Cellular and Molecular Biology - Term Test 1

Lectures 1-9

bacteria

• unicellular (mostly)

• gram negative and positive

• huge diversity of survival strategies

archaea

• unicellular 

• many extremophiles

eukaryotes

• unicellular and multicellular

• protists, fungi, plant, animals 

evolution of cellular life 

• cellular life evolved from a common ancestor 

• gene passed vertically from parent to child and horizontally within and between species 

• mitochondrion endosymbiosis gave rise to eukaryotes 

• primary endosymbiosis gave rise to photosynthetic eukaryotes 

  • photosynthesis spread to other eukaryotes via secondary endosymbiosis

common characteristics of life

• growth

• metabolism

• response to stimuli

• movement reproduction

genome

the storage material containing the organisms genetic information

• contains instructions for self-replication (genes)

• all cellular life has double stranded DNA (dsDNA) genome

• viruses can have a DNA or RNA genome, single-stranded or double-stranded (depends on the viral species)

• all things necessary to survive (behaviours) come from genomes

central dogma 

the underlying molecular mechanisms that controls life 

• controls all cellular processes such as growth, metabolism and response to stimuli

• gene on the DNA genome is expressed through transcription, producing an RNA copy of the gene 

what does transcription produce

depending on the gene, transcription produces mRNA or functional RNA

• mRNA gets translated into proteins

• functional RNAs do not get translated, they function as is

  • they take part in cellular processes as an RNA molecular

• proteins and functional RNAs performs activities to regulate cellular processes

what are cells filled with 

cells are a sack made of lipid membranes and are filled with aqueous solution containing various organic and inorganic molecules 

• macromolecules (nucleic acids, proteins, carbohydrates, lipids) 

• variety of other organic metabolite 

• inorganic ions  

• water 

• organelles (for eukaryotic cells)

prokaryotes

• no membrane bound organelles

• usually smaller than eukaryotes

• mostly singled cellec

eukaryotes

• has membrane bound organisms such as a nucleus

• compartmentalizes biochemical reactions using organelles

• usually larger than prokaryotes

• single or multicellular

presence/absence of nucleus has a huge implication for transcription and translation

size of prokaryotic cells

1-2µm

size of eukaryotic cells

5-20 µm

size of mitochondria 

1-2 µm

size of ribosome

20 nm

size of one atom

fraction of a nano-meter

the two types of microscopes 

light and electron microscope 

light microscope

uses photons to image structures

• accessible and easy to use (at least of the simpler kinds)

• can image live cells in real time

• can image in colour

• lower resolution

electron microscope 

uses electrons to image structures

• very high resolution 

• no real time or colour imaging 

• much more expensive and difficult to operate 

• archives a much higher resolution and can visualize organelles/large macromolecules 

• electron microscopes are harder to purchase and operate compared to a simple light microscopes  

macromolecules

they are teh major structural, enzymatic and regulating components of cells

• about 30% of bacterial mass/total volume are chemical compounds (dry meat)

• about 80% of bacteria sage dry mass consists of macromolecules

types of macromolecules

• sugars,

• fatty acids

• amino acids

• nucleotides

phospholipids 

• two hydronic fatty acids, covalent bond to a hydrophilic head 

• phospholipid s non-covalently associated with themselves as via hydrophobic/hydrophilic.

  • phospholipid bilayer (heads facing up, tails facing down 

sugars 

polymerize into polysaccharides 

• polysaccharides used as components of cellular structures, energy storage, ect.  

amino acids

polymerize into polypeptides (proteins) 

• proteins represent various cellular functions as much as metabolism, structure, regulation, signal transduction, etc

nucleotides

polymerize into nucleic acids

• DNA is the storage material of genetic information

• RNA can be na intermediate between DNA and protein (mRNA) or can have biological function by itself (function RNA)

• sugars, animo acids and nucleotides always polymerize covalent bonds

nucleic acids are one of the four macromolecules 

• nucleic acids are long, linear polymer of nucleotides 

• RNA is a polymer of ribonucleotides 

  • transient storage of genetic information (mRNA)

