BIOL 200

What is life? (6 things)

1. high degree of chemical and structural complexity

2. a capacity to extract, transform, and use energy from the environment

3. Defined functions for the organism’s components and regulated interactions among them

4. a capacity to sense and respond to environment

5. a capacity to self-replicate

6. a capacity to evolve

Life is built by

complex macromolecules

to function, life requires large organic molecules (and structures formed by them)

Without energy, molecules

decay toward more disordered states and to come to equilibrium with its surroundings

what does life require from the environment?

continual imput of energy

cells are open/closed systems

open

what is an open system

they require the import or energy sources and raw materials, and export of waste

lipid membrane

• selective barrier

• greater concentration of nutrients and synthesized products inside the cells

• allow cells to have a distinct intracellular environment very different from its surroundings

  • to be outside of a chemical equilibrium with its surroundings

Most molecular function is accomplished by

proteins

each protein serves a specific function

Proteins serve (3 things)

1. structural roles

2. generate movement

3. sense and detect signals

Many proteins are capable of

catalyzing reactions (enzymes) including those needed to obtain and use energy from the environment

Genetic information is stored in

DNA

genetic information = information to self-replicate

DNA (and RNA) have __ building block

4 (nucleotides), making them much simpler than proteins

proteins have __ building blocks

20 (amino acids)

Central Dogma of Molecular Biology

• DNA contains information to make proteins

• DNA → RNA → proteins

• cells cannot go in the opposite direction of this flow

• DNA is also required as a template to synthesize DNA

First Step of Making Protein

1. Genetic information is copied to a third type of molecule, RNA

2. Process is called Transcription

mRNA

• messenger RNA is the result of transcription

• exported from the nucleus and used by large molecular machine called ribosome

ribosomes

• synthesize proteins using the information in RNA

• called translation

translation

nucleotides are read in triplets (codons)

greater information is conveyed in RNA (4³ = 64 combinations)

requires two other types of RNA (tRNA and rRNA)

tRNA

transfer RNA

coupled into an amino acid residue, pairs with the sequence in mRNA during translation to convert the sequence in codons into specific amino acids

rRNA

ribosomal RNA

make up ribosomes

composed of proteins and RNA

catalyze protein synthesis

catalytic side that synthesizes is the rRNA part

Three important polymers in molecular biology (macromolecules)

1. DNA

2. RNA

3. Proteins

information in cells is carried by polymers

polymer

• large molecules composed of multiple covalently linked building blocks (small molecules) called monomers

• ex. PET, most common type of polyester

  • monomer is ethylene terephthalate

How to cook PET

• requires mixing two precursors

• input of energy, in the form of heat (150-290 C)

• removal of byproducts (such as methanol)

proteins are formed by amino acids

• monomers that form proteins are the amino acids

amino acid structure

central carbon atom (alpha carbon), an amino group, a carboxyl group, and a side chain (R group)

polymerization of amino acids

• amino acids are joint together by the reaction of carboxyl group in one of them with the amino group of a second amino acid

• reaction results in the formation of a peptide bond

a polymer of amino acids is called

polypeptide

backbone of protein

the linear chain of carbon and nitrogen atoms that contain the peptide bonds

side groups stick out of the backbone

protein orientation

an amino group at one end (N-terminus) and a carboxyl group at the other (C-terminus)

Side chains

• 20 different side chains = 20 different amino acids

• differ in their size, shape, charge, hydrophobicity, and chemical reactivity

essential amino acids

the 9 of the 20 amino acids humans and other mammals can’t synthesize and must consume in diet

3 groups of amino acids

hydrophobic, hydrophylic, and special amino acids

hydrophobic amino acids

• poorly soluble in water

• contain linear or branched hydrocarbons or contain aromatic rings

hydrophylic amino acids

• readily soluble in water

• can either be positively (basic), negatively (acidic), or polar with uncharged groups

special amino acids

• cysteine: reactive sulfhydryl group (SH)

  • serves in catalysis of enzymes

  • reacts with other cysteins to form disulphide bonds to join two polypeptides

• glycine: smallest amino acid

  • fits in small spaces

  • side chain H

• proline: side chain that forms a covalent bond to the N in th eamino group attached to the alpha carbon

