Chemistry Flashcards
What is the difference between peptides and Proteins
Peptieds are usually less than 50 amino amino acid residues linked by peptide bonds whereas proteins are more than 50 amino acid residues
What potential problems can come from dipeptide synthesis?
There are 3 main problems,
Firstly, Peptides are directional, so in for example the peptide we wanted to synthesise had alanine an the N-terminus and Phenylalanine at the C-terminus there is always the possibility of the peptides being swapped around so Alanine at the C-terminus as although this is not the peptide we were looking for there is nothing that stops it from ocurring
Secondly, In a reaction vessel, Peptides are able to self-condense. This simply means they are able to react with other peptides of themselves as so to form Ala-Ala or Phe-Phe instead of reacting with each other.
Finally, there is a possible problem in peptide synthesis which Depends on the side chains. Reactive side chains are able to participate in reactions. For example Lysine, containing a secondary amine group can participate in the reaction in the place of the other amine group.
How do we use protecting groups?
Protecting groups can be used to protect amino acid residues. They are able to be easily removed under mild conditions when required, though will still fufill the role of preventing something like unwanted side reactions that might occur. It makes it so that only the groups we want to react can occur.
After that condensation reaction between the 2 peptide we remove the protecting group potentially for a new cycle of reactions
With amnio acids that have reactive R-groups, we also need to protect these so hence we need more Protecting groups but otherwise the cycle is the same
Since peptide bonds do not just spontaneously form, how do we make it so that this bond formation does infact occur?
We need to chemically activate and then couple amino acid derivatives
We activate the carboxyl group by attatching a leaving group to the Acyl carbon of the carboxyl component to facilitate the attack of the amino acid component
This is necessay as otherwise the two amino acids at ambient room temperature will just form a salt
How do we activate the carboxyl Group?
We can convert it into an acyl halide which provides a good leaving group to ensure that this happens rapidly under mild condition
Describe how Resonance plays a role in the amide bond
The reso structure of the Amide bond has partial double bond character. This involves charge splitting with N being positive and O being negative
As a result the bond is rather chemically inert. The amide group is also planar
What is a dihedral angle
A dihedral angle involves for angles and are measured clockwise
Angles of 0 or 120 are called eclipsed and angles of 60 or 180 are called staggered
Essentially, we are looking through the B and C angle
What are the Dihedral angles in proteins
Phi, Psi and Omega
Phi is the angle between the NH and the R group The 4 atom which it involves are The C carbon on C=O, N, The C on the R group and The C on the Carbon on the C=O
Psi is from Angle between the C=O and R group. Its dihedral angle is One NH TO NH
Omega is the peptide bond
Describe the influence of steric facors on condormaiton
Steric factors influence the conformation, when the R groups are on 2 different sides, and is staggered(180), instead of being eclipsed(0) the peptide bond is preferred as a Cis conormation of the peptide bond can result in steric clashes which are unfavourable
We say that the Phi and Psi angles aer co-dependant. When one is 0 the other cannot be 0 and this is simlplt to avoid steric clashes
Describe the role in H bonding in protein structure
Back-bone hydrogen bonding is associated with Scondary structure, whereas side chain Hydrogen bonding is associated with tertiary structure
What is sequence conservation and why is it useful
In general, evolution conseves amino acids which are important to structure and function across protein species, we are able to determine this by the alignment of multiple homologue sequences of a particular protein in different species which will reveal the residues which are conserved and those which are not.
Conserved residues tell us the important funcional parts of the protein sequence. Typically only a limited amount of a protein’s sequence is absolutely conserbed, Howeber insulin is a proein which is highly conseved which makes insulin therapy with different species a possiblity
Describe protein structure stabilising forces?
These are essential to keep ptoreins stable.
They can be grouped into covalent interactions(peptide bonds and disulfide bridges)
Non-covalent bonds(Pi-Pi interactions, VDW forces, Hydrogen bonds, Electrostatic interactions, Hydrophobic interactions)
What are the surface characteristics on a protein?
Hydrophillic residues are predominantly on the surface with hydrophobic regions predominantly being in the core, an exception of this may be membrane proteins which are in a lipohillic environment so will tend to appear more on the surface.
If soluble proteins have hydrophobic surface region, they have a particular function, for example for ligand binding in the acive site of an enzyme.
Describe Folding and conformational changes in a protien
A chain of amino acids fold into their tertiary structure with its main driving force being that the non-polar groups want to clump into the middle to avoid contact with water. Drom this, it will maximise the other interactions.
Proteins fold to their native process through a directive pathway going through several intermediate states. A molten globule can for by hydrophobic collapse.
Chaperone proteins assist in the folding of proteins in the cell which are unable to do this by themselves
Descibe me 3 differernt ways we can determine protein structure
X-ray crysollogaphy, where the proteins are growin in crystals and by exposing it to an X-ray source, we can detect it with a detector to see its electron density and get a model of the Structure
NMR spectroscopy
Cryo-electron microscopy
Describe Covalent bonds in proteins
Peptide bonds are very stable and require harsh conditions with 6M concentration of acid or allkali to break them apart
Disulfide bridges can form between cysteine residues via thiol groups. They can be broken down into cysteines via B-mercaptoethanol to re-form thiols.
Describe Non-covalent bonds in proteins
Hydrogen bonds vary in strength but contribute most to protein stability in the core as they are the firsthest away from water which would disrupt it. They can be disrupted by heat
VDW forces are generally weak, short range interactions due to dipole dipole interactions between close atoms
Pi-Pi oceralp occurs between Pi elecron clouds delocalised over rings and bonds. These can also be disrupted by heat.
