BIOC0014

Properties of the ribosomal proteins

-55 proteins in the 70s ribosome- most proteins have more than one domain -contain long N-/ C- terminal extensions (rich in Arg/Lys) highly basic - the globular domains are at the surface whilst the extensions and loops are buried within the ribosome - the proteins interact with the rRNAs by shape and charge complementarity -most proteins interact with multiple helices

Properties of molecular chaperones

-Molecular chaperones interact with unfolded proteins or partially folded protein subunits -They stabilize non-native conformations and facilitate correct folding of protein subunits -some chaperones are non-specific, and interact with a wide variety of polypeptide chains, but others are restricted to specfic targets -many chaperones are ATPases and uses the energy from ATP binding or ATP hydrolysis

Name 3 scenarios which can affect structure formation of nascent polypeptide chains during their biosynthesis within the cell.

-Rare codon usage -mRNA structure -Post-transcriptional modification of the mRNA -Interaction of the nascent chain with the tunnel -interaction of the NC with the ribosome surface -interaction of the NC with ribosome associated molecular chaperones

How is in vivo protein folding different from in vitro

-Vectorial synthesis -The presence of the ribosome -The crowed cellular environment

Facts about the 30s subunit

0.8 MDa 21 proteins 1500 nucleotides rRNA (15s)

How to obtain structures of the same process as it occurs in vivo- on the ribosome

1. Design a folding snapshot (Ribosome NC complex-RNC) by adding a stalling sequence at the end of the peptide sequence- make a construct using a expression vector 2. Allow cell to express the construct 3. Purify out the RNC 4. Western blot and NMR to study the structure of the RNC compare to the isolated peptide

Facts about the 50s subunit

1.5 MDa 34 proteins 3000 nucleotides rRNA (23s and 5s)

What is the composition of the ribosome (protein and rRNA)

1/3 protein 2/3 rRNA

What is the rate of translation?

10-20 aa per second

How is the ribosome stabilised?

12 intersubunit contacts (mostly RNA-RNA interactions) Mg2+ ions is concentrated within the folds of the 23s rRNA, creating a core at the center similar to a hydrophobic core in proteins

What is a trigger factor

A trigger factor is a protein that binds to the ribosome near the ribosome exit tunnel to facilitate protein folding

What happens in the A, P, E sites

A- binds incoming amino-acyl tRNA P- contains the polypeptide E- where the deacylated tRNA exits

Alzheimer's Disease

Abeta aggregation

What is the A-minor motif

Adenosine is the most abundant nucleotide in the rRNA and it is mostly buried within the ribosome. The adenosines will form important structural motifs to stablise the 23s rRNA.

Where are the aggregates collected

Aggresome

Advantages of cryo-EM

Allows visualisation of the native, hydrated state of the sample, near physiological conditions, 3D structure preserved, rapid freezing can trap transient states

Structure characteristics of amyloids

Amyloids forms a fibrillar structure 10nm in length Amyloids binds Congo Red (dye) The fibres are made up of different numbers of component beta strands (protofilaments) Protofilaments are twisted around each other in either compact or ribbon like arrangements

Why are unfolded/misfolded proteins bad

Because they are: -aggregation prone -protease sensitive Non-functional The exposed hydrophobic residues don't like to be in the water, when a protein is misfolded or unfolded, the hydrophobic sites might fold with the hydrophobic sites of another protein forming aggregates. If the aggregates are not reversible, they form into amyloids which are toxic.

Functions of dynein 1

Cargo transporters- during interphase, the minus ends of MT is localised at the organising centre near the nucleus so dynein 1 is moving cargo towards the nucleus Cell migration- dynein 1 tethered to the plasma membrane and pulls the MT network, pulling the whole cytoskeleton forward Cell division- Organising the minus end of the MT into the mitotic spindle Also found at the chromosome surface, at the kinetochore- once the MT is attached to the chromosome, dynein removes signalling molecules at the kinetochore, signalling the cell that this chromosome is ready for cell division

Processes MT are involed in

Cell division, Cell (Neural) migration

Explain how protein folding, a highly specific process dependent on the precise sequence of the protein, can be assisted by general chaperones with little or no sequence specificity for their substrates.

