Protein (Lecture 3-6)

basic concepts in sequence analysis

sequence similarity, homologous sequences

sequence similarity

degree of matching between sequences which is generally measured in percentage

homologous sequence

two or more sequences that are derived from a common ancestral sequence

looking for matching in sequences

there are multiple possible ways to align sequence, looking for the position with the most matching letters, matching becomes more difficult as length increases and when sequences are not the same length so gaps are introduced

AlphaFold

uses sequence analysis and protein structure databases and not calculations to estimate the configuration of a protein

aa rotation

the axis of the backbone can rotate at the bonds connecting the alpha carbon to the carbonyl and amide groups allowing limited rotation

type of configuration aa take

most take a trans configuration giving a linear backbone but 5-7% peptide bonds with proline are cis

isomerization

an aa can transform between trans and cis but very slowly and it dramatically changes function of a protein

chaperones

proteins that can fold newly made proteins, refold misfolded or unfolded proteins and disassemble potentially toxic proteins

how do chaperones work

through ATP dependent cycles that cover hydrophobic aa which are exposed in order to isolate protein folding or refolding while productive folding events occur

types of chaperones

chaperones and chaperonins

chaperone example

Hsp70 (Heat-shock protein) which binds to newly created short segment proteins

chaperonins

specialized, large, barrel-shaped subfamily of chaperones

chaperonins example

GroEL is composed of two stacked rings, each composed of seven subunit that interacts with GroES where the middle forms an open space where misfolded proteins can enter

Ubiquitin/proteasome system

Ubiquitin-tagged proteins are fed into a multisubunit chamber in which the subunits form inward-facing proteases

ubiquitin

small protein that tags damaged or misfolded proteins for degradation

proteases

an enzyme that breaks down proteins into smaller peptides or single amino acids by cleaving peptide bonds through hydrolysis

Ubiquitylation

a three step reversible post-translational modification where ubiquitin is covalently attached to a target substrate protein

protein aggregation

process where misfolded, partially unfolded, or intrinsically disordered proteins that aren’t degraded clump together

Amyloid fibrils

end product of protein aggregation and a marker for disease

formation of amyloid fibrils

the generation of short segments that form long arrays, or filaments, of β-sheets where β-strand is oriented nearly perpendicularly to the axis of the filament and two stacks twist about one another, forming protofilament

ligand

The molecule to which a protein binds to

two important properties of protein binding

specificity and affinity

specificity in protein bonding

the ability of a protein to bind to only one particular ligand

affinity in protein binding

the strength of binding between protein and ligand and is expressed as dissociation constant (Kd). The stronger the interaction, the lower the Kd

antibodies

proteins in the immune system to identify, neutralize, and eliminate foreign invaders

structure of antibodies

y shape with 2 identical structures held together by disulfide bond of a heavy chain and light chain combined and a carbohydrate underneath

antigens

a molecule that an antibody recognizes as foreign

complementarity determining region (CDR)

where antibodies and antigens connect by multiple protein loops from both the heavy and the light chain with lots of variability

enzymes

accelerate the rate of a reaction by reducing the free energy of the transition state

Ligands of enzyme

includes the substrates of the reactions they catalyze

active site of an enzyme

the complete region where a substrate binds and undergoes a chemical reaction

substrate binding site

the sub-region in the active site where the substrate binds to

Vmax of enzyme

the maximum rate of catalysis with most amount of substrate

Km of enzyme

the substrate concentration that supports a rate of catalysis equal to half of the Vmax

meaning of high vs low Km

low Km: High affinity. The enzyme binds the substrate very easily and reaches half-maximal speed at a low substrate concentration

high Km: Low affinity. The enzyme needs a lot of substrate around to reach half of its maximum speed

serine protease

enzymes that cleave peptide bonds in proteins

trypsin

a type of serine protease which uses a 2-step catalytic mechanism to hydrolyze peptide bonds

mechanism of trypsin

step 1: cleavage of peptide bond with formation of a covalent substrate-enzyme complex

step 2: hydrolysis of acyl enzyme complex

what acid-base catalysis is dependent on

pH: catalytic activity relies on the protonation and deprotonation states of functional groups

ex. Pancreatic serine protease requires ph<9

allosteric regulation

when a molecule binds to a different site called the allosteric site which creates a different shape in the active site and therefore changes the activity of the enzyme

post translational modifications of proteins

changes in the chemistry of proteins that occur after synthesis

examples of post translational modifications of proteins

ubiquitinylation, phosphorylation, dephosphorylation

protein kinases

the enzyme that attaches a phosphate group from ATP to a target protein during phosphorylation

protein phosphatase

an enzyme that removes a phosphate group from a phosphorylated amino acid residue on a target protein during dephosphorylation

ubiquitinylation

process of marking a protein for degradation

ubiquitinylation mechanism

E1 (Ubiquitin-activating enzyme): : activates ubiquitin using ATP and forms a high-energy thioester bond with it

E2 (Ubiquitin-conjugating enzyme): receives the activated ubiquitin from E1 onto its active cysteine residue