  • functional RNA

• DNA is a polymer of deoxyribonucleotides

  • storage of genetic information in genomes

four types off deoxyribonucleotides used to build DNA

all four deoxyribonucleotides have similar composition with ‘bases’ that are slightly different from one another

• adenine

• thymine

• guanine

• cytosine

nucleotides structure

nitrogenous base + pentrose (five-carbon) sugar + one or more phosphate group

different kinds of nucleotides 

• nitrogenous bases 

  • adenine, guanine, cytosine, thymine/uracil

• pentrose sugar

  • ribose or deoxyribose

• number of phosphates

  • 1, 2, or 3 phosphates attached

purine (A and G)

• purines have nine atoms constituting its two rings, numbered 1-9

• nitrogen in the larger ring furthest away form the smaller ring is nitrogen 1

• nitrogen in the smaller ring is connected to carbon 5 is nitrogen 7

• nitrogen in the smaller ring without a double bond is nitrogen 9

the above is true at least for the two common purines used in the central dogma

pyrimidines (T, C, and U)

• have six atoms constituting its ring, number 1-6

• two nitrogen’s at positions 1 and 3 

• carbon 2 is always connected to an oxygen with a double bond 

• carbon 4 is always connected to an extra atom outside the ring 

the above is true at least for three common pyrimidines used in the central dogma

two types of pentose sugars (both a ribose) 

• carbons are numbered 1’ to 5’ 

ribose

• has OH groups attached to its 2’ and 3’ carbons

• found in RNA

deoxyribose

• has an OH group attached to its 3’ carbon

• missing the OH group at its 2’ carbon, compared to ribose

• found in DNA

up to three phosphates are added 

• phosphate groups are added to the 5’ carbons found of the pentose sugar 

• the phosphates are named alpha (a) and beta (b) and gamma (v) 

  • alpha is the closest to the 5’ carbons

  • gamma is the furthest away

• a lot of energy is stored inside the phosphoanhydride bonds between gamma-beta and beta-alpha phosphates

nucleoslide

nitrogenous base + pentose sugar (without any phosphate) 

nucleotide

nitrogenous base + pentose sugar + at least one phosphate

nucleotide nomenclature

• we follow the IUPAC code to name and abbreviate nucleotides systematically

• the letter ‘N’ is used to represent ‘aNy’ nitrogenous base in DNA

• in a DNA sequence, writing ‘N’ means that the position can be any one of A, G, C, or T

• abbreviation for other common combinations of nucleotides:

  • ‘R’ = A or G (puRines) 

  • ‘Y’ = T or C (pYrimadines) 

nucleotide triphosphates

the incoming nucleotide must always be a triphosphate 

• the 3’ OH attaching to the alpha-carbon of incoming nucleotide trisphosphate breaks the high energy phosphoanhydride bond between beta-alpha phosphate 

breaking bond between beta-alpha phosphates release

1. energy

2. pyrophosphate

• get broken to release more energy

• this energy is then used to catalyze the formation of the 3’ - 5’ phosphodiester bond

nucleotides are energy currencies

• phosphoanhydride bonds in nucleotides can be broken

nucleotide polymerization is a special case 

the reagent of reaction (NTP or dNTP) carries the energy to polymerize itself 

therefore nucleic acid synthesis must use triphosphate nucleotides as reagents 

• mono- and di- phosphate nucleotides cannot be used during nucleotide polymerization since they don’t carry enough energy 

nucleotide has many functions in the cell

• being the building block of DNA and RNA is a major function of nucleotides 

  • storage of genetic information, various activities as functional RNA

• other critical function is energy currency

enzyme currency 

• breakage of high energy phosphoanhydride bonds are coupled to energetically unfavourable reactions  

• ATP and GTP are major nucleotides used as energy currencies 

• ATP and GTP are generated using energy released by digesting food 

coenzymes for various metabolic pathways

coenzyme A

• adenosine derivative with a 5’ di-phosphate and a 3’ monophosphate

• 5’ di-phosphate connected to more structures

coenzyme A is used to facilitate many metabolic pathways

• fatty acid metabolism

• gets added to private to Acetyl-Coft, which is the starting point into the citric acid cycle (TCA cycle)

electron carrie for metabolism 

NAD+ is dinucleotide 

• first nucleotide has an unconvential base, nucliotinamide (this is NOT a pyrimidines) 