Chaperones can bind to different cofactors and it is the cofactor that determines the specificity for substrates

Overview of decoding and elongation

Codon recognition (EF-Tu-GTP brings the correct amino-acyl tRNA to the A site) Activation of GTPase activity- binding of amino-acyl tRNA to the A site GTP hydrolysis- EF-Tu release leaving the amino-acyl tRNA at the A site Accommodation- as the amino-acyl tRNA binds to the A site, other tRNAs are rejected to enter the A site Peptidyl transferase- the polypeptide on the P site will move to the A site and form peptide bond with the aa on the tRNA at the A site EF-G-GTP binding- EF-G-GTP will the bind to the tRNA on the A site (now containing the polypeptide) EF-G-GTP hydrolysis- translocate the tRNA on the A site into the P site (the tRNA that is originally at the P site will move to the E site and eventually leave the ribosome) EF-G-GDP then dissociates from the ribosome and the whole cycle restarts

Disadvantages of cryo-EM

Complex preparation, longer time for checking samples, low contrast

In single particle analysis, how do you compare the starting model with the sample?

Cross-correlation- if the correlation is high when the two samples overlap, it suggests that they are really similar

How does DCX interact with MT

DCX binds at the corner of 4 tubulin dimers (between protofilaments) allows stabilisation of both longitudinal and lateral connections

What is the mechanism of DCX MT stabilisation

DCX have little effects on MT growth rate but it effects the depolymerisation rate Increase in DCX concentration--> decrease rate of depolymerisation At very high DCX concentrations--> no depolymerisation, MT can form from GDP tubulin

What happens when GTP hydrolysis> MT polymerisation

Depolymerisation, leaving with GDP tubuilin which are not polymersing competent and it is only when the GDP is exchange to GTP can the tubulin get re-incoporated into the MT

Systemc amyloidosis

Deposits of aggregates in the abdomen The aggregates have a amyloid structure Amyloids can be deposited in many different organs because they cannot be cleared Deposits disrupts cell function and have cytotoxic effects Point mutation in lysozyme is the cause of systemic amyloidosis

Kinetic studies of protein folding

Determine the speed at which protein folding happens- tells the rate and number of steps and energetic barriers in real time/ to what degree does a particular folding pathway occur in the biological time scale

Where on the 23s rRNA is the peptidyl transfer centre?

Domain V- it is the most common site for antibiotics binding that inhibits translation

What are the heat shock proteins responsible for?

During a stress response, the cell will turn off normal protein synthesis and turn on the synthesis of hsps. The hsps are responsible for binding to the crucial proteins that are unfolded or misfolded to prevent them from aggregating. The hsps will also help the misfolded proteins to refold into native state or send them to the proteolysis machinery for degradation.

Describe the longitudinal interactions between tubulins

Each tubulin have a plus end and a minus end The minus end if the alpha tubulin always forms contact with the plus end of the alpha tubulin the long string of tubulin interacting head to tail is called a protofilament tubulin incorporated at the plus end

How are heterogeneous complexes detected and sorted in cryo-EM

Eigen image analysis Heterogeneous complexes such as the ligand bound/un-bound form of receptors They can form because each machinery is at a different stage of the process Eigen image analysis finds the similarity and differences between samples- identify localised strong signal- find variation

Biophysical approach to study co-translational folding

FRET- compaction of the N terminus (tagged with fluorescent probe) shows that intermediates are formed Optical tweezers CryoEM NMR

Different methods to study protein folding

Fluorescence spectroscopy- Tryptophan (intrinsically fluorescent- sensitive to environment) is a reporter for changes in the protein as it unfolds: in the core in the folded stage, at the surface when unfolded- change in emission wavelength- increase as it unfolds Circular dichroism- allow the study of secondary interactions within a protein, conformational changes in the protein during folding and unfolding will alter the CD signal. NMR

Describe the three different states of proteostasis

Healthy unstressed- everything is balanced Healthy stressed- More aggregation but also overexpression of chaperones so everything is balanced out Aged or diseased- too much aggregation that overwhelms the chaperone system and misfolding takeover folding

Hsp100

Hexameric, located on either side of the protease- recognise proteins for degradation and drag them into the protease chamber

Describe the lateral connectivity between tubulins

Homolytic connectivity- alpha interacts with adjacent alpha, beta interacts with adjacent beta the interactions start to build the wall of MT

Hsp90

Hsp90 exist as a dimer- it is constantly dimerised at one end and when ATP binds the other end is also dimerised. Its function is to stabilise steroid receptors and kinases

Function of the 30s subunit

Initiate the binding of the mRNA (binding happens at the platform region- Shine Dalgarno sequence)

What are the major steps of translation

Initiation, decoding, elongation, termination

What causes the delay in protein folding?

Interactions between the NC and the ribosome

Functions of IFT dynein-2

Intraflagellar transport Involved in the assembly of cilium and signalling function not generating motion Moves cargo in the interior of the axoneme- IFT involves long train structures that move back and forth of the MT in the cilium, outward transport by kinesin-II and backward transport by dynein-2

How does the formation of intermediates assist protein folding?

