molecular biology 1

cell

basic unit of life

simple organisms can consist of a single cell (autonomous cells)

living organisms can be viewed as a collection of cells organised in specialised structures (e.g. organs)

cells and evolution

all organisms are related and most probably evolved form a common single-celled ancestor

they can be arranged in the evolutionary tree - eubacteria branch then arches then eukaryota

conservation through evolution

key cellular components are conserved through evolution

example: conservation of the globin protein:

hemoglobin and myoglobin in vertebrate and leghemoglobin in other species all from an ancestral oxygen-binding protein

example of key motifs being conserved throughout evolution

haem and chlorophyll have the a similar structure with the porphyrin ring but different central metals coordinated (haem has Fe2+ and chlorophyll has Mg2+)

what is the porphyrin ring

4 pyrrole rings (each has a nitrogen)

linked by methine bridged (-CH-)

to form a flat, conjugated ring

• the nitrogen atoms can form a coordinate bond with a metal atom

central metal in haem

Fe2+

the iron can switch oxidation stated (between Fe2+ and Fe3+), which is good for its function for electron transfer

(primary function of haemoglobin is oxygen transfer but also sometimes involved in electron transfer, cytochromes are involved in electron transfer)

central metal in chlorophyll

Mg2+

prosthetic group

a non-peptide (protein) component tightly associated with a protein and required for its function

can be metals or organic

acts to support the structure of the protein

can also act as an electron carrier

• haem = prosthetic group in haemoglobin

• chlorophyll = prosthetic group in photosystems

why has the porphyrin ring been conserved evolutionarily

the conjugated ring has electron delocalisation which makes it stable and chemically versatile so evolution keeps it and saps the metal for different functions

key defining feature of a eukaryotic cell

presence of a membrane-bound nucleus and organelles

what replaces the nucleus in prokaryotes

the nuceloid (DNA not enclosed by a membrane)

how is DNA organised in prokaryotes vs eukaryotes

prokaryotes - circular, free in cytoplasm

eukaryotes - linear, enclosed in nucleus

what membranees are present in prokaryotic cells

inner (plasma0 membrane

cell wall

often an outer membrane (gram-negative bacteria)

periplasmic space between membranes - contains enzymes and transport proteins

why are prokaryotes more limited in complexity

lack of compartmentalisation - all reactions occur in the same space, less regulation of biochemical processes

size of prokaryotic cells

~1-5 micrometers

size of eukaryotic cells

~10-100 micrometers

plasma membrane

selective barrier controlling ion, nutrient and signal exchange

phospholipid bilayer with proteins embedded - transport via channels, carriers, pumps

mitochondria

ATP production via oxidative phosphorylation

lysosomes

degrade macromolecules and organelles

acidic lumen (pH of 4.50) and hydrolytic enzymes

nuclear envelope

double membrane separating nucleus from cytoplasm

contains nuclear pores for regulated transport

nucleolus

ribosome production

rRNA synthesis and ribosomal subunit assembly

nucelus

DNA storage and gene regulation

transcription occurs here - mRNA is exported out through pores in the nuclear membrane

smooth ER

lipid synthesis, detoxification, Ca2+ storage

enzymatic reactions in membrane

rER

protein synthesis for secretion/membranes

ribosomes translate proteins into ER lumen

Golgi apparatus

modify, sort and package proteins

glycosylation (enzyme-cata;ysed post-translational modification process of attaching carbohydrates to proteins or lipids) and vesicle trafficking

secretory vesicles

transport proteins to the membrane for secretion

fuse with the plasma membrane - exocytosis

peroxisomes

detoxification and fatty acid oxidation

produce and break down H2O2 via oxidases/catalase

cytoskeletal fibres

structural network for shape, transport, movement

microtubules, actin, intermediate filaments

microvilli

increase surface area for absorption

actin-based protrusions of membrane

cell wall

provides structural support and protection

made of cellulose in plants, peptidoglycan in bacteria and chitin in fungi

vacuole

storage and maintenance of turgor pressure

water influx keeps cell rigid

chloroplast

photosynthesis

light reactions (thylakoids) and Calvin cycle

which organelles are involved in the protein trafficking pathway

nucleus

rER

Golgi

vesicles

membrane.lysosome/secretion

which organelles are involved in detox

sER and peroxisomes

which organise generate energy

mitochondria - ATP

chloroplast - glucose (plants)

what is the fundamental purpose of cellular compartments?

to separate and control biochemical reactions within distinct environments

what defines a cellular compartment

a membrane bilayer enclosing a distinct internal environment

structure of a membrane bilayer

hydrophilic heads facing the aqueous environment

hydrophobic tails facing inwards

this forms a selective barrier

this si formed spontaneously due to the hydrophobic tails

why is compartmentalisation essential in eukaryotic cells

allows separation go incompatible reactions, increased efficiency, independent regulation

do all organelles ahem identical membranes

no, each organelle has a distinct lipid composition and specific proteins

why do membranes differ between organelles

membrane composition is tailored to function

for example mitochondria membrane contains ETC proteins, lysosome membrane is acid resistant and contains proton pumps

what determines membrane permeability

molecule size

polarity

charge

which molecule diffuse freely across membranes

small non-polar molecules (O2, N2, CO2)

which molecule are slightly permeable across membranes

small uncharged polar molecules (e.g. H2O, urea)

which molecules cannot cross without help

ions (K+, Ca2+, Cl-, etc.)

