Part 1 Flashcards
What is protein folding and missfolding?
Folding= Acquisition of the correct structure of preform designated functions Missfolding = the failure to acquire the correct structure hence the failure to function
How do we depict protein structure if even one aa is a complex structure?
Alanine= 12 atoms
Protein consists of 20 different aa and average 500 aa
simplifying structure allows scientists to understand how mutations affect cell function
2 major protein structures- Alpha and beta
What are the different representations of alpha helix?
- Backbone
- Sticks
- Space filling
- Ribbon
Same for b sheet
Beta representations
Sheet- flattened arrows pointing to the c terminus= Anti-parallel
H bonds between polypeptide chains lying parallel- adjacent region to beta sheet
Loop between b sheets provides structure to protein
What is the alpha heical structure?
A spiral conformation in which every backbone N-H group donates a H bond to the backbone C=O group which of aa located approx 4 residues earlier along protein structure
7 transmembrane helical receptor
What is the structure of beta helical sheet?
Beta sheets consist of b strands connected laterally by backbone hydrogen bonds
Different conditions consist of different bonding type
Aromatic residues often found in b strands in the middle of b sheets
What interactions hold protein structures together?
Hydrogen bonds= Hold a shared H between O and N atoms
Ionic bonds= attraction between +ve and -ve charged ions
Weak van der walls= short range hydrophobic interactions
Disulfide bonds= involve a chemical link between 2 adjacent cystines
Folding of polypeptide chain in aqueous environment
Inside= Hydrophobic- Non polar side chains contained Outside= Hydrophillic- Polar side chains form H bonds with water
What is the quaternary structure?
Relationship between individual proteins in a multimeric complex (often duplication and multimersiation occurs)
Protein said to contain 2 Alpha and 2 beta
What methods can be used to sequence aa directly?
Edman degradation- each aa has own MW remove sequence of a, react peptides using PIT- label amino-terminal residue without disrupting aa bonds
Mass spectroscopy
How can secondary structure be predicted?
From primary by either de nova (the beginning) or alignment with other proteins of known structure using protein device- hydrophobic interacts with hydrophobic so align next to each other
What is coil coil?
When 2 alpha helix wrap around each other to form helical structure Amphatic side (leu and val) pi Triple coil- 3 helices twist around central aspect
What are the several techniques to obtain ‘real’ protein structure?
Circular Dichroism (CD)
- circularly polarising (190-250nm), give characteristic shape of CD
- different temps show stability- less stable faster lose CD characteristic
- cooling shows if it will refold
X-ray Crystallography
- x ray beam fired through protein crystal, most goes through few deflected
- structure traced from defraction
Nuclear Magnetic Resonance (NMR)
- Calmodium- binds activating proteins/kinases
- protein of interest= highly pure and labelled with isotopes
- C13 and N15 wobble spin
- Introduce into proteins recombinantly (grown on media)
Electron Microscopy
- Uses negative stain on actin filaments
- dried on grid, lightly coated with heavy metal
- see deposits of metal around protein synthesis
Cryo-electron microscopy
- liquid nitrogen to freeze
*For all these techniques you need to purify first
Pros and cons of various structural techniques
CD- cheap, quick, no size limit, low resolution but limited dynamics
X-ray- costly, crystals slow, data fast, high resolution, no size limit but bigger is harder, some artefacts, limited dynamics
NMR- costly, data fast, analysis slow, reasonable resolution, size 50KDA, good dynamics, repetitive process
EM- costly, medium speed, good resolution, no size limit, no dynamics
Where is most eukaryotic DNA is contained in?
Nuclear chromosones
Nuclear chromosomes comprises linear DNA and proteins which have specialised functions, these are:
- Packaging and unfolding of DNA repair and genetic recombination
- controlling DNA replication, DNA repair and genetic recombination
- maintaining chromosome integrity by preventing loss of end sequence
- governing proper chromosome segregation during cell division
- Regulating gene expression
- Mitochondria and chlorplast also contain small, circular chromosome
What are diphoid eukaryotic cells?
Contain two copies of each chromosome
Chromosome pairs differ by?
Size
DNA sequence content
During interphase which chromosomes occupy distinct sub nuclear territories?
The nuclear periphery is composed of transcriptionally inactive DNA
Inside= active
Transcriptional activation of gene accomplished by movement from periphery to centre of nucleus
Which fibre highly coats chromosomes?
chromatin
Biochemical anaylsis of nucleosomes reveals a protein core around which DNA wound, like cotton on a bobbin
What info do we know?