• second nucleotide is an AMP 

• two nucleotides attached directly via alpha phosphates 

•  NAD+ receives electrons released during glycolysis and citric acid cycle 

  • this converts to NAD+ and NADH

  • electrons donated to the electron transport chain to generate ATP

cAMP is made from ATP 

• bond between alpha-beta phosphates broken 

• the open end of alpha phosphate gets connected to the 3’ carbon of its own ribose sugar, making a cyclic structure 

formation of double stranded DNA (dsDNA)

• nucleic acids are synthesized via polymerization of nucleotides

• polymerization of dNTPs generate single-stranded DNA (ssDNA)

  • synthesis in the 5’ to 3’ direction

  • long, unbranched chain of nucleotides

• in our cells, DNA rarely exists as ssDNA

• ssDNA attached to form a second, complementary ssDNA to form double stranded DNA

dsDNA bonds

• covalent bonds hold nucleotides in ssDNA (3’ - 5’ phosphodiester bonds) 

• hydrogen bonds hold two ssDNA together in dsDNA 

  • no covalent bonds occur between two strands of ssDNA

• the hydrogen bonds of dsDNA occur between nitrogenous bases of ssDNA

  • nitrogenous bases are held inside the middle of dsDNA

  • sugar phosphate backbone occur on the outside

• two ssDNA in dsDNA associate in an anti parallel orientation, where their 5’ - 3’ directionally is reversed

base pairing 

• nitrogenous base always pair with a specific partner 

• cytosine pairs with guaine via three hydrogen bonds occur 

• thymine pairs with adenine via two hydrogen bonds 

• these are called base pairs (bp) 

• base pairs are written as G.C and A.T 

full chemical structure of dsDNA 

• these G.C and A.T base pairs are part of a longer stretch of dsDNA 

  • 5’ and 3’ ends of this dsDNA are connected to more nucleotides (dashed lines)

• numbers are shown for important atoms in red

• distance between two C1’ carbons in a base pairs is about 1.1 nm

• in the true 3D structure, nitrogenous bases and their sugars are held perpendicular to each other

physiological implications of base pairing 

• the G.C has one more hydrogen bond compared to the A.T pair 

  • it takes more energy to break apart a G.C pair

• G.C pair holds the two strand of dsDNA more strongly compared to the A.T pair

  • for example, dsDNA with higher GC% is harder to break into ssDNA using heat (desaturation)

• GC% of the genome is species dependent and is highly variable

  • prokaryotes have gemones with GC% of 8-75%

  • organisms living in a higher temperature tends to have genomes with a higher GC% presumably to resist denaturation by heat

calculating GC% and AT% of dsDNA 

since dsDNA has complementary base pairing 

• for every adenine, there is a corresponding thymine

• for every guaine, there is a corresponding cytosine

therefore A% = T% and G% = C%

base pairing is the basis of DNA…

• during DNA replication, two strands of dsDNA are broken apart and used as a template to help synthesize new strands of DNA

• base pairing is also the basis for transcription (generates ssRNA using ssDNA as a template)

writing DNA/RNA sequences 

• nucleotide sequences are ALWAYS written in the 5’ to 3’ direction 

  • DNA/RNA sequences written without a label are assumed to be written in this direction

  • if you must write them in a 3’ to 5’ direction you must make this very clear

• for dsDNA, chose on of the ssDNA sequences to represent the whole molecules

  • it is not necessary to indicate both sequences as they are complementary to each other

  • show the sequence of the more important ssDNA (for example, the coding strand of a gene as opposed to the non-coding strand)

discovery of the dsDNA structure 

• phoebus aaron theodore levine 

• erwin chargaff 

• x-ray crystallography

• under supervision of rosalind franklin, raymond gosling took x-ray diffraction images of crystallized dsDNA (early 1950s)

