BIOC2306 Sequences and conformational changes

how are proteins dynamic

atomic vibrations and rotational motions

large conf change on ligand binding

undergo transient unfolding

breathing motions too

Evidence of breathing motions

fluorescence quenched when not on surface with quenching agents therefore interact with solution and things in it

hydrogen-deuterium exchange (monitor by NMR or mass spec)

which protons can be exchanged rapidly in HD exchange

NH protons

but dep on pH and location/degree of burial

freq of unfolding

explain aromatic ring flipping

diff environ of protons in aromatic ring of residues observed to flip, change environ of proton

rate of flipping observed as two peaks or single weighted average

buried residues still flip very frequently, suggests atoms move out way (dynamic)

e.g. Phe and Tyr, but Trp and His cant rotate as bigger and not symmetrical

what else shows dynamic nature in crystallography

blurring of e density due to local flexibility

extent of smearing expressed as temperature factor ‘B’

high B = v dynamic

sometimes no e density observed as so flexible

binding shows reduced flexibility (initial flex facilitates complex formation)

small proteins can show diff conformations, crystal traps in diff states

why does high temperature appear to give smoother E landscapes

protein with more E so easier to jump between states, especially in roughness of native well

the 4 main classes of proteases

serine

cysteine

aspartic acid

metallo-

nature of proteases

some unspecific for substrate, just cleave at consensus sequence

many function in extracelluar environ

some are compartmentalised

how protolytic cleavage shows dynamic areas of proteins

req consesus seq to be accessible

surface and dynamic loops less resistant to degred

role of E dependent chambered proteases

allow reg of degred (selective as narrow entry)

allow degred of very stable proteins (use E dep unfoldases)

how using ATP allow degrad v stable proteins

chem E gen mechanical force to pull apart proteins for degrad

waht is the 26s proteosome

a chambered protease

Clp P structure

2 stacked ring of heptamers

each monomer has serine protease active sites

an bind 2 diff AAA+ proteins

Clp A and Clp X structure

ring like hexamer that can bind at each end of Clp P

has 2 and 1 AAA+ domains, respectively

AAA+ superfamily structure

form hexameric rings

function independently or with other proteins

what is the AAA+ superfamily involved in

Protein degrad

disagg (Clp B)

complex remodelling (Clp X if not in complex with Clp P)

how does ClpXP recognit sub

bind N or C term tag

e.g. ssrA tag added to polypep on stalled ribos

AANDENYALAA seq has 2 binding motifs

LAA recognit by loop regions in pore of ClpX

AANDENY recognit by SspB (adaptor protein) that increase affinity for Clp X, also prev dissoc to allow for several cycles

how does the AAA+ unfold substrate

the AAA+ domain utilises ATP to unfold sub

what are the two most common single molecule manipulation methods to measure forces applied onto mols

atomic force microscopy (cantilevers) and laser traps/tweezers

how is atomic force microscopy and laser traps/tweezers similar

both measure displacement of a spring of known stiffness to measure force applied

when are atomic force microscopy and laser traps/tweezers better suited

AFM good for over short distances

Laser tweezers better for small forces over large distances

uses of Atomic force microscopy

surface topology

unfolding

Pros and cons of contact mode of AFM

easy to use, use any type of tip, contact force can be changed to optimise contrast

sample damage, displace molecules

pros and cons of tapping mode of AFM

less sample damage/displacement as reduced lateral force, difference in phase between the driving oscillation and tip oscillation gives additional information

specific tips req, more difficult

how tapping mode of AFM works

tip moved along surface and the height is moved up or down to maintain a preset contact force

how tapping mode works in AFM

a high freq sinusoidal oscillation is applied to the tip, cantilever is moved up and down to maintain the amplitude of this oscillation

how does cantilever affect AFM resolution

sharpness/aspect ratio determines resolution, use carbon nanotube tip for highest res

low aspect ratio tip underestimates depth of features

what resolutions can be achieved by AFM

ideal conditions (vacuum and adhered on atomically flat surface) can resolve individual bonds

Biological samples generally globular and softer, get best image from 2D crystals get 1 nm res

Non crystalline samples res varies

how is AFM useful

used to study structure

pot dynamics one day e.g. myosin walking