lecture 1

what makes up most of cell's dry mass?

- proteins

protein major functions

- storage

- transport

- movement

- structure

- signal transduction

- growth and development

- immune protection

- enzyme activity/catalysis

structure determines...

function

medicinal use of 3D protein structure

- drug design

- predict function of mutated protein

biochemical use of 3D protein structure

- predict MOA

- develop tools to manipulate MOA

biotechnological use of 3D protein structure

- design new enzymes

David Baker

- won the Nobel Prize in Chemistry 2024 for computational protein design

- generated tools to design proteins

Demis Hassabis

- won nobel prize in chemistry 2024 for protein structure prediction

John M. Jumper

- won nobel prize in chemistry 2024 for protein structure prediction

- AI tools to predict the function of a protein without its structure, so that you can eventually figure out the structure

amino acid

- the building block of proteins

- called residues in an amino acid chain

- 20 exist in nature

classifications of amino acids

- polar acidic/negative

- polar basic/positive

- uncharged polar

- nonpolar

what differentiates amino acids

- R group

parts of amino acids

- alpha carbon

- amino group

- carboxyl group

- hydrogen atom

- R group or side chain

primary structure of proteins

- linear sequence of amino acids

primary structure bonds

- peptide bonds between C-terminus and N-terminus of 2 amino acids

- lose a water molecule in the process

direction of reading amino acid sequence

- N to C terminus

secondary structure of a protein

- regular stretches of a polypeptide chain

- alpha and beta pleated sheets

secondary structure bonds

- Hydrogen bonds between amino acids that are near one another in the linear sequence

alpha helix

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

- hydrogen bond every 4th peptide bond

- ferritin

beta sheet

- forms from neighboring segments of the polypeptide backbone that run at the same orientation -parallel- or in the opposite direction to the immediate neighbor -antiparallel-

- fatty acid-binding protein

tertiary structure of a protein

- full 3D organization of a polypeptide chain

tertiary structure bonds

- disulfide

- hydrogen

- ionic bonds

disulfide bonds

- covalent bond between 2 thiol groups of Cys residues in one polypeptide chain

- oxidation of 2 SH groups

disulfide bonds extracellular

- often have several

intracellular disulfide bonds

- lack of them

hydrogen bonds

- an electrostatic attraction between a hydrogen atom, which is covalently bound to a high electronegative atom, to another electronegative atom of the same or different molecules of their close neighborhood

ionic bonds

- chemical bond formed between 2 oppositely charged R groups found close to each other

how many amino acid residues in most natural polypeptide chains/proteins

- 50 to 2,000

average molecular weight of an amino acid

- 110 grams/mol

- 110 daltons Da

average molecular weight of proteins

- 5.5 to 220 kDa

importance of knowing amino acid sequence

- can determine function by predicting its 3D structure

what can alterations in primary structure lead to

- abnormal protein function

- disease

- ex: sickle-cell anemia and cystic fibrosis

types of tertiary structures

- globular

- fibrous

globular proteins

- spherical, water-soluble proteins.

- hydrophobic inside and hydrophilic amino acids outside

- myoglobin

fibrous proteins

- 2 large alpha helices intertwine to make a stronger structure

- more extended compared to globular

- alpha-Keratin

proteins domains

- large proteins consist as a set of these

- a unit in the protein that can fold independently

- 40 to 350 contiguous amino acids

- target specific domain for drug

different domains associated with...

- different functions

cytochrome b562

- a single-domain protein involved in electron transfer in E. coli

NAD binding domain of lactate dehydrogenase

- single binding domain

- binds NAD

antibody light chain

- one domain

- where the antibody will bind to different foreign substances

SH2 domain

- 100 amino acids

- involved in signal transduction

Src protein

- formed from 3 domains

- 536 amino acids

- SH2

- SH3

- catalytic

quaternary structure of a protein

- the complete conformation of a protein that is formed as a complex of more than one polypeptide chain

- each chain is a subunit

dimer/homodimer

- a protein complex containing 2 subunits

what do drugs target is quaternary structure

- dimerization of a protein

- due to some proteins being functional only if their subunits come together and dimerize

human hemoglobin

- composed of alpha2beta2 tetramer

- since each subunit can hold a heme group, it increases oxygen capacity

complex assemblies

- some proteins may come together to form different ones

- myosin interacts with actin to form sarcomere

- many sarcomeres come together to form myofibril

- many myofibrils come together to form muscle fiber

native protein

- a protein in its folded and active form

denatured protein

- when a protein is converted into a randomly coiled peptide without its normal activity

- primary structure is not affected

how do proteins get denatured

- any treatment that disrupts weak, non-covalent bonds stabilizing the tertiary structure

- happens with heating, chemical denaturants, oxidizing, or reducing agents

Chemical denaturants

- urea

- guanidinium chloride

protein biosynthesis

- translation of nucleotide sequences into amino acid sequences

- a conserved process

ribosomes

- responsible for protein biosynthesis

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

- on the rough ER but not smooth

- large subunit

- small subunit

membrane bound vs free ribosomes

- structurally and functionally identical

A of ribosome

- aminoacyl-tRNA

- where the tRNA brings the amino acid

P of ribosome

- peptidyl tRNA

peptidyl transferase center

- region of the large ribosomal subunit that catalyzes peptide bond formation between the aminoacyl tRNA in the P site and the aminoacyl tRNA in the A site