It helps to reduce search for the right conformation, whether an intermediate is required during folding depends on the complexity of the protein's structure

What is EF-Tu

It is a GTPase elongation factor that delivers the amino-acyl tRNA to the ribosome

What is EF-G

It is a GTPase elongation factor that promotes translocation of the tRNA during synthesis

What is the stalk region of the ribosome

It is a protein region where ribosomal factors bind, it contains L7/12 and L10/11. L7/12 don't directly interact with the rRNA but they bind to L10 which binds L11 and the rRNAs

Function of the axonemal dynein

It is found exclusively in motile cilia- powering the wave like motion. There are multiple copies of the axonemal dyein in the axoneme. The axonemal dynein forms stable attachment to the MT through the tail domain and use their motor domain to generate motion along the adjacent MT, this generates sliding between the MT of the axoneme

Why is there limited folding in the ribosome exit tunnel?

Limitied size, hydrophobic wall minimises interaction

What protein is a good model to study the major difference between in vitro and in vivo protein folding?

Luciferase- removal of denaturants the protein doesn't refold into the native state- multidomain protein Luciferase folds co-translationally, during translation, the translated N-terminus will start to fold when the C-terminus is still being synthesised- the formation of the N-terminus stable domain (folding intermediates) facilitate the folding of the rest of the protein

What regulates the MT dynamics?

MT associated proteins e.g. Doublecortin (DCX)

Properties of Kinesin

MT-based movement ATP dependent Movement in one direction (+ OR - end) Motor domain contains ATP and MT binding sites

When does misfolding happen?

Misfolding can happen at any stage of protein folding: Intermediates during folding Unstable native state protein --> unfolding --> misfolding Misfolding can also happen during synthesis

alpha-synuclein

Misfolding leads to PD Function unclear intrinsically disordered with no native structure

Doublecortin (DCX)

Mutation are clustered in the NDC and CDC domains- these domains are important for binding MT DCX binds and stabilises MT GTP is still hydrolysed when DCX binds to MT

What is the eukaryote equivalent of the trigger factor?

NC associated complex

When does the protein start folding after synthesis

NMR studies showed that the NC only start folding when linked to the ribosome by ~45aa- delayed folding on the ribosome

Where does elongation factor bind on a tRNA

On the accepter arm/T arm

Function of the 50s ribosome

Peptide bond formation

What else can the base of the ribosome bind?

Peptide deformaylase, methionine aminopeptidase, SRP. These factors bind to the ribosome at different timings to vacillate synthesis and folding.

Where is the tRNA binding site?

Peptidyl transfer centre on the 50s subunit

What does the 50s subunit contain?

Peptidyl transfer centre, Ribosome exit tunnel

How can protinase K distinguish folded and unfolded proteins?

Proteinase K cleaves after aromatic residues If a protein is folded, by adding small amounts of proteinase K, the structure of the protein will just be nicked but largely resistant If a protein is unfolded, the addition of proteinase K will cause the peptide chain to be cleaved into fragments as there is no structural protection for the protein thus the protein is proteolytically sensitive If a protein is a mixed structure (e.g. multidomain) the addition of proteinase K will lead to the protein being cleaved into individual stable subdomains which show some resistant to the protease

Sample preparation for cryo-EM

Put samples on the EM grid into liquid ethane (vitrification); Take multiple images (projections) from different angles to generate a 3D reconstruction of the sample

How can changes in ribosome composition lead to diseases?

Qualitative change in the ribosome such as r-protein loss, r-protein missense mutations, r-protein paralogue exchange, rRNA modifications can lead to translational infidelity or alter the pattern of translation which can lead to cancer development and progression

What is the role of the anti-codon loop?

Recognise and bind to the mRNA in a complementary manner

Building of the protofilament

Requires tubulin dimers and GTP, when reaching a critical concentration (~10uM for mammalian tubulin) a microtubule nucleus will form spontaneously in solution (nucleation) and from these nuclei, MT will polymerise

Equilibrium studies of protein folding

Tells the potential of protein folding/ allows measure of protein stability and characterise highly populated intermediates- take a protein and denature it using denaturant. Let the sample equilibrate over time and measure the fraction of the unfolded/folded structure

Where does the aa bind on a tRNA

The accepter stem loop- ester bond at the CCA motif

How is the net stability of a protein defined?

The difference in free energy between the native and denatured state, for most proteins the difference in free energy is 5-15kcal/mol

What is the major component in thermodynamics that drives protein folding?

The hydrophobic effect- In the unfolded state, water orders itself around hydrophobic residues in a polypeptide chain In the native state, the hydrophobic residues are buried but the water molecules are random So even though the process of protein folding is losing entropy, there is an increase in system entropy as water molecules are liberated from the polypeptide chains.