• the 51st image (photo 51) was shown to Maurice Wilkins, who then showed it to James Watson

• James Watson and Francis Crick solved the structure of dsDNA using photo 51 and published in 1953

phoebus aaron theodore levene

• multiple discoveries between 1910s - late 1920s

• showed that DNA contains equal proportion of nitrogenous bases, deoxyriboses, and phosphates

• hypothesized that a nitrogenous base, deoxyribose and phosphate combine to make the building block of DNA, and named this unit ‘nucleotide’

Erwin Chargaff

• discovered the Chargaff’s rule, late 1940s 

• in dsDNA, around of guanine = amount of cytosine, and amount of adenine = amount of thymine

X-ray crystallography 

• method to analyze the atomic structure of a crystal 

• can be used to analyze structures of macromolecules like proteins and nucleonics acids 

• purity and crystallize molecules 

• shoot x-ray through the crystal 

• the crystal diffracts x-ray and the pattern is recorded on a film/screen 

• diffraction pattern represents the structure of the crystal = atomic structure of molecule in the crystal 

physical dimensions of dsDNA

• an antiparallel, double stranded helix with a right handed twist

• dimeter = 2nm

• length occupied by one base pair = 0.34 nm

• length of one turn = 3.4 nm

• therefore = 10 base pairs per turn

B-DNA is physiologically relevant form of dsDNA 

• dsDNA can fold in different shapes under different conditions 

• B-DNA is the physically relevant, hydrated from 

  • right-handed helix

  • occurs in physiological conditions (chemical conditions inside our cells)

• A-DNA is the ‘dried’ form

  • right handed helix

  • occurs at <75% humidity

• left handed Z-DNA may occur in cells, but function is not well understood

dsDNA structure 

• base pairs in the middle 

• sugar phosphate backbone on the outside 

base pairs in the middle are not completely hidden by the backbone 

• openings between the backbone exposes some parts of base pairs 

• openings between sugar-phosphate backbones are not evenly spaced 

• nitrogenous base pairs are laid flat when you look down at dsDNA along its length 

• two deoxyribose stick out from the base pairs in a non 180º angle 

• one side of the base pairs has a wider opening (257º) 

• opposite side has a narrower opening (103º) 

• these openings become the major and minor grooves of dsDNA, when these base pairs stack onto each other (while rotating 36º every bp since 10 bp = 1 turn)

major groove

larger gap between the backbone exposes

• expose the bases more due to its wider opening

minor groove

smaller gap between the backbone and

major and minor grooves

• major and minor grooves always occur in a pair, and on opposite sides of dsDNA 

• major and minor grooves expose some atoms of nitrogenous bases to the environment 

  • ‘environment’ = nucleus, cytoplasm, etc.

• major and minor grooves allow other macromolecules (like proteins) to directly touch the nitrogenous bases 

• every nitrogenous base has a unique shape 

• proteins can tough and READ the sequences of dsDNA without opening the dsDNA 

• enables efficient protein DNA interaction when DNA binding proteins are searching for specific DNA sequences to bind to 

  • it takes time and energy to open up dsDNA to completely expose the bases 

discovery of the basic feature of RNA 

• in the 1953 paper, Watson and Crick described dsDNA structure without surprisingly occuract 

  • they even correctly, hypothesized the machinist of DNA replication, just based on the dsDNA structure

• however, they made some claims that turned out to be incorrect

• watson and crickets hypothesized that RNA can not fold into a structure similar to B-DNA

  • in reality, dsDNA looks very similar to B-DNA

basic features of RNA 

• RNA folding into double stranded structures are the basis of many molecular mechanisms 

• chemical composition and polymerization mechanism or RNA and DNA are nearly identical 

• structural difference between RNA and DNA 

  • ribose is used as the pentose sugar in RNA

  • uracil is used in the place of thymine in RNA

• RNA usually exists as ssRNA in the cell

• ssRNA can fold onto itself by complementary bases-pairing

ssRNA folds by base pairing 

• ssRNA is a long, flexible string 

• ssRNA can base pair onto itself using complementary base pairing 

• unique shape of folded ssRNA gives it biological function (RNA) 

