Chapter 4: Protein Structure and Function

BIO380

proteins

very important and gives function to the cells

range of functions of proteins

enzymes, structural, transport, motor, storage, signal, receptors, transcription regulators, special-purpose

enzymes

function: catalyze covalent bond breakage or formation

structural proteins

function: provide mechanical support to cells and tissues

transport proteins

function: carry small molecules or ions

motor proteins

function: generate movement in cells and tissues

storage proteins

function: store amino acids or ions

signal proteins

function: carry extracellular signals from cell to cell

receptor proteins

function: detect signals and transmit them to the cell’s response machinery; to recognize what’s going on

transcription regulators

function: bind to DNA to switch genes on or off (occurs in nucleus)

special-purpose proteins

function: highly variable (everything else)

how proteins are studied

proteins can be purified from cells or tissues

different ways cells can be grown in culture

in vitro vs. in vivo

primary cell culture (from tissue)

cell line (immortalized)

in vitro

in glass

in vivo

in body

primary cell culture (from tissue)

cells can be separated into their component fractions

purification methods

4 different ways of homogenization

high frequency sound (ultrasound), mild detergent, high pressure hole and forcing cells through, shearing cells between plunger and wall

two types of ultracentrifugation

fixed motor vs hinge

fixed motor centrifugation

test tubes don’t move; pellet that is formed at the bottom is at an angle; refrigerated to keep constant temperature; vacuum to decrease friction and heat from movement (no protein lysis)

swining/hinge rotor centrifugation

rotor not circular; individual holders for the test tubes and buckets extend out; pellet is more flat compared to other method; refrigerated to keep constant temperature; vacuum to decrease friction and heat from movement (no protein lysis)

centrifugation products

supernatant and pellet

supernatant

smaller and less dense components

pellet

larger and more dense components (larger fragments of a cell)

differential centrifugation

separates parts of a cell depending on speed of centrifugation

may be repeated to ensure isolation → pellet is resuspended and centrifuged to get more isolated products

low speed centrifugation (from homogenate)

larger parts of the cell in pellet

whole cells, nuclei, cytoskeletons

medium-speed centrifugation of supernatant 1

transfer supernatant from tube

high speed creates a new pellet with new products broken down

mitochondria, lysosomes, peroxisomes

high speed centrifugation of supernatant 2

closed fragments of ER, other small vesicles

very high speed centrifugation of supernatant 3

ribosomes, viruses, large macromolecules

cells can be separated into component fractions with two methods

equilibrium sedimentation and velocity sedimentation

velocity sedimentation

separates on basis of size and shape; fast-sedimenting component comes out first, slow-sedimenting component last

based on density compared to sucrose gradient (5-20%)

equilibrium sedimentation

separates on basis of buoyancy; sample is evenly distributed at first and placed in steep sucrose gradient (20-70%)

through centrifugation → will see bands in light

high buoyant-density component will be at bottom, low-buoyant density will be at top

protein separation by chromatography

sizes, charges, shape differs → will pull through based on characteristics through chromatography

column chromatography

basis technique used for protein separation

sample is applied and passed through a solid matrix, things go through porous plug and into test tube

waste products come out first → constantly using solvent to elute for wanted product

3 types of column chromatography for protein separation

ion-exchange

gel-filtration

affinity