biochem topic 3

globular proteins

compact, rounded up balls

fibrous proteins

long, rope like

domain

polypeptide segment that folded into a single structural unit with hydrophobic interior

zinc fingers

20-60 residue strcture containing 1 or 2 Zn2+ ions

homodimer

protein containing identical peptide chains

hydrophobic effect

in protein folding, hydrophobic regions are located in the protein interior. unfolding the protein exposes these regions to water which is energetically unfavourable due to the disruption of hydrogen bonding network

all proteins are ____

flexible due to bonds within the peptide chain that allow for rotation, bending, stretching

metamorphic protein

have multiple possible conformations they can easily switch between, and are energetically equal

• alternate structures may exhibit different degrees of functionality or entirely different function

intrinsically disordered protein

have no fixed 3d shape

• exist as dynamic , flexible protein

• made of disordered protein segments

disordered protein segment

stretch of protein that doesn’t fold into a fixed 3d shape (alpha or beta)

jobs of disordered segments

1. Flexible Linkers & Hinges: They connect two rigid domains together like a rope, allowing the domains to move independently (just like the hinge in an antibody).

2. Molecular Springs: They can stretch under physical force and recoil back, acting like tiny rubber bands.

3. Shape-Shifting Binders: Because they are floppy, they can wrap around multiple different partner molecules, flexing into whatever shape fits each target.

4. Biomineralization (Bone & Teeth): They wrap around mineral ions (like calcium phosphate) to keep them dissolved so they don't form brittle, dangerous crystals where they shouldn't.

5. Dynamic Scaffolding ("Fuzzy Complexes"): They act as flexible backbones that hold multiple proteins together while allowing the whole complex to shift and fine-tune its activity.

6. Droplet Formation (Membraneless Organelles): Lots of disordered segments can entangle with each other like a web, causing the liquid inside the cell to separate into tiny, concentrated gel-like droplets (like oil droplets in water) to concentrate RNA and enzymes.

domain

subunits of protein, made of the 3-d arrangement of polypeptide chains

• can have different properties in one protein

protein famillies

group of evolutionarily related proteins that share common ancestor , charecterized by similar amino acids and 3d folding

conserved residues

residues that are critical for fnction that dont change on an evolutionary time scale

chromatography

• essential for purification of proteins from complex mixtures

• typically a column packed with a matrix of beads is used along with a buffered solution that is filtered through

size exclusion chromatography (gel filtration)

• each porous bead has a gel matrix inside

• small molecules can enter internal spaces of bead but not large

• small proteins will be trapped in the gel, while large ones exit the column early

• size and shape affect speed of exiting

ion exchange chromatography

• ion exchanges rely on attraction of opposite charges

• column is packed with porous charged beads

• pH of buffer determines protein’s net charge

• To get the stuck (red) protein off, you add a buffer with high salt concentration

Isoelectric point (pI)

the specific pH at which a molecule—most commonly an amino acid, peptide, or protein—carries a net electrical charge of zero.

Affinity Chromatography

• Separation based on molecular interactions

• ligand' = a compound that binds to a protein

• When the ligand is immobilized on the column matrix, only proteins with specific affinity for it will bind, while others do not

• The flow of buffer washes away unbound proteins, leaving only the specifically bound protein. • A powerful method of chromatography but requires prior knowledge of which ligand to use

Analysis of protein samples

• gel electroprosis

• NMR spectroscopy

• X-ray Crystallography

gel electrophoresis

• Charged molecules move toward the positive (+) electrode when current applied • Separates on the basis of polypeptide chain size

• A gel acts as a molecular sieve

• Smaller molecules migrate faster toward the bottom of the gel

Mass Spectrometry

• Using mass spectrometry to characterize proteins in complex mixtures (proteomes)

• Can provide info on identity (sequence), abundance, and various modifications

X-ray Crystallography

• Determine three-dimensional structures of proteins

• Purify protein of interest to homogeneity

• With X-ray crystallography, the protein must first be crystallized.

• When a crystal is bombarded with a narrow beams of X-rays, the electrons in the atoms scatter the X-rays, producing a diffraction pattern

• Computer analysis of the diffraction pattern generates a electron density map, which can then be used to build a 3D protein structure model.

NMR spectroscopy

-The 3D structures of relatively small proteins can be determined using NMR.

• Protein samples of high purity are required for NMR studies

• crystallization of the protein is not required

• With NMR spectroscopy, an ensemble of structures is obtained

poryphin

organic molecule that binds metal directly in its central pocket

ex. heme

Kd

ratio of free ligand and protein/bound complex

• measures affinity of bonding

Kd close to 1/big

indicates a higher ratio of unbound reactants, and a looser bond

Kd close to 0 /small

indicates more complex than free reactants, and a tighter bond

invariant residues

amino acids that are identical among famillies of protein, any changes would alter function

Y

ratio of substrate to complex (how many spaces are occupied?)