large polar molecules (e.g. glucose)

charged molecules (e.g. ATP, amino acids)

why are ions impermeable to membranes

hydrophobic core repels charged species

how do impermeable molecules cross membranes

via membrane proteins: channels, carriers, pumps

integral membrane proteins

embedded within the bilayer (often span membrane)

peripheral membrane proteins

loosely attached to membrane surface

lipid-anchored proteins

covalently attached to lipids within membrane

why is selective permeability critical

maintains ion gradients, metabolic control, cell signalling

how do membranes enable biochemical specialisation

by localising enzymes, creating gradients, controlling substrate access

significance of different head groups and tails within phospholipids

alters packing, shape and length

three main classes of membrane lipids

phosphoglycerides (phospholipids)

sphingolipids

sterols

basic structure of a phosphoglyceride

glycerol backbone, 2 fatty acid tail, phosphate group, variable head group

what distinguishes sphingolipids from phosphoglycerides structurally

sphingolipids have a sphingosine backbone instead of glycerol and usually one fatty acid chain attached via an amide bond

sterol and general structure

a lipid with a rigid four-ring structure that inserts between phospholipids in membranes

sterol found in animal membranes

cholesterol

sterol found in plant membranes

stigmasterol

sterol found in fungal membranes

ergosterol

head group attached fro phosphate of phospholipid

determines the identity of the phospholipid

common phospholipid head groups

• PC: phosphatidylcholine

• PE: phosphatidylethanolamine

• PS: phosphatidylserine

• PI: phosphatidylinositol

how do different head groups affect membrane properties

they alter charge, size and hydrogen bonding

this affects membrane curvature, interactions and protein binding

how does cholesterol affect membrane fluidity

buffers fluidity:

decreases fluidity at high temperatures

prevents tight packing at low temperatures - increases fluidity

cholesterol … membrane fluidity at low temperatures

increases

cholesterol … membrane fluidity at high temperatures

decreases

why do sphingolipids make membranes thicker

longer, more saturated hydrocarbon chains - tighter packing

membrane thickness in presence of cholesterol

more ordered and slightly thicker

why does PE promote curvature

small head group - cone-shaped - induces membrane bending

which lipid head promotes flat vs curved membranes

PC - cylindrical - flat bilayer

PE - cone-shaped - curvature

why is membrane curvature important

essential for vesicle formation, endo/exocytosis, organelle shape

how does lipid composition influence membrane function

controls fluidity, thickness, curvature and protein activity

bright-field (light) microscopy

light passes through the sample - contrast appears from the H&E staining

Haematoxylin

basic dye

binds acidic structure (DNA, RNA, acidic amino acids such as aspartate and glutamate) - blue/purple

Eosin

acidic dye

binds basic proteins (lysine, arginine) - pink

what information does H&E staining provide in tissues

reveals cellular and tissue structure, allowing distinction between norma vs pathological (e.g. cancerous) tissues

florescence microscopy

uses flourescently labeled moleculesthat bind specific cellular targets

why are cells fixed and permeabilised in flouresecne microscopy

fixation - preserves structure

permeabilisation - allows dyes/anitbodies to enter cell

what does the fluorescent probe phalloidin label

actin (microfilaments)

what does the fluorescent probe antibody label

tubulin (microtubules)

phalloidin dye colour

labelled with Rhodamine

red

antibody dye colour

green

confocal microscopy over conventional microscopy

uses optical sectioning to image only a single focal plane, eliminating out-of-focus light

why does confocal microscopy produce sharper images

excludes out-of-focus light, improving resolution and 3D reconstruction

compare bright-field, fluorescence and confocal microscopy

bright-field - general structure (low specificity)

fluorescence - specific molecules

confocal - high-resolution and depth (3D imaging)

why can’t electron microscopy be used on living cells

electron beams damage biological molecules (break bonds, generate free radicals) and require a vacuum

why does electron microscopy have higher resolution than light microscopy

electrons have a much shorter wavelength than visible light - higher resolving power

why are samples coated with heavy metals in electron microscopy

to increase electron scattering (contrast) and reduce damage, but it can reduce resolution/detail

how does cryo-EM reduce damage and improve imaging

rapid freezing preserves structure and reduces radiation damage (~6 fold) allowing near-atomic resolution (~0.2-0.3nm)

how does TEM (transmission) work and what does it show

electrons pass through thin specimen

shows internal ultrasound (2D image)

how does SEM (scanning) work and what does it show

electron beam scans surface, detects scattered electrons

shows 3D surface topography

key difference between TEM and SEM

TEM - internal structure - 2D

SEM - surface structure - 3D

why is cryo-EM a major advancement over conventional EM

preserves native structure without heavy metal staining and achieves near-atomic resolution

compare light vs electron microscopy

light - lower resolution, can image living cells

electron - very high resolution but requires dead, fixed samples