Biochemistry- Proteins

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Define Genome
all genes encoded in the genetic material i.e. the complete DNA sequence
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Genomics
The comprehensive study of whole sets of genes and their interactions
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How can genomics be studied?
Through micro-array
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Describe the process of a microarray (step 1 and 2)
1) ssDNA for many genes of interest are deposited on a solid surface in a defined grid 2)labelled target molecules (DNA/RNA) are eluted over the surface and bind specifically and quantitatively.
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Step 3)
detection of bound and labeled molecules is carried out to see which ssDNA is active.
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Why is the information we get from genomics bias?
the gene can be turned on, RNA produced, yet no protein made i.e. it only looks at the gene at an RNA level (hence also ignoring post-translational modifications)
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What is the C-value?
The quantity of DNA in a haploid nucleus- related to genome size
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Why do variations in c-values not bear any relationship to the complexity of the organism?
Due to non-coding DNA
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What does the c-value correlate with? (8 things)
•cell size •cell division rate •body size •metabolic rate •developmental rate • organ complexity •geographical distribution • extinction risk
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What is the model organism used for studying genomes and why?
E.coli- easy and cheap to grow, short generation time ,• Were first isolated in 1885- hence have lots of background info on genetics, metabolism and physiology.
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What is the proteome?
-the entire complement of proteins expressed or sometimes …the particular set of proteins expressed under specific conditions
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What is proteomics?
The study of the full set of proteins encoded by the genome
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How can it be studied?
Through gel electrophoresis
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Describe the process of gel electrophoresis
This separates the proteins according to size Proteins of interest can than be isolated form the gel and analysed by Mass-spectrometry/by c-terminal sequencing of a small stretch from the protein, allowing us to identify its sequence.
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Why is the proteome more complex than the genome? (4 reasons)
Alternative splicing of premRNA, post-translational modifications, protein interactions, different protein activities
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What is the central problem for biochemists?
The function of a protein depends on its 3D structure. The genetic code specifies only the amino acid sequence of a protein- from which we can’t tell what shape the protein will be.
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What is neuraminidase? (sialidase)
a protein that cleaves sialic acid from the surface of a cell in order to release the virus.
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What would an image of the protein show?
Its electron density, subunits and ligand binding sites
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How can we simplify the image?
Rotate the image, remove most atoms, show polypeptides, and emphasise structural features
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How can we study the structure further?
Zoom in on subunit, simplify representation as before and highlight binding site.
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And finally?
Zoom into active site regions and identify essential amino acids
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AMINO ACIDS
..
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What are their common features?
1. Carboxyl group 2. Amino group 3. Side chain (R group) 4. H atom
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What distinguishes them? How many different ones are there?
The structures of the R group -20 different R groups found in proteins
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What is the smallest and biggest amino acid?
Smallest = glyceine, biggest = tryptophan
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What 4 amino acids have aliphatic side chains?
Alanine (A) Valine (V) Leucine (L) Isoleucine (I)
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Aromatic?
Phenylamine (F) Tyrosine (Y) Trptophan (W)
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Hydrophobic (i.e. non-polar)
A,V,L,I, but also F, P, W, C, Y, T, G and K, R.
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Polar
N,Q,S,T,C but also Y,W
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Charged- what does this depend on?
D,E,H,K,R, but also Y,C - depends on pH
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Give 4 key properties of amino acids.
Acidic, aromatic side-chains absorb UV light, chiral (except glyceine) and possess atleast two ionising groups (amino and carboxyl as well as some side chains)
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What are the three main naming conventions?
1) By optical activity: operational classification 2+3)By configuration: D- and L- (FISCHER CONVENTION) and R- and S- (CAHN-INGOLD-PRELOG SYSTEM)
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Describe operational classification
Observe which direction the molecule rotated plane-polarised light… then classify by either (+ or d) if clockwise and (- or l) if anticlockwise
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What do d and l stand for?
Dextrarotatory and laevarotatory
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What is wrong with this system?
