containing protein disulfide isomerase(PDI) catalyzes the rearrangement of polypeptides non-native disulfide bonds via disulfide interchange reactions to yield native disulfide bonds

what do protein chaperones do

• more alpha chain genes than beta (2:1)

• means excess of a chain

• excess a chains can form aggregates

• aggregates=damaged red blood cells

• AHSP chaperones a chains to b chains to prevent aggregates and so that there will be enough a chains for b chains

why is the hemoglobin stabilizing protein AHSP important

planar, atoms, plane

the ____ character of the peptide group limits the conformational flexibility of the polypeptide chain and means ___ of the peptide bond are located in a single ____

trans, cis, steric hinderance

peptide groups with few exceptions assume the ___ confirmation in which successive C atoms are on opposite sides of the peptide bond joining them. ___ confirmation can result in _____ in which the atoms would orient closer than their van der waals distance which results in repulsion

psi (Ѱ)

angle between alpha carbon and the carboxy group can rotate

phi (ᵩ)

angle between alpha carbon and amino group

Type I reverse turn

third residue rotated so that the R group (gold) faces outwards and any amino acid can occupy this position

Type II reverse turn

Type I is rotated 180 degrees so that the R group faces inside, the only R group that can fit in this space is the H in Glycine so only Glycine can occupy this 3rd residue position

• proline can also occupy the second residue position and help with the turn

Coiled coil

2–7 alpha-helices are twisted together like strands of a rope, facilitating protein-protein interactions.

• View down the coil axis shows the alignment of nonpolar residues along one side of each helix. The helices have the pseudo repeating sequence a-b-c-d-e-f-g in which residues a and d are predominately nonpolar

• 2 keratin polypeptides form a dimeric coiled coil

• protofilaments are formed from 2 staggered rows of head-to-tail associated coiled coil

• protofilaments dimerize to form a protofibril, four dimers form a microfibril

typically high with Ala, Leu, Arg, and Cys

found in hair, nails, horns, etc

describe alpha- keratin structure

• One polypeptide chain folds to form four beta strands which twist to form the distorted beta sheet. Two sheets assemble to form a beta keratin filament 

• more rigid and water proof

describe beta-keratin structure

irregularities in secondary strucutres

any place where a protein's backbone does not adopt one of the regular repeating patterns of an α-helix or β-sheet.

• loops: connect a-helices

• turns: allow reverse in direction for alpha or beta sheets (mostly beta)

• beta bulge

• kink in an a-helices(usually proline)

name some irregularities in secondary structures

certain amino acids tend to exist more in an alpha or beta sheet

what are alpha helix and beta sheet propensities

separated: Nonpolar residues tend to occur in the protein interior and polar residues on the exterior

• In an a-helix from sperm whale myoglobin the nonpolar residues are primarily on one side of the helix with polar on the other (A)

• An antiparallel beta-sheet from concanavalin A. The protein interior is to the right and has nonpolar amino acids and the exterior to the left with polar AA (B)

where do the locations of polar and nonpolar R groups tend to exist on alpha helix and beta sheet

symmetrical

The majority of soluble and membrane bound proteins in modern cells are _______oligomeric complexes with two or more subunits. Cyclic, dihedral, and tetrahedral

Hydrophobic effect

• nonpolar substances minimize their contacts with water

  • Major determinant of native protein structure

• The more positive the value the more hydrophobic are the amino acids located in that region of the protein

• A large positive hydropathic index indicated a hydrophobic region of the polypeptide, whereas a large negative value indicates a hydrophilic region

• Upper bars denote the proteins interior and lower bars denote the proteins exterior regions as determined by X-ray crystallography

describe hydropathy value

Intrinsically disordered proteins

unstructured in solution but becomes folded structure when interacting with another protein or substance

Ex) CREB

• The backbone of the kinase-induced domain (KID) of CREB is drawn as a purple worm. It is unstructured when free in solution but becomes two perpendicular a helices when it forms a complex with the KID- binding domain of another protein 

high, high, low, low

folding follows a pathway from ___ energy and ___ entropy to ___ energy and ____ entropy. Driving force of the process is noncovalent interactions (h-bonds) that give off energy= lose energy when folding

hierarchical

Protein folding is a cooperative _______ process in which small elements of structure form coalesce into larger elements which coalesce to yield larger elements (primary—> tertiary)

• GroES cap binds to GroEL ring to induce a conformational change in the cis ring that moves the improperly folded protein into the cis cavity to be refolded

• catalyzes the hydrolysis of 7 ATPs to 7 ADP and release 7 Ps in order to refold the protein

• second molecule of improperly folded protein binds to the trans ring as well as 7 more ATP

• cis ring releases GroES cap, ADP, and the properly folded protein

• molecule turns 180 degrees such that trans ring becomes the cis ring that is to be capped by GroES and the previous cis ring becomes the new trans ring

describe the GroEL/ES protein chaperone process

Alzheimers

• Forms plaques of amyloid deposits of Amyloid betta protein that are surrounded by a halo of neurites (axons and dendrites) from dead and dying neurons

• Amyloid-beta accumulates in the brain 10-15 years before tau, a protein that stabilizes microtubules(in cells), aggregates filaments in neurons (tangles) (tau=intracellular neurofibrillary)

Creutzfeldt_Jakob Disease (CJD)

degenerative neurological disorder that is incurable and invariable fatal caused by prions, or misfolded proteins that replicate by converting their properly folded counterparts in their host to the same misfolded structure