biotech exam 2: lectures 10-13

• alpha-carbon

• amino group

• carboxylic acid group

• hydrogen atom

• R group or side chain

Components of amino acids

peptide bond

bond that links amino acids to form polypeptide chains

carboxyl of one amino acid and amino of the other

A peptide bond forms between….

N-terminus —> C-terminus

Directionality of polypeptide chain synthesis

disulfide bond

a covalent bond formed by the oxidation of 2 cysteine residues in a polypeptide chain

primary structure

amino acid sequence of a protein

secondary structure

a regular pattern of hydrogen bonds between peptide amino and carboxyl groups of amino acids that are near one another in linear sequence

• alpha helix

• beta pleated sheet

alpha helix

A single polypeptide chain twists around on itself to form a rigid cylinder

beta sheet

forms from neighboring segments of polypeptide backbone that run parallel or antiparallel to its immediate neighbor

tertiary structure

full 3D spatial arranegment of a polypeptide chain

• disulfide bonds

• hydrogen bonds

• ionic bonds

• hydrophobic interactions

Bonds present in tertiary structure

protein domains

compact modular units of 40-350 contiguous amino acids that fold more or less independently, resulting in different functions

quarternary structure

complete conformation of a protein that is formed as a complex of 1+ polypeptide chian subunits

native protein

a protein in its folded and active form

denatured proteins

a randomly coiled peptide that loses its normal activity due to disruption of weak, non-covalent bonds that stabilize tertiary structure

• Its primary structure is not affected

• heating

• chemical denaturants (urea and guanidium chloride)

• oxidizing/reducing agents

What causes protein denaturation?

protein biosynthesis

translation of nucleotide sequences into amino acid sequences that occurs at the ribosome

genetic code

set of rules defining how the four-letter code of DNA is translated into the 20-letter code of amino acids

codon

a set of 3-letter combos of nucleotides that correspond to specific amino acid or stop signal

tRNA

adaptor molecule in protein synthesis

amino acid recongition site

template-recognition site (anticodon)

Components of tRNA

attached to a hydroxyl group of the adenosine residue in the tRNA amino acid attachment site of the CCA sequence at the 3’ end

What does an activated amino acid look like?

anticodon loop

makes the 3 bases that constitute the anticodon accessible for interaction with an mRNA anticodon

1. Formation of an adenylated amino acid (has adenine)

2. Attachment of amino acid to corresponding tRNA, making aminoacyl-tRNA

2 steps to attaching amino acid to tRNA to make aminoacyl-tRNA

aminoacyl-tRNA synthetase

enzyme that catalyzes the amino acid reaction and linkage of aminoa cid to tRNA

when an aminoa cid is coupled ot its respect tRNA molecule

When does a tRNA become activated?

• synthesized from N-terminus to C-terminus, attaching peptidyl-tRNA

• mRNA: from 5’ to 3’

• Stepwise addition: 1 codon at a time

How are amino acids added to synthesize protein?

ribosome

molecular machines that coordinate the interplay of aminoacyl-tRNAs, mRNA, and proteins

• composed of large and small subunit that contain proteins and rRNAs

A site: aminoacyl-tRNA, where amino acids with tRNAs are delivered

P site: peptidyl-tRNA, where new peptide bond is formed between aminoacyl-tRNA in P site and aminoacyl-tRNA in A site

E site: exit, where tRNA is released form the ribosome

small ribosomal subunit: mRNA binding site

Components of ribosome

antibiotics

inhibit protein synthesis

Tetracylcine

binds to 30S subunit and blocks binding of aminoacyl-tRNA to A site (bacteria)

Streptomycin

prevents transition from initiation to elongation, and causes miscoding (bacteria)

Chlormaphenicol

blocks peptidyl transferase reaction on ribosome, inhibiting 50S subunit (bacteria)

Erythromycin

binds in exit tunnel of ribosome/50S subunit, inhibiting elongation of peptide chain (bacteria)

Puromycin

causes premature chain termination by acting as an analong to aminoacyl-tRNA (bacteria and eukaryotes)

Cycloheximide

inhibits translocation (eukaryotes)

posttranslational modifications

Alterations in protein structure after it has been synthesized in cell

• covalent attachment of groups to amino acid side chains

• cleavage/modification of polypeptide backbone

Types of posttranslational modifications

stabilizes fibers of newly synthesized collagen

Hydroxylation of Proline

Vitamin C deficiency = insufficient hydroxylation of collagen

Abnormal collagen fibers are unable to maintain normal tissue strength

What causes scurvy?

fluorescence is formed by the spontaneous rearrangement and oxidation of Ser-Tyr-Gly sequence at center of protein

• serve as markers within cells

Green Fluorescent Protein (GFP)

chaperone

a protein that interacts with, stabilizes, or helps another protein to acquire its functionally active conformation without being present in its final structure

heat-shock proteins (hsp)

chaperones that respond to any increase in misfolded proteins and help proteins refold

Imporperly folded proteins and deposition of protein aggregates (amyloid fibrils/plaques)

• Normally soluble proteins are converted into insoluble fibrils rich in beta-sheets

What causes neurological diseases like Alzheimers and Parkinsons?

defects in a membrane-bound protein CFTR chloride ion channel

• due to mutation in CFTR (F508del) —> improper protein folding, causing CFTR to not reach cell membrane

What causes cystic fibrosis?

1. Small molecule pharmacological chaperones: Stabilize specific misfolding-prone or mutant proteins, pulling them towards a lower free energy minimum

2. Inhibition of aggregation of specific amyloid-prone protein: stabilizes native state or partially folded state, preventing amyloid formation

What are some drug discovery strategies that restore protein folding and function?

stabilize CFTR by filling internal cavity, preventing premature degradation

How do CFTR correctors like Lumacaftor and Tezacaftor treat CF?

L1/2

concentration of ligand L at which half of the receptor R is bound to L and half is free

antibodies/immunoglobulin

protein produced by beta lymphocytes in response to a foreign antigen

variable region

part of antibody that binds to an antigen

Estorgen binding is strong, while testosterone binding is weak

How does estrogen binding to estrogen receptor compare to testosterone?

monoclonal antibodies

designed to target specific molecules in disease

12 domains that adopt the Y-shaped fold structure

2 light (L) chains and 2 heavy (H) chains linked by disulfide bonds

Structure of antibody

through specific hypervariable loops

• immunoglobulin fold: has 2 beta sheets linked by disulfide bonds

How do antibodies bind antigens?

hypervariable loops or complemetarity-determining regions (CDRs)

3 loops at end of structure tht