This provides no indication of absolute configuration of chemical groups around the chiral centre + If a molecule has 1< asymmetric centre, could have an optical rotation not obviously linked to rotatory power of individual centres
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What does configuration refer to?
the arrangement of substituents at a chiral centre
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Describe the Fischer convention
2. This involves chiral centres being assigned to D- or L- configuration by comparison with glyceraldehyde
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Why is glyceraldehyde used?
because it is one of the smallest commonly used chiral molecules, and certain chemical manipulations can be performed without affecting its configuration
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Why is the difference between D and L forms important?
compounds are different shapes and enzymes recognise substrates partly by their shape. →Wrong shape means no reaction!
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What is the general rule of thumb for determining the D/L isomeric form of an amino acid
"CORN" rule. The groups: COOH, R, NH2 and H (where R is the side-chain) are arranged around the chiral center carbon atom- with the hydrogen atom away from the viewer. - if CO-->R-->N = clockwise, its D form, if anticlockwise, its L form
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Describe the CAHN-INGOLD-PRELOG SYSTEM
Substituent atoms are assigned priority by atomic no. of atom directly attached to the chiral carbon (High → Low).. View chiral centre towards lowest priority substituent (H) ...clockwise = R (rectus) anticlockwise = S (sinister)
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What is beneficial about this system?
labels each chiral center in a molecule (and also has an extension to chiral molecules not involving chiral centers). Thus, it has greater generality than the d/l system, and can label, for example, an ( R, R) isomer versus an ( R, S) — diastereomers
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What is the relationship between R and S and + and -?
no fixed relation to the (+)/(−) system. An R isomer can be either dextrorotatory or levorotatory, depending on its exact substituents.
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Why does the D/L system remain in common use in some areas of biochemistry?
it is convenient to have the same chiral label for all of the commonly occurring structures of a given type of structure in higher organisms.
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What form are naturally occurring amino acids compared with that of carbohydrates? (in D/L system)
naturally occurring amino acids are nearly all l (note some d-amino acids exist in short peptides in bacterial cell walls), while naturally occurring carbohydrates are nearly all d.
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What are naturally occurring amino acids in the S system? what is the exception?
mostly S, but there are some common exceptions e.g. cysteine which is R because of its C-S side chain, which unlike all other side chains, has a higher priorty than the CO2H group
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What is the significance of (+/-) nomenclature?
It is a measured optical rotation depending on experimental conditions and cannot be predicted from structure- therefore rarely used.
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PROTEIN STRUCTURE...
...
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How do peptide bonds form?
between a carboxyl and an amino group in a condensation reaction. (water produced)
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What does the dipeptide produced contain?
• Amino terminus (free amino group) • Carboxy terminus (free carboxyl group) • Two side chains
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All polypeptides have C and N terminus's. By convention, which is numbered as the first residue? Hence how can polypeptides be read?
the amino-terminal (N terminal) is numbered as residue 1. The sequence is therefore written from amino-terminus (left) to carboxy-terminus (right)- order matters.
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How many peptide bonds does a polypetide with n residues have?
n-1
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The peptide bond is planar in which plane?
Amide plane
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Why do peptide planes tend to be rotated away from eachother?
so that side-chains don’t clash
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What does a ramachandram plot show?
That protein structures aren't random
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When amino acids appear outside the areas of common in the plot, they tend to be...
...glyceine, because there is no sidechain attached to the carbonyl
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In secodary structures...
each Cα is in a regular arrangement with respect to its neighbours. Secondary structures stabilised by hydrogen bonds,
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The polypeptide chain adopts a.. (what two options?)
• α-helix: polypeptide chain adopts a helical (spiral) structure in 3-D • ß-sheet: polypeptide chain adopts a pleated structure (strand) and interacts with adjacent strands
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Who was the alpha helix discovered by? How many residues does it have per turn? How far does it rise per turn?
Linus Pauling. 3.6 residues per turn. 5.4A per turn (1.5 per residue)
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Why is there a preference for amino acids in alpha helix?
Side-chains are nicely spread out and do not clash.
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What is the direction of the amide plane? What does this create?
N-terminus (positively charged) at bottom, C-terminus (negatively charged) at top. If go from N to C side chains hang downwards and carbonyl groups point upwards. dipole moment created
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Why is there no dipole moment in a beta sheet?
charges alternate directions, and hence cancel out
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Which is more common, left or right handed alpha helix?
Right handed alpha helix
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Which is rarer, an alhpa pleated sheet or a beta helix? Where can beta helices be found?