  • in HIV< a portion of its mRNA folds into a unique shape (RRE) to bind proteins that guide the mRNA for translation and packing

RNA Function 

• not for permanent storage of genetic information in cellular organisms 

  • some viruses such as HIV, coronavirus and Ebolavirus have RNA genomes

• multiple physiological functions in the form of mRNA, tRNA, rRNA, snRNA, etc

• functional RNAs do not get translated into proteins

  • fold into unique 3D shapes to perform their functions

common characteristics of life

polypeptides are long polymer of animo acids

• polypeptides fold into proteins

proteins participate in diverse cell functions

• enzymes

• structural components

• signaling

• receptor

• motor proteins

• transport systems

• gene regulation

• immune systems and antibodies

amino acids are the building blocks of polypeptides 

• 20 types of amino acids are used to build polypeptides

• all 20 amino acids have similar compositions with different side chains attached to the amino acid backbone 

• the 20 bases can be connected in any number/order 

• action acid sequence of the polypeptide determines how it folds 

amino acid structure

amino acids has a central carbon (alpha carbon, Ca) connected to 4 ‘groups’

• amino group

• carboxyl group

• side-chains group (R-group)

• hydrogen

R-group is the variable component between different amino acids

• all other components are the same (except for proline)

at physiological pH, amino group and carboxyl group becomes positively and negatively charged, respectively

side chains determine properties of amino acids

r group varies in

• size and shape

• hydrophobicity/polarity

• charge

• chemical reactivity

amino acids used by cells to build proteins `

10 amino acids are non polar 

10 amino acids are polar 

• 5 of these are not charged 

• 2 of these carry a negative charge 

• 3 of these carry a positive charge 

amino acids with non polar side chains 

10 in total 

• R group is usually a hydrocarbon (carbons and hydrogens) 

• pure hydrocarbons are not polar 

amino acids with polar, uncharged side chains 

5 in total 

• R group contains an amide or hydroxyl (OH) group 

• amines and hydroxyls have electrons unevenly distributed within their structure

  • this makes one part of the structure more positive, and other parts more negative (polar)

• 2 amino acids ahve polar, acidic side chains attached physiological pH

  • donates a hydrogen and becomes negatively charged

  • in both cases, the acidic group is a carboxyl group

• 3 amino acids have polar, basic side chains at physiological pH donates

  • accepts a hydrogen and becomes positively charged

  • amino, guanidum or an imidazole group

amino acids a re categorized mainly by their polarity

• polarity determines how each amino acid side chains interaction with water

• water is a polar molecule

  • have uneven distribution of electrons

  • one side is slightly negative, on their side is slightly positive

• polar side chains like to be beside water since they also have an uneven distribution of electrons, creating a positive/negative surface that attracts water

• non polar side chains dislikes being beside water because these side chains has an even distribution of electrons

• there is a varying degree of hydrophobicity even between amino acids in the same category 

• some amino acids have hydrophobicity that are in-between polar and non polar 

amino acids with unique properties: one that influence folding very strongly 

how a polypeptide fold into a protein is determined by its sequence of amino acids residues 

• polarity of each amino acid residues have huge influence in this process 

certain amino acids have additional features that strongly impacts protein folding 

• glycine: smallest amino acid, makes the polypeptide backbone very flexible 

• proline: has a side chains that connects to its nitrogen, makes the polypeptide backbone very inflexible 

• cysteine: can form di sulphide bonds with other cysteine residues to covalent link tow polypeptide backbones 

amino acids with unique properties: ones with aromatic rings

• aromatic rings are larger and bulkier compared to other side chains

  • takes up more space when proteins fold

• tyrosine, phenylalanine and tryptophan has side chains that absorb ultraviolet at 270 - 280 nm

  • polypeptides that contain these amino acid residues can be analyzed by UV-spectrophotometer

• histidine has an imidasole ring that has a pKa of ~6.0

  • therefore, only some histidine residues are positively charged at physiological pH

