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Protein S&F
Protein Structure and function revision
| Question | Answer |
|---|---|
| What is meant by the term 'proteomics' | The study of proteins, their structures and functions |
| List five different functions of protein in a eukaryotic organism | Transport, Storage, structure, signalling/receptor, enzymes |
| Isoelectric focusing separates proteins based on what characteristic? | Charge |
| Deduce the pI of a protein which moves in an isoelectric gel to pH 3 | pI would be 3, stops moving at pH that equals pI as the protein has become neutral. |
| In SDS-PAGE proteins lose their 3d shape, what level of protein structure is disrupted here? What type of chemical interaction was the main stabilising force at this level? | Tertiary/Hydrophobic |
| Explain the relationship between time and mas when interpreting Time of Flight Mass Spectrometry Data | TOF MS measures how long it takes ionised peptide fragments to travel from one end of a tube to a detector at the other end. Lighter fragments move faster, so fragments separate out on the basis of mass and are detected at different times (time of flight |
| Why is trypsin the usual protease used in peptide digestion? | It cleaves after arginine and lysine, which are common amino acids. |
| Explain how N-terminal amino acid can be liberated in Edman degradation without destroying all other amide links in a peptide chain? | Reaction with PITC at N-terminal makes the first bond unstable. Adding a strong acid hydrolyses this bond but not the others. |
| Proteins 3D' structure is determined by which property? | Initial amino acid sequence (Primary Structure) |
| Describe the effect changing pH can have on protein structure. | Acid and basic side chains are affected, causing their charge to change. Changes interractions within the protein, can lead to changed conformation of active site. Protein may unfold and denature. |
| Explain the role of beta-mercaptoethanol in maintaining activity of functional sites. | Acts to protect thiol group, keeps them reduced. If thiol groups become oxidized they can form disulphide bonds and no longer available for interaction with substrate. |
| Explain the difference between a holoprotein and an apoprotein | Holoprotein is with cofactor, apoprotein is without. |
| Describe three ways of limiting damage due to proteolysis? | Work quickly, work at low temperatures <4oC, use a cocktail of proteases. |
| Explain why glycerol is commonly added when storing protein samples? | Acts as a cryoprotectant, forms H-bonds with water molecues, preventing formation of ice crystals which might damage the protein. |
| Explain why binding to a protein's functional site is specific? | Lock and key: Molecule binding site must be complementary in nature, the right shape, charge and amino acid structure for the binding molecule. |
| Name an enzyme that uses induced fit | Hexokinase - Protein changes shape after substrate binding, only then coming to the correct conformation for the substrate |
| Explain why elastase and chymotrypsin have different substrate specificites even though they are both serine proteases? | The active site of elastase is larger, which means it can accomodate proteins with larger side chains |
| Discuss the difference between divergent and convergent evolution in terms of serine proteases. | Divergent proteins share a common ancestor and catalytic mechanism, but different specificity due to gradual changes; i.e. chymotrypsin and trypsin. Convergent proteins have no common ancestor but shared mechanism; i.e. chymotrypsin and subtilisin |
| If a protein was found to be optimally active at pH 7.4 what would this tell you about glutamate residues in the active site and what type of non-covalent bonds are occuring? | Glutamate side chains would be fully ionised. Electrostatic/ionic bonds would occur. |
| In terms of protein-ligand interactions, explain why water is usually excluded from binding sites? | Substrate would form hydrogen bonds with water rather than to protein, i.e. it would weaken non-covalent interactions holding ligand to protein. |
| Explain why molecules such as TPCK are particularly useful when trying to chemically modify residues within a binding site. | Looks like natural ligand so is directed to binding site. This gives specific modification within binding site rather than modification in other areas of protein. TPCK mimics substrate and modifies residues in active site, allowing identification. |
| Briefly describe the technique eof X-ray crystallography. | Need crystals of protein Bombard with X-ray beam Atoms in crystal scatter X-rays Diffraction pattern captured Pattern analysed to deduce 3D structure of protein (need prior knowledge of primary sequence) |
| Explain what is meant by a 'structural analogue'. | Similar structure to natural ligand but not identical. Usually means it cannot be hydrolysed or modified by protein in same way. |
| Describe the use of a named structural analogue in identifying important interactions within binding sites, | GDPS used to determine how GTP binds to nucleotide binding site of G-proteins |
| Justify the use of non-covalent bonding, rather than stronger covalent bonding, in binding ligands to proteins. | Need interactions between proteins and ligands to be reversible. Non-covalent bonds are strong enough to allow temporary interaction but also to allow dissociation too. |
| Describe what is meant by 'allosteric' modification of protein activity. | Allosteric modulators are regulatory compounds that bind to sites other than the functional site, but which lead to a change in conformation and activity of the functional site. |
| Explain, in terms of protein conformation, why binding of oxygen to haemoglobin molecules becomes progressively easier with each molecule bound? | Binding triggers conformational changes which disrupt haemoglobin electrostatic interactions. Protein changes from 'tense' to relaxed form, less energy required to interact with haem, co-operative binding. |
| Describe the role of consensus sequences in regulation of activity by phosphorylation | Structural motifs recognised by specific protein kinases to highlight particular serine or threonine residue to phosphorylate. Kinases transfers phosphate to OH group of this amino acid side chain. Phosphate addition influences H-bonding and ionic bonds |
| Explain what is meant by the term 'Zymogen'. | An inactive precursor format of an enzyme. Requires proteolytic cleavage to become active. |
| Describe the role of calmodulin in regulation of protein activity. | Calcium sensitive eukaryotic dimer. Binding of Ca2+ is required at two sites. Calmodulin then binds to numerous other proteins in the cell to modulate activity. - Link between protein activity and calcium levels in a cell. |
Created by:
Cmccubbin1
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