Electrostatic bonds are sronger interactins which are stronger when two charged groups are closer. They can be broken down high ionic strengths which may compete with the existing salt
Hydrophobic interactions are interactions between hydrophoic sife chains which are generally forced to gether to minimise the exposure to water
Describe The structure of insulin
Insulin is originally produced in the cells as preproinsulin. Once it enters the Endoplasmic reticulum it becomes Preonsulin as the signalling peptide is cleaved. It is then processed through the Golgi apparatus to become the mature form of insulin.
In its mature form,Insulin consists of 2 Chains. Its A chain is linked to the B chain with 2 disulfide bridges, with there being an additional bridge in the A chain on its own.
The A chain consists of 21 residies, with he B chain containing 30.
Insulin is a Globular hormone which normally exists as a Monomer. When it however is in higher concentrations insulin dimerizes and in the prescence of zinc further associates into hexamers. However only the monomer is the biologically active species
Under conditions where the native state is desyabilised, insulin aggregates to form amyloid fibres which can cause biomedical complications as it can interfere with the requirements of functional insulin in each dose. This mainly occurs at hugh insulin concentration
Explain protein Engineering in Insulin
There are a number of insulin analogues which have been liscenced for treatment including
Rapid acting insulin analogues. After SC injection the proportion that is bound in the for of Hexamers and dimers is lower which means that the monomeric form of the molecule can be absorbed at the point of injection faster- insulin lispro, insulin aspart and insulin glulisine are examples
In the Insulin Aspart the proline is substituted with an aspartic acid residue which as increased charge repulsion and prevents the formation of hexamers
Describe the ionisation states in the Histidine side-chain
Histidine contains an imidazole ring which hsa a pKa value of approximately 6. This means that in Physiological conditions, relatively small shifts in pH values below the pH of 6 will cause the imidazole ring to become protonated and carry charge equally between the two structures, This can be represented with two equally important resonance structures
Describe how Post-translational modifications to proteins can alter them
Post-translational modification is the process of modifying a protein after translation
An example of modification is the Phosphorylation of Hydroxyl groups of Ser, Thr or Tyr with the result being an added charge. This is believed to be key to some signalling pathways in the cell
Glycosylation is additional Sugar moieties to Ser, Thr or Asn resideus which can alter the solubility of the protein
Hydroxylation is the addition of OH groups to Pro it Lys residues and can alter Hydrogen bonding
Disulfide bridge formation. The joining of two cysteine residues to produce a more stable protein with additional covalent linkage
Methylation, is the addition of a methyl group to an Oxygen or Nitrogen. This is the addition of a hydrophobic group
What is a possible consequences of too many groups
A protein with too many basic groups, will have a net positive charge in physiological conditions as the groups will all be protonated
A protein with too many acidic chains will have a net negative charge.
What is the Isoelectric point
The isoelectric point is where the overall protein carries no net charge.
The individual amino acids may be charged but the Overall protein is not.
It depends on the amino acids present(How many basic or acidic groups the protein has) and the pH of the solution environment
Describe the role of the isoelectric point in Long-acting insulin
Insulin glargine is a long-acting analogue which contains 2 extra arginine residues at the end of the B chain to alter the Isoelectric point. This increases the IEP of insulin and alters the solubility. By increasing the IEP it is more soluble in lower acidic pH conditions which are used in formulations but less soluble upon injections
This works as the inulin is soluble in solution but when injection ist becomes less soluble and begins to form dimers and hexamers which form by precipitation. This then means when they enter the body they will take a longer time to monomeric forms of insulin which are active thus producing a longer acting effect.
Describe Gel electrophoresis
This is a method of separation and analysis of macromolucules like proteins
The principle is that molecules can be separated according to their size and charge.
Proteins are electrophoreised within a matrix or gel and then in an electric field, charged molecule migrate either towards an anode or athode dependent on their charge
The force of attraction to the electrode depends on two different factors. The size of the charge on the molecule, and the size of the electic field
We also need to take into account forces which prevent movement of molecules such as Friction and/or repulsion in medium
So overall Ion velocity =electrometric mobility x applied electric field
What is absorbtion spectrophometry
In absortion spectrophotometry, a dissolved substance will absorb light of a specific wavelength characterises by that substance.
We can via the extinction coefficient determine the Concentration
What is the use of UV absorbtion in measuring protein concentration
UV absorbtion is a good way in detecting protein concentrations as no additional reagents or incubates are required. Furthermore, No protein standard is needed to be prepared. The assay also does not consume protein. The relationship between the absorbance to protein Conc is linear.
However, any non-protein component in the solution that absorbs UV light will interfere with the assay
What protein detection methods can we use post Gel-electrophoresis
- Typical detection methods include
- Coomassie Brilliant Blue dye staining
- Western blot(Protein immunoblot)
- Transfer of gel contents onto a membrane and detection via labelled antibodies
Describe SDS-PAGE
The native structure of proteins is maintained during electrophoresis. Separation is done according to size and charge. The Acrylamide gels serve as a size-selective sieve during separation. As proteins move through a gel in response to an electric field, the smaller molecules travel more rapidly in comparison to the larger proteins. In addition to this, Highly negative molecules will migrate faster than less negatively charged molecules towards the anode. These 2 effects in combination mean that a highly negatively charged larger molecule will migrate faster than a less negatively charged smaller molecule. We might do this to assess the quaternary structure of the protein to see if it is a hexamer, a dimer, or a monomer.