  • other histidine residues are uncharged

  • small changes in pH dramatically affects this behaviour of histidine

  • the ‘flexibility’ of histidine side chains are utilized by enzymes to handle protons on/off the other

amino acids with unique properties: ones with a hydroxyl group 

• serine, threonine, and tyrosine has a hydroxyl groups exposed at the end of their side chains

• exposed hydroxyl groups are great targets for phosphorylation 

  • kinase: any enzyme that adds phosphate group to target

  • phosphate: any enzyme that removes phosphate group from target (dephosphorylation)

• phosphorylation is used to regulate protein function after it has been made

  • for example, some proteins are only active when they are phosphorylation and vice versa

  • cells phosphorylation/dephosphorylate their proteins to switch them on and off in response to their physiological needs

amino acids polymerize into polypeptides 

• the end of a polypeptide has a free carboxyl groups exposed 

  • this is called the carboxyl terminus (C-terminus) of the polypeptide

• next amino acids is placed beside the polypeptide

  • amino group of the amino acid is facing the carboxyl group at the C-terminus of polypeptide

• condensation reaction joins the carboxyl carbon of the polypeptide to the nitrogen of the next amino acid

• this newly formed bond is called the peptide bond

polypeptide has a N-C-C backbone 

• individual amino acids (before polymerizing have nitrogen, alpha carbon, and carboxyl) upon polymerization, these three atoms form a long chain with a repeating ‘NCC patter’ 

• this is the peptide backbone 

N-terminus (amino-terminus)

the front of the peptide backbone with a free amino group 

C-terminus (carboxyl-terminus)

the end of the peptide backbone with a free carboxyl group

amino acid vs amino acid residue

• the chemical structures of amino acids change when they polymerize 

  • inside the polypeptide they are no longer ‘amino acids’ are still recognizable inside the polypeptide 

• individual amino acids are still recognizable inside the polypeptide 

• these ‘remainders’ of amino acids are called amino acids residues 

• the polypeptide in this figure is made out of four amino acid residues 

polypeptide vs proteins

• polypeptide is any chain of amino acids polymerized via peptide bond

• protein is defined more vaugely

• protein is a longer polypeptide that folds into a unique 3D shape and obtains a biological function

• proteins are usually over 100s of amino acid residues long

peptides fold into proteins

conformation: a proteins final folded structure

• polypeptide must fold in a shape that satisfies the needs of its amino acid residues

  • polar sides chains facing water

• polypeptide folding is determined by its sequence of amino acid residues 

• protein structure is dynamic 

  • a protein may have multiple stable conformations

  • switches from one conformation to another while it performs its biological activity

polypeptide folding in aqueous environments 

• proteins that function in aqueous environment are surrounded by water 

  • inside the cytoplasm, nucleolus, ER, golgi, mitochondria, etc

• these proteins usually fold into a sphere

• place hydrophilic amino acid residues on the surface of the protein, exposing them to water

• police hydrophobic amino acid residues inside the protein, away from water

  • becomes the hydrophobic core

polypeptide folding is supported by three non-covalent interactions 

van der waals attractions 

• very weak interaction when atoms are placed closer to another

hydrogen bonds

• bonding between two polar molecules

• electrostatic interactions - bonding between two charged molecules

• these interaction are weaker than covalent bonds, but they occur in large number and accumulate into a strong force

unique amino acids the influence protein folding: cystine 

• no covalent bonds contribute to protein folding except for one example 

• a pair of cystine side chains can form coavalent disulfied bonds to connect the polypeptide backbone 

• holds protein structure very strongly 

• antibodies are produced by our immune system to bind and remove potentially harmful materials (antigens) in our body 

• two antigen binding sites form at the ends of the antibody 

• one antibody is made of four individual polypeptides 

  • two units of ‘heavy chain’ 

  • two units of ‘light’ chain’ 