• incorporation of proline into proteins generates kinks in the linear chain

DNA and RNA are formed by

nucleotides

the monomer of nucleic acids (DNA and RNA) are called nucleotides

nucleotides

composed of pentose (5-C sugar), a phosphate group, and a ring-shaped molecule containing nitrogen called nitrogenous base

negative charge in phosphate makes them acidic

DNA + RNA have an overal -ve charge

pentose in RNA and DNA

• the pentose in RNA is ribose

• the pentose in DNA is 2’-deoxyribose

• the difference is the loss of an OH group at position 2’ in deoxyribose

RNA and DNA use 4 bases each

• purines: pair of fused rings

• pyrimidines: contain a single ring

Thymine is only present in

DNA

Uracyl is only present in

RNA

Bases are connected to the ribose through

the nitrogen atom at position 9 (in purines) or 1 (in pyrimidines)

nucleotides have a ribophosphate backbone

• nucleotides are joined through two phosphodiester covalent bonds where a phosphate group links two (deoxy)ribose at positions 5’ and 3’

• phosphate and (deoxy)ribose groups generate the backbone of nucleic acids

• DNA and RNA have an orientation with a 5’ end (containing a phosphate) and a 3’ end (containing a hydroxyl group)

• each monomer can contain a different base, therefore generating a unique sequence

RNA is more prone to breaking than DNA

• hydroxyl group at position 2’ in the ribose of RNA can spontaneously react with the phosphate group, break the RNA molecule

• DNA lacks an OH at the 2’ of ribose, making it much more stable and the choice for the genetic material in cells

• some viruses have genomes of RNA

• DNA molecules in our genome are hundreds of millions of base pairs in length

Purines and Pyrimidines pair in DNA

• the purine Adenine (A) pairs with the pyrimidine Thymine (T)

  • kept together by 2 hydrogen bonds (weak non-covalent bonds)

• the purine Guanine (G) pairs with the pyrimidine Cytosine (C)

  • interact through 3 hydrogen bonds

DNA is double stranded

• in cells, two DNA polymers ‘anneal’ (pair) to form a double-stranded following the pairing of bases (A-T and G-C)

• the sequence of bases in the two strands is complementary

• pairing between the two strands is antiparallel

• individually weak, the sum of many H-bonds help keep the two strands together

• double helix structure

• ribophosphate backbone in the outside and the bases inside

• bases are regularly spaced 0.34 nm appart

• on the outside of the helix, the spaces between the strands form two helical grooves of different widths: the major and minor grooves. Atoms on the edges of each base are exposed

Double-stranded DNA (dsDNA) shapes into a

right-handed double helix

Some proteins recognize specific DNA sequence motifs

• structure of DNA allows DNA-binding proteins to “read” the sequence in it without unwinding double helix

• major groove is wide and contains unique chemical information

• most protein-DNA interactions use the major-groove

• minor groove is shallow and cannot distinguish all bases

Evidence of Common ancestry

1. shared use of amino acids and nucleotides across life

2. the use of only L amino acid stereoisomers to form proteins

3. conservation of the codons that code for the same 20 amino acids

machinery for protein synthesis is similar across organisms

• catalytic site of ribosomes is in the rRNA

protein function is determined by…

their structure

hierarchy of protein structure

what folds into a specifc 3D structure to acquire a function?

linear polypeptide chains

the properties of amino acids direct the folding to specific structures

primary structure (sequence)

• the sequence of a protein has a direction, with a beginning (N-terminus) and an end (C-terminus)

• properties are given by the side chains

• number of amino acid residues and their mass in daltons is what protein size is expressed as

• smallest proteins are ~40 amino acids in length. the “average” protein depends on the organism. In yeast, the average is 466 aa residues

• largest protein has 34,000 aa residues

oligopeptides

small chains <30 amino acids

polypeptides

larger chains, single chain, often 200-300 amino acids

amino acid residue

an amino acid that has been incorporated into a peptide or protein chain through a dehydration reaction

green flurescent protein example

• 238 aa in length

• 26.9 kDa

• absorbs blue light and transforms it into green light

secondary structure

• local conformations of the peptide chain backbone, generating stable arrangements of the aa residues

two major peptide chain backbone conformations

alpha-helix

beta-sheet

both are based on H-bonding (stabilizes) between peptide bond carbonyl O atoms and one amino acid residue, and amide H atoms on a different amino acid residue

~60% of the length of the average polypeptide chain consists of segments of alpha-helix and beta-sheet. multiple secondary structure elements per polypeptide

structure of alpha-helix

• don’t contain proline

• side chains can be hydrophobic, hydrophilic, or charged

• H-bonds occur between AA in same chain (C=O and N-H)