Ribosomes each have a "ribosomal exit tunnel". Name the tunnel's main purpose(s) and outline three characteristics of the tunnel's structure.

The main purpose of the tunnel is to allow the NC to exit the ribosome Characteristics of the tunnel structure- -Contains a hydrophobic wall which keeps the NC largely unfolded -100A in length, 20A at its widest, holds 30-35aa -The tunnel is lined with 23s rRNA and constricted by loops -The upper tunnel, lower tunnel, exit vestibule -allow the formation of small secondary structures such as the Zn+ binding motif

Organisation of the 23s and 5s rRNA

The two rRNA forms a important scaffold for communication

Why are the cryo-EM images in low contrast?

There is little contrast and scattering between protein and water Weak exposure to electron beam because it needs to be low to prevent damage to the sample

In protein folding, what are the roles of thermodynamics and kinetics?

Thermodynamics affects how stable the protein structure is whilst kinetics affects the rate at which the protein is being synthesized and fold which can impact whether the protein folds into the native state or become misfolded. Thermodynamic properties indicate whether folding is favourable

Which AAs are intrinsically fluorescent

Tryptophan, tyrosine, phenalalaine

How to measure protein folding/unfolding over time?

Unfolding- put protein in denaturant refolding- put protein in solution with no denaturant then monitor the folding/unfolding process by a change in CD or fluorescence.

How to measure folding on the ribosome

When the NC emerge from the ribosome, they exert a force. To monitor folding induced by force, a secM stall motif is added to the end of the NC to induce arrest when interact with the ribosome The arrest can be overcome by properties of the NC chain, when the NC chain emerge from the ribosome and start folding into some structure, it generates a force that is strong enough to overcome the stall motif and pull the NC off the ribosome.

How does the ribosome distinguish between the wobble vs non-cognate tRNAs

When the right tRNA binds, there will be conformational changes in the G530 located on the 16s rRNA. The conformational change will allow the G530 to interact with the minor groove of the codon-anticodon double helix

What is vitrification?

absence of crystallization on cooling and solidification- the ice doesn't have the chance to crystallize, prevent dehydration, prevent damage of the native structure of the sample

Parkinson's disease

caused by alpha-synuclein aggregation (Lewy bodies)- due to alpha-synuclein point mutations

Hsp70

cofactors- hsp40- dimer that has a domain to activate the ATPase of Hsp70, it is also the substrate recruitment factor, it brings the substrate to the Hsp70 hsp110- the NEF- takes out ADP after hydrolysis by ATP, have the same fold as Hsp70 but doesn't perform its function responsible for disaggregation

How do we know if the nascent chain can fold on the ribosome

compare natively folded isolated proteins with proteins on the RNC using NMR- if the resonance overlap, it suggests that the NC can fold on the ribosome into the native state

How does averaging improve signal: noise ratio

noise is random, signal is not, by averaging the noise will be averaged out making the structure more prominent to see

What are chaperones?

proteins that regulates the folding states of protein

Is the catalytic events of the ribosome carried out by proteins or rRNA

rRNA- the ribosome is a ribozyme

What is the ribosome exit tunnel lined with

the 23s rRNA and constricted by loops

What are the difference between eukaryotic and prokaryotic ribosomes?

the eukaryotic ribosome is more complex, contains more rRNA in the form of extension segments as contains additional ribosomal proteins- the presence of these ES is due to the increase in complexity of the translation regulation in eukaryotic cells. The additional rProteins function to stablise the rRNA ES structures and act as binding sites for eukaryotic specific regulatory factors

Compare cytoplasmic ribosome and mito ribosome

the mito exit is wider, the mito ribosome have a protein shell, the mito ribosome contains minimal set of conserved rRNA and are largely single stranded

What are the different regions of the ribosome exit tunnel and what activities happen there

the upper tunnel- peptide bond formation/ targets of antibiotics/ arrest peptides the lower tunnel- formation of secondary structures exit vestibule- formation of secondary and some tertiary stuctures Complex folding happens outside the ribosome exit tunnel

Explain codon usage and how it is related to translation rate

there are 61 tRNAs coding for 20aa, this is due to redundancy in the genetic code where multiple codons will code for the same aa. The different types of tRNA does not exist in equal amounts in the cell, when there is a rare codon, translation will become slower by 5-8 aa. This will have impact on protein folding

How does trigger factor facilitate protein folding?

two models: 1. holds the NC prevent it from folding during synthesis to prevent misfolding 2. act a s a scaffold to accelerate the process of co-translational folding