The protein subunits of the nucleosomes are core histones
The N terminal tails of the 8 core histone subunits project out from the nucleosome core and are free to interact with other proteins, facilitating regulation of chromatin structure
What are linker histones?
Example H1
Strap DNA onto histone octamers and limit movement of DNA relative to the histone octomer
Stabalizes formation of 30nm fibre
What is DNA packaged by?
Histone octomers
Packaged into a compact, flexible 30nm chromatin scaffold then can be remodelled to accommodate protein complexes involved in gene transcription and DNA replication
What is a telomere?
DNA sequence at ends of linear chromosome maintain chromosome integrity- stops it getting shorter
Repeat array eg TTAGGG are synthesised by telomere
Define chromosome ends to maintain identity
What is the replication origin?
DNA sequence where DNA replication is initated
What is the centromere?
DNA sequence on which kinetochore assembles and mediates chromosome segregation at mitosis and meiosis
contain specialised proteins and DNA sequences
Contain alpha- satelite DNA repeats
What is the kinetochore?
Protein complex that binds microtubules in the mitotic spindle
What are the different plates of the kinetchore?
Inner- bind to alpha satellite DNA
Outer- binds protein components of mitotic spindle
What is centromeric chromatin?
Contains specialised histones that mediate attachment of the chromosome to the kinetochore and modified histones that help to hold sister chromatids together
What is the centromere in yeast like?
Centromere is a basket that links a single nucelosome of centrameric chromatin to a single microtubule (not big array)
What percentage of the DNA sequence of eukaryotic genome encodes for cellular proteins?
1.5%
What percentage of the human genome is made up of repeated DNA sequence elements?
50%
What are the 3 different types of transposons (mobile genetic element that relocate across the genome)?
- DNA transposons- move by cut and paste without self duplication eg. chromosome A-B, powerful mutant
- Retroviral retrotransposon- Replicate via RNA intermediate, producing new DNA copies that integrate new genomic location using self-encoded reverse transcriptase.
- Non-retroviral polyA retrotransposons- in vertebrae genomes and replicates via an RNA intermediate using its own retrotransposon encoded reverse trranscriptase- copy and paste
Non retroviral polyA retrotransposon sequence
- L1 in chromosome
- L1 RNA synthesis
- Reverse transcriptase binds L1 RNA
- Cleavage of first strand of target DNA
- DNA primed reverse transcriptase
- 2nd strand produced
- L1 DNA copy of new position @genome
What is DNA replication?
Semi conservative
DNA synthesis occurs in which direction?
5’ to 3’
By formation of phosphodiester bonds- formed by hydroxyl grouo carrying out a neuclophillic attack
Why is DNA synthesis effectively an irreversible reaction?
It is coupled to breakdown of PPi to 2pi by pyrophosphatase.
The free energy required for DNA synthesis is provided by the breakdown of 2 high energy phosphate bonds
dNTP + (dNMP)n - (dNMP)N+1 + 2Pi
How does it go from triophosphate to pyrophosphate?
Incoming deoxyribonucleoside triphosphate in the 5’ to 3’ direction of chain growth
What is the direction of DNA synthesis and what does this mean?
DNA synthesis can only proceed 5’ to 3’ direction and the DNA strands are oriented antiparallel
This means that both strands cannot be synthesised continuously
Template strands for leading strands and lagging strands synthesis are orientated antiparallel to one another at the replication fork
What are the leading and lagging strand?
Leading= continuous 5' to 3' Lagging= discontinuous 5' to 3' contains okazaki fragments from breaks in replication
What is all DNA synthesis initiated by?
Extension of a short primer or RNA
Short primer is synthesised by DNA primase and only requires a DNA template and NTPs
RNA initiates DNA synthesis
What does the lagging strand require?
- DNA primase- makes RNA primer
- DNA polyermase- extends RNA primer, requires primer template junction
- ribonuclease H- removes RNA primer
- DNA polymerase- extends across gap
- DNA ligase-seals the nick- to convert Okazok fragments into a continuous strand of DNA
How does DNA ligase work?
Uses the energy of ATP hydrolysis to ligate newly synthesised, adjacent DNA fragments in a 2 step catalytic reaction
ATP+ 5-P - P-P + 5-P AMP
P-P - 2pi + free energy
Why is the ligand process energetically favourable?
Bc of conversion of ppi and zpi by pyrophasphatase- coupled reaction
strong and stable