  • happens in the position n+4

• tilted axis generates a periodicity of 3.6 aa residues per turn

• backbone has a straight rod structure with side chains pointing outward

• surface properties depend on side chains. so does the alpha-helix formation and its interactions with other parts of the protein and other molecules

structure of beta-strand

• short (5-8 aa)

• H-bonds formed between two adjacent Beta-strands oriented perpendicularly to the chains of the backbone

• alignment of 2 or more beta strands generate nearly 2-D sheets

• side chains protrude above and below the beta-sheet plane. they determine the interactions within the protein and with other molecules, and the propensity to form

beta-strand organization

• B-strands in the same polypeptide can be parallel or antiparallel

• B-strands from two different proteins can also interact to form sheets

motif

combination of two or more secondary structures that form a distinct 3D structure found in multiple proteins

associated to a specific function

tertiary structure

• the overall conformation of the polypeptide

  • the spatial organization of the multiple secondary structure elements

• stabilized by hydrophobic interactions (most important one), van der walls, and Hbonds

• forces stabilizing the tertiary structure are weak, resulting in fluctuations in the structure

structure acquisition is partly driven by

the effect of hydrophobicity

• water molecules surrounding hydrophobic molecules dispersed in water adopt a constrained, cage-like organization (low entropy)

• if hydrophobic molecules coalesce, the total number of low entropy, constrained water molecules is reduced, and this net increase in entropy ultimately drives the formation of separate hydrophobic and aqueous phases

• coalescence of hydrophobic molecules also favoured by weak non-covalent van der Waals intermolecular interactions

oil drop model of protein folding

hydrophobic and hydrophylic side chains are distributed throughout the linear sequence

• when the protein folds, the hydrophylic side chains tend to be exposed to water and hydrophobic residues tend to cluster together in the inner core, hiding form the aqueous surroundings

conformations

each protein adopts only one or a small number of similar structures

domains

distinct regions of the proteins structure

how tertiary structures are divided

they can represent a particular funciton, structure, or refer to the spatial relationship with the rest of the protein

functional domains can be seperated from the rest of the protein and remain functional

modular nature of proteins

similar domains can be found in diverse proteins, and also as multiple similar copies within any given protein

shapes/colours show a variety of distinct domain types, including EGF domain (green) and membrane-spanning domain (blue)

tertiary structure of GFP (green fluorescent protein)

• explains activity

• the typical structure of FP consist of a barrel containing 11 beta sheets and central helix that contains the chromiphore

  • interior of the barrel is highly crowded by side-chains of amino acid residues, limiting movement inside

  • alpha-helices at the ends of the protein serve to stabilize it

  • chromophore is formed by a post-translational modification that result in the cyclization of peptide backbone

quaternary structure

• multimeric proteins can contain any number of identical or different polypeptides

supramolecular complexes

large “molecular machines” made up of multiple distinct proteins, each of which may itself contain multiple subunits

ex. transcription initiation complex

techniques to determine protein structure

X-ray crystallography and cyroelectron microscopy

both require the purification of individual proteins

generally representing months or years of work

motifs and domains

each domain can contain multiple motifs

unstructured regions

regions in DnaA without a fixed structure (more flexible). This is the case for most proteins.

DnaA

a bacterial protein that initiates DNA replication

contains 4 domains

the domain IV contains two motifs that allow the interaction with specific sequences on DNA:

• A Helix-Turn-Helix and a Basic Loop

the domain III contains motifs for the binding and hydrolysis of ATP:

• Walker A and Walker B

• it contains also other motifs for multiple other functions

motif

the combination of two or more secondary structures that form a distinct 3D structure found in multiple proteins. Associated to a particular function

protein folding and hierarchy of protein structure

• polypeptide first folds regions with secondary structure

• secondary structure elements group into motifs then into domains

• then the tertiary structure is acquired

each amino acid residue can rotate on its axis

• at aa residue position of the polypeptide, the axis of the backbone can rotate at the bonds connecting the alpha carbon to the carbonyl and aminde groups

• rotation is limited due to steric constrains imposed by the backbone and the side chains

proline confers special conformation to proteins

• most amino acid residues in the polypeptide chain orient in a trans configuration relative to the peptide bond, resulting in a linear backbone

• in contrast, between 5-7 percent of the peptide bonds with proline acquire a cis-configuration

proline helps direct protein folding

• isomerization between trans and cis configurations can occur spontaneously, but it is slow

• peptidyl-proline isomerases (PPlases) are enzymes that speed up the process during folding