• Y closer to 1 indicates an abundance of substrate thus less space available

• Y close to 0 indicates less substrate and more space available

conservatively substituted residues

amino acids can be swapped for structurally similar ones while maintaining properties

cooperative binding

when protein with multiple binding site (like hemoglobin) increases its affinity for ligand, the more that ligand attaches

• sites talk to each other as ligand attatchment changes protein shape

deoxyhemoglobin

hemoblogin without oxygen

hemoglobin contains

2 alpha and 2 beta groups

T -state

tense state of hemoglobin

• low affinity for oxygen

• deoxyhemoglobin state

• fe lies out of heme plane(too large)

• rigid structure

R-state

• relaxed state of hemoglobin

• oxy form

• confirmation that’s formed once o2 has been bound

• O2 causes a histidine group to shift an alpha helix slice, causing the fe atom to move and lock into place

the backbone unit lies in a

single plane

• can only rotate about the alpha carbon

• rotation occurs between C-N and C-C

rotation about the C alpha - C is described by

psi angle

rotation about C alpha - N is described by

phi angle

primary structure

• amino acid sequence

• covalent bonds (peptide)

secondary structure

• H-bonding between backbone (dipole-dipole intramolecular force)

• alpha helix or beta-pleated sheet

tertiary structure

• non-covalent: H-bonds (backbone-backbone, backbone-side chain, side-chain-sidechain), Ionic bonds,Van der Waals interactions

• covalent:Disulfide bonds

• 3d shape

quaternary

multiple domains come together to form protein

• Stabilized by same bonds as for tertiary structure

Molecular Chaperones

Proteins (e.g., Hsp70, GroEL/GroES chaperonins) that prevent improper aggregation of exposed hydrophobic surfaces during folding in the crowded cellular environment

Hydrophobic Effect

Nonpolar/hydrophobic side chains aggregate in the interior of the protein to minimize ordered clathrate (water cage) structures, increasing overall solvent entropy

• entropy must increase so protein folding can occur on its own

alpha helix proteins produce structures resembling

something like a bottle brush or a round hairbrush

• desirable for small amino acids

• larger amino acids will sterically clash

irregular secondary structures include

turns, loops, helices and irregular conformations

many proteins have a mixture of

α-helices, β-sheets, and other types of folding patterns to form various overall shapes

beta pleated sheets are preferred by

larger amino acids which are too bulky for alpha helix

regarding proline and secondary structure

• its unique structure (ring that attaches itself to main body) forms bond angles that are too rigid

• since it closes on itself, there is no open spot for hydrogen bonds to form

glycine is unique for

• its tiny side chain

• is extremely flexible

• can’t stay in same position, also doesn’t have secondary structure

one major covalent bond that is also involved in tertiary structure, and that is the disulfide bond that can form between

cysteine residues

• This bond is important only in non-cytoplasmic proteins since there are enzyme systems present in the cytoplasm to remove disulfide bonds

domain

• an independently folded part of a protein that folds into a stable structure

• A protein may have many domains or consist only of a single domain

• Larger proteins generally consist of connected structural domains

• separated by loosely folded region

• can have different structure and functions

• a single polypeptide chain can fold into many domains

a fully folded, functional single polypeptide chain can be considered a protein

true

native state

conformation with least free energy (more stable)

subunit

separate polypeptide chains

Proteins can be unfolded or “denatured” by treatment with solvents that

disrupt weak bonds

examples of things that cause denaturation

• high concentration of urea (breaks H-bonds)

• disruption of H-bonding and hydrophobic core

• extreme pH

• extreme temp. (high)

Denatured proteins have a ______ conformation and lack _____

random, flexible. biological activity

denatured proteins often

aggregate and precipitate due to exposed core

membrane proteins

Proteins that are inserted into or tightly bound to membranes

proteases

proteins that break down other proteins

almost all proteins arise by

an evolutionary process

• new ones are derived from old ones

homologous proteins

proteins that come from the same ancestors

Studying the sequences of homologous proteins can give clues to

the structure and function of the protein

• conserved amino acids are usually vital for function and active site

• properties give insight of structure and folding and function!

the only covalent interaction between amino acids

cysteine bonds

hydrophobic amino acids bond through

vaan der waal forces

homodimer

protein made of 2 identical subunits

monomeric

single monomer

DEAE

• anion exchanger

• contains positively charged beads

CM

• cation exchanger

• negatively charged beads

disulfide bonds

• formed between cysteine side chain

• in oxidizing conditions

alpha helix secondary structure may be disrupted by

• adding urea

• steric hinderance of side groups (space)

• proline or glycine

• electrostatic repulsion of side groups

secondary structure is formed by H-bond of

amide and carbonyl group on the peptide ebackbone

hydrogen bond involves a donator and a

acceptor.

• in peptide bonds the donator is the nitrogen from amide (covalented bonded to H). the acceptor is the carbonyl

alpha helix is residues per turn and ___ A rise per turn

3.6 residues, 5.4 A

DEAE chromatagraphy targets ______ proteins, elutes with ______

negative (anion). elutes with Cl - (competes for positive beads)

CM chromatagraphy targets ______- and elutes with

cation . elutes with Na+ (also wants negative beads)

how do chaperones help protein folding

mask hydrophobic groups to prevent aggregation

• important for newly made proteins

SDS -PAGE

gel electroporsis

SDS detergent denatures and gives protein negative charge

electrode attracts proteins to positive end

smaller proteins move faster

seperate by size

cryo-em

study structure of large proteins that can’t crystalize

chaotrophic agents

like urea increase solubility of hydrophobic residues, denaturing

due to resonance between _____ bonds, the peptide bond has some double bond charecteristic

C-O and C-N

• carbonyl and amide