Beta helix, found in antifreeze protein in beetles...
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How is the beta helix beneficial to the beetle?
mimicry of ice structure by surface hydroxyls and water in a beta helix antifreeze protein (TmAFP). It can line up H2O molecules as if they’re in a an ice crystal- hence water structured without actually forming ice, so the beetle doesnt freeze!
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What does beta sheet format entail?
2 polypeptide chains interacting with eachother via alternating hydrogen bonds.
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Describe the two types of beta sheet...
a)antiparallel= polypeptide strands going in opp directions= v. stable b)parallel= polypeptide strands in same direction= less stable (due to longer H bonding distance- To compensate, they become wonky in shape. )
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Describe tertiary structure
• Segments of secondary structure (here α-helix) • Linked by less regular segments - turns (loops) • Mainly held together by hydrophobic residues in the centre • Results in a compact, globular structure
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Summarise all 4 stages of protein structure
• Primary- amino acid sequence • Secondary- Regular, local arrangement of the polypeptide chain • Tertiary- Complete folding of a single chain • Quaternary- Association of multiple chains
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CONFORMATION....
...
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Define conformation
the complete 3-D structure of a protein molecule
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Conformation is stablised by?
Non covalent interactions including: • hydrogen bonds • ionic interactions (salt bridge)- seen in alpha helices • hydrophobic interactions • van der Waals interactions
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Disulphide bonds may also stabilize the conformation of a protein. They exist as covalent bonds between cysteine side chains ( R = -CH2SH . However:
-not all proteins have cysteine residues -not all cysteine residues are involved in disulphides (some are free cysteines)
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What can be used to reduce disulphide bonds and what does it produce?
2-mercaptoethanol (or anything with an SH) is a reagent used in excess to reduce disulphide bonds Produces free –SH groups (cysteine side chains)
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What happens when a protein becomes unfolded?
All interactions remain intact except for H bonds, causing protein to lose its 3D shape and hence its function
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Decribe the post-translational modification of insulin in 4 steps. (1st and 2nd step on this slide)
1. Synthesised as inactive precursor preproinsulin -pre-sequence is required for membrane transport 2. Pre- sequence removed by proteolysis
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(3rd and 4th step)
3. Proinsulin folds and disulphides form -forms inactive precursor -pro-sequence required for correct folding 4. Activation involves removal of pro- sequence
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What is this an example of?
Proteolytic cleavage
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Give 7 other examples of post-translational modifications (first 5 on this slide)
glycation, phosphorylation (addition of phosphates to proteins- involved in cell signalling), glycosylation (addition of complex sugars to the surface of a protein), acetylation, ubiquitination (important for destruction of proteins)
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(6 and 7th on this slide)
sumoylation (similar to ubiquitination) , methylation (activation/inacivation of genes)
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What are the three steps for experimentally studying proteins?
1) purify a sample of protein 2) determine amino acid sequence 3)determine conformation
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How can we determine conformation?
• X-ray crystallography or NMR • Homology model if there is a closely related protein of known structure
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For soluble, globular proteins
Stage 1 – extract proteins from tissues/cells into solution Stage 2 onwards –remove unwanted contaminants (proteins + biomolecues)
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What are the essential requirements for these processes? (2)
• An activity assay for the target protein • An assay for total protein concentration
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What is the basis for calculation of specific activity?
units of activity / amount of protein (units/mg protein)
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What can be used for stage one of protein study (purification of protein sample)
Column chromatography =Separations of soluble proteins based on differences in molecular characteristics
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What 3 different types of filtration can be carried out using column chromatography?
Size (gel) filtration, ion exchanve, and affinity chromatography
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Describe size (gel) filtration
Solution pushed through column of gel beads. large molecules do not get trapped- come out first. small molecules enter pores in the gel and hence come out later.
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Describe ion exchange
Proteins move through the column at rates determined by their net charge at the pH being used. With cation exchangers, proteins with a more negative net charge move faster and elute earlier.
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Separation influenced by... (3 factors)
1. Choice of beads (+ve or –ve charge) 2. pH of protein-containing solution 3. Ionic strength of solution
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Describe Affinity chromatography
separation depends on specific binding interactions. It involves antibodies which recognise some proteins and not others
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