• multiple disulphide bonds contribute to this structure 

disulfide bonds can occur 

• within the same polypeptide backbone 

• between two different backbones 

flexibility of polypeptide backbone

• the polypeptide backbone is flexible

  • allows it to hold into many shapes 

• not all parts of the polypeptide backbone are equally flexible

• the bonds before and after the central carbon are flexible

  • N-Ca bone and the Ca-carboxyl

• peptide bond is inflexible

• protein folding is limited by these properties

unique amino acids for folding: glycine

• glycine is the smallest amino acid

  • side chains is a single hydrogen

• glycine increases the flexibility of surrounding area since it provides more ‘open’ space for other atoms to occupy

• mutations that replaces amino acids into a glycine may destabilize protein structures because it introduces flexibility

unique amino acids for folding: proline

• proline has its side chains covalently attached to its backbone nitrogen

  • its technically not an ‘amino’ acid since it does not have an amino group 

  • proline is an amino acid 

• prolines N-Ca bond is completely inflexible as its rotation is locked by the side chain 

• mutations that replaces amino acids into a proline may disrupt protein structure by removing the flexibility that was necessary to accomodate the original fold 

protein structures can be represented in many ways 

• use appropriate method to best represent what you want to show 

A. peptide backbone represented as lines 

B. peptide backbone represented as ribbons to show secondary structures 

• α helix: curled ribbons 

• β sheet: ribbon with arrows

C. all atoms shown as sticks to display the side chains 

• clutters the image but can represent molecular interactions using a specific side chains groups 

D. all atoms shown using a space filling model 

• represents the surface shape of the protein 

protein data bank

scientists have been analyzing atomic structures of biological molecules for about a century

• x-ray crystallography

• nuclear magnetic resonance (NMR)

• cryogenic EM

structural data deposited in the protein data bank

• open access

• structure catalogued with a four letter code

• over 200,000 structures are currently deposited

protein structure is described by four level of ‘complexity’

• primary structure (1º) 

  • linear amino acid sequence of the protein

• secondary structure (2º)

  • short regions of the peptide backbone fold into individual 3D structures that compose the tertiary structure

• tertiary structure (3º)

  • the fully-folded, 3D structures of the protein

• quaternary structure (4º)

  • in many cases, multiple individual proteins combine into a larger, mega structure

primary structure (1º) 

• primary structure is the raw sequences of amino acid residue of the protein secondary structure

  • also called ‘primary sequence’ ‘amino acid sequence’

  • written from N-terminus to C-terminus

• primary structure determines how the polypeptide folds

• sometimes, a change in single amino acid change have a large effect on protein function

multiple sequence alignment (MSA) 

• compare primary sequences of different proteins 

  • mutant protein vs. wild type

  • homologous proteins from different organisms

  • can also compare nucleotide sequences

• this example compares two mutant proteins against the wild type

  • mut_1 has the 4th valine substituted with leucine (non polar to non polar)

  • mut_2 has the 4th valine substituted with glutamic acid

  • we can hypothesize the mut_2 is affect more because its mutation substitutes valine into something completely

nomenclature of MSAs and Mutation 

• in MSA an asterisk (*) indicates that all proteins have the same amino acid residues at that position 

• writing mutation 

  • for example, mutation is mut_1 is written V4L because it has valine at position 4 changed to leucine

  • mut_2 has a V4E mutation

• mutations in DNA can be written the same way

secondary structures (2º)

• proteins fold into their final shape (conformation) in a cell

  • this is their tertiary structure

• tertiary structure contains multiple substructures that have distinct shapes

  • these substructures are called secondary structures

• proteins do not fold first into secondary structures followed by tertiary

  • they fold directly from primary structure into tertiary structure

  • secondary structures do not occur independently

• secondary structures are the ‘building blocks’ that combine into a tertiary structure

  • dissecting tertiary structures into secondary structures enable us to understand proteins better 

folding of the peptide backbone 

secondary structure is the folding of the peptide backbone 

• primary sequence (= sequence of side chains) determines what type of secondary structure that backbone becomes 

• however, side chinas do not directly stabilize the secondary structure by forming bonds, etc 

hydrogen bonds between backbone atoms hold the secondary structure together 

• α-helix and β-sheet are the two major secondary structures 

  • 310 helix, π-helix, various turns and loops also exist