• isomerization of a single proline can dramatically change protein structure, and affect its activity

how long does folding take

micro- to milliseconds

why can proteins fold when isolated in test tube

because the information required for the acquisition of structure is in the sequence

despite the limitations in rotation at each residue, the combination of many small changes in the orientation of proteins allow them to twist and turn

where are the hydrophobic aa residues

• inside the correctly folded proteins

• hydrophobic patches at the surface of a protein is a sign of misfolding

chaperones

proteins that help guide protein folding along productive pathways, by permitting paritally misfiled proteins to return to the proper folding pathway

recognize exposed hydrophobic residues

upregulated in conditions where misfolding is increased (ex. heat-shock)

can fold newly made proteins, refold misfolded or unfolded proteins, and disassemble potentially toxic protein aggregates that form due to protein misfolding

work through ATP-dependent cycles of binding to, and release from, misfolded “client” molecules, at exposed hydrophobic patches. by blocking the exposed hydrophobic patches the chaperaone keeps the folding or refolding protein isolated, while productive folding events occur

two types: chaperones and chaperonins

ex. Hsp70 (Heat-shock protein)

→ binds to short segments of an unfolded proteins such as those newly synthesized as they emerge from the ribosome

→ binding and hydrolysis of ATP results in changes in the conformation of Hsp70 and are needed for its function

chaperonins

large complexes that isolate unfolded proteins

ex. GroEL is composed by two stacked rings, each composed of seven subunits

→ each ring interacts with a seven-subunit co-chaperone that acts like a lid, GroES, also containing seven subunits

→ in the center of GroEL there are chambers where all or part of a protein enters

chaperonin mode of action

• proteins of less than 60 kDa in mass are captured by hydrophobic residues near the entrnce of the GroEL chamber

• ATP hydrolysis regulates the cycle

• protein folding in the two rings is coordianted

• multiple cycles may be requried for proper folding

what does degredation target?

irretrievably misfolded proteins

protein degradation

ubiquitin/proeasome system

• poly-ubiquitin “tags” damaged or misfolded proteins for degradation

• ubiquitin-tagged proteins are fed into a multi-subunit chamber in which the subunits form inward-facing proteases

ubiquitylation

important protein regulator

small ubiquitin protein (76 aa residues, 8.6 KDa) becomes covalently linked to the lysine residue of target proteins

ubiquitin conjugating system

1. carboxyl terminus is activated

2. ubiquitin is transferred to a ubiquitin conjugating subunit (E3 ubiquitin ligase)

   1. 600 E3 coded in our genome, each with specific substrate binding

polyubiquitinylation

• marks protein for degradation

• ubiquitin ligases recognising exposed hydrophobic reisudes add multiple ubiquitins to proteins, forming chains of 4 or more ubiquitins

• polyubiquitinylated proteins are recognized by Ub receptors in the proteasome

• Deubiquitinases (Dubs) hydrolyze bonds between ubiquitous to recycle them

• ATPase driven auxillary proteins unfold proteins and transports to the core for degradation

• once inside the inner chamber, polypeptides are digested into short fragments of 2-24 aa in length

• peptide bonds of hydrophobic, acidic, and basic residues are cleaved at the active site

• the resulting polypeptides are further degraded into single amino acids in the cytoplasm

what leads to protein aggregation?

accumulation of misfolded proteins

due to the imperfect system of refolding with chaperone assistance or multiubiquitination/proteasome mechanism

protein aggregation

• misfolded proteins or incompletely degraded proteins can interact with each other, hiding their hydrophobic residues, forming aggregates

• high protein concentration and changes in environmental conditions leads to formation of aggregates

• can be amorphous or well-organized, such as in the amyloid state

amyloid fibrils

• formed by the generation of short segments (6-12 residues in length) that form long arrays, or filaments, of B-sheets

• each B-strand is oriented nearly perpendicularly to the axis of the filament. two stacks stwist about one another, forming protofilaments, many of them forming fibrils

amyloids of makers for disease, why?

• found in tissue

• neurodegenerative diseases (ex. alzheimers and parkinsons) all contain amyloids

• amyloid formation is associated to age, but it is also more prevalent in mutant proteins

Binding

diverse functions performed by proteins are based on their ability to engage in binding

proteins bind to one another, to other macromolecules, to small molecules, and to ions

ligand

the molecule to which a protein binds

specificity

important to protein binding

the ability of a protein to bind to only one particular ligand, even in the presence of a vast excess of irrelevant molecules