E3 (Ubiquitin-ligase): Recognizes the specific target protein and transfers ubiquitin from E2 to a target residue

common ancestor evidence

Shared use of amino acids and nucleotides across life, the use of only L-amino acid stereoisomers to generate protein

oligopeptides

small chains of <30 aa

polypeptides

larger chains of >30 to hundreds of aa

primary structure of proteins

the exact linear sequence of amino acids

expression of size of protein

number of aa or their mass in daltons (1 Da= 1 atomic mass unit)

smallest protein length

40 aa

average human protein length

476 aa

secondary structure of proteins

local conformations of the peptide chain backbone

three major peptide chain backbone conformations

alpha helix, beta sheet, and beta turn

peptide conformation bonding

h-bonding between the carbonyl oxygen atom on one aa and amide H atom on a different aa

what structures are in the average polypeptide

alpha-helix and beta-sheet make 60% the length of the average protein

bonding of alpha-helix

the NH of one aa at position n forms a hydrogen bond with the CO of a second aa at position n+4

structure of alpha-helix

tilted axis generates a periodicity of 3.6 aa per turn and backbone has a straight rod structure with side chains pointing outward

*proline is not usually in alpha-helices

bonding of beta-sheet

hydrogen bonds are formed between two adjacent β-strands oriented perpendicularly to the chains of the backbone

structure of beta-sheet

alignment of 2 or more beta-sheets generate nearly 2-dimensional sheets with side chains sticking up or down

*sheets can run parallel or antiparallel

Definition

bonding of beta-turn

the first and the fourth alpha carbon in the structure are maintained close to each other through h-bonding

structure of beta-turn

composed of four amino acids that form a sharp U-shaped bend in the protein backbone structure

*glycine and proline are common in beta-turn

tertiary structure in proteins

the overall conformation of the polypeptide from multiple secondary structures

bonding in tertiary structures

hydrophobic interaction, van der Walls, and hydrogen bonds

motifs

a combination of two or more secondary structures that form a distinct three- dimensional structure found in multiple proteins that are associated to a specific function

three example of motifs

alpha helix-based coiled-coil, helix-loop-helix, zinc-finger

alpha helix-based coiled-coil motif

up to 4 different αlpha-helices coil about one another with aliphatic residues at positions 1 and 4 in a 7-aa repeat that bury between between the helices

*in fibrous proteins and transcription factors

helix-loop-helix motif

2 alpha-helix join together in a loop and is used to bind calcium

*looks like a right angle

zinc-finger motif

1 alpha-helix and two beta-sheets use their sidechains to bind zinc

oil-drop method of protein folding

hydrophobic molecules hide inside hydrophilic molecules

domains

distinct regions of the proteins structure

*tertiary structures are divided into domains

functionality of domains

most domains can be separated from the rest of the protein and remain functional and similar domains are found in diverse proteins

quaternary structure in protein

the arrangement of two or more individual polypeptide chains, called subunits, into a single, larger functional protein complex

supramolecular complexes

large "molecular machines" made up of multiple distinct proteins, each of which may itself contain multiple subunit

*can include other molecules like lipids

X-ray crystallography to determine protein structure

X-rays pass through a protein crystal, X-rays are diffracted as they interact with electrons in the proteins atoms generating a pattern on the detector that represents the distribution of electrons in the sample

chromatography

separation of proteins based on their differential interactions with a immobile material where the mobile material moves up the immobile material at rates that depend on how much they interact with the solid phase

gel filtration chromatography

separation of proteins based on size and shape where the column is packed with specialized gel beads containing tiny pores so the big proteins that can’t enter the beads leave the column first and the little ones leave later

ion-exchange chromatography

separation of proteins based on electric charge where proteins with a charge with attach themselves to the oppositely charged immobile phase and then be released at different rates using sals or pH

antibody affinity chromatography

separation of antibodies based on which proteins can be covalently coupled to the immobile phase and all other proteins are washed away by lowering the pH

electrophoresis

separation of macromolecules based on an electric current which passed through a buffer solution with a cathode (negative charge) at one end an anode (positive) at the other so negatively charged molecules (DNA/RNA) travel to the cathode and proteins go either depending on properties

SDS-PAGE

separation of proteins based on size where SDS denatures proteins by disrupting the oil drop structure of proteins and giving them a negative charge which are then pulled through an electric current where smaller proteins move quicker

immunoblot (western blot)

proteins in an SDS-PAGE gel are transferred to a membrane where they are accessible to primary antibodies and then secondary antibodies that are fluorescence making it easier to visualize where specific proteins are

co-immunoprecipitation

technique to identify protein-protein interactions where an antibody attaches to a protein and if other proteins are attached to the initial protein, they get pulled with the antibody aswell

immunofluorescence

technique to identify cellular localization of proteins by using specific antibodies that are chemically attached to a fluorescent dye

• cells are dead

how fluorescence works

an incoming photon usually from ultraviolet or visible light excites an electron in the fluorescence molecule and as the electron goes back to its ground state, it releases light

4 important elements of a fluorescence microscope

1. light source for proton

2. filter cube that separates excitation light from emission light

3. objective lens which aims light onto sample

4. sensitive camera

gene fusions

combines the coding sequences of a gene of interest and a fluorescent protein to produce a single fusion protein for live-cell imaging and localization studies