E ribosome

- where the mRNA enters

- where the tRNA is released from the ribosome and exits

Francis Crick 1961

- described a 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 combinations of nucleotides, each of which corresponds to a specific amino acid or stop signal

methionine

- AUG

- all amino acids start with this codon

stop codons

- UAA

- UAG

- UGA

transfer RNA

- the adaptor molecule in protein synthesis

- contains an amino-acid-recognition site

- template-recognition site/anticodon

what pattern can each tRNA be rearranged into

- cloverleaf

- secondary structure RNA

where is activated amino acid attached to in tRNA

- hydroxyl group of the adenosine residue in the amino-acid-attachment site of CCA at the 3' end

anticodon loop

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

step one in amino acid to tRNA

- formation of adenylated amino acid

step 2 amino acid to tRNA

- the attachment of the amino acid to the corresponding tRNA

activated tRNA

- when an aa is coupled to its respective tRNA molecule

animoacyl-tRNA synthetase

- enzyme that catalyzes the aa activation reaction and linkage of aa to tRNA

- most cells have a different synthetase for each amino acid

what direction is mRNA read through ribosome

- 5' to 3'

bacterial vs eukaryotic ribosomes

- Bacterial: 70S

- Eukaryotic: 80S

- mitochondria and chloroplast have ribosomes more similar to bacteria

antibiotics and ribosomal differences

- the differences between eukaryotic and bacterial ribosomes can be exploited for the development of antibiotics

- bind and block protein synthesis in bacteria, so they could also have an effect on mitochondria

streptomycin and other aminoglycosides

- inhibit initiation and cause the misreading of mRNA

- bacteria

tetracycline

- binds to the 30S subunit and inhibits the binding of aminoacyl tRNAs

- bacteria

chloramphenicol

- inhibits the peptidyl transferase activity of the 50S ribosomal subunit

- bacteria

cycloheximide

- inhibits translocation

- eukaryotes

erythromycin

- binds to the 50S subunit and inhibits translocation

- bacteria

Puromycin

- causes premature chain termination by acting as an analog of aminoacyl-tRNA

- bacteria and eukaryotes

posttranslational modifications

- alterations in the structure of a protein after it has been synthesized in the cell

- covalent attachment of groups to amino acid side chains

- cleavage or modification of the polypeptide backbone

Hydroxyproline

- hydroxyl groups in many proline residues stabilize fibers of newly synthesized collagen

vitamin C deficiency

- results in insufficient hydroxylation of collagen because it is a cofactor essential in hydroxylating proline

- abnormal collagen fibers unable to maintain normal tissue strength

- scurvy

Green Fluorescent Protein (GFP)

- a protein found in certain species of jellyfish that glows green when excited by certain wavelengths of light (fluorescence)

- source of fluorescence is a group formed by spontaneous rearrangement and oxidation of the sequence Ser-tyr-gly within the center of the protein

- serve as markers within cells, as they have been engineered to absorb and emit light across the entire visible spectrum

folding of a newly synthesized protein

- protein begins to fold while it is still being synthesized

- chaperones

- heat shock proteins

chaperone

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

- the most abundant protein

heat shock proteins hsp

- Chaperones upregulated under stress conditions

- when the temperature is too high and denatured proteins need to be refolded

what is protein misfolding and aggregation associated with

- neurological disease

- Alzheimer's, Parkinson's and Huntington's disease

amyloid fibrils

- also called amyloid plaques

- improperly folded proteins and deposition of protein aggregates in extracellular space

- seen in Alzheimer's, Parkinson's and Huntington's disease

- when normally soluble proteins are converted into insoluble fibrils

what are fibrils rich in

- Beta-sheets

improper folding of protein can lead to

- protein failing to achieve its functional conformation or reach required location in the cell

cystic fibrosis

- the most common lethal genetic disease affecting Caucasians

- caused by defects in CFTR - cystic fibrosis transmembrane conductance regulator

CFTR

- channel for chloride ions

- comprised of 1480 amino acids

deletion of Phe residue at position 508 in CFTR

- most common mutation in greater than 90% of CF patients

- improper folding of the protein and CFTR does not reach the cell membrane

drug discovery strategies that restore protein folding and function

- small molecule pharmacological chaperones

- inhibition of aggregation

small molecule pharmacological chaperones

- stabilizing specific misfolding-prone or mutant proteins and pulling them towards a lower free energy minimum

- CFTR correctors

CFTR correctors

- Lumacaftor

- tezacaftor

Lumacaftor

- the first medicine to trear underlying cause of CF in people 12yrs or older with 2 copies of F508del mutation

- FDA approved in 2019

Tezacaftor

- FDA approved in 2019

- stabilize CFTR by filling an internal cavity

- prevent CFTR premature degradation

CF treatment inhibition of aggregation

- of a specific amyloid-prone protein that stabilizes the native state or the partially folded state

- prevents the formation of oligomers or amyloids