Tutorial 05 Protein chemistry and structure determination Flashcards
State the levels of protein and mention the main structural features at each state
- Primary structure: Linear Amino acid sequence linked by peptide bond/ amide bonds
- Secondary structure: Alpha Helix, Beta Sheet, Turns and Loops.
- Tertiary structure: Hydrophobic interior and hydrophilic exterior that interact with the aqueous environment. Arrangement into compact soluble structure. Can be subunits in the quaternary structure.
- Quaternary structure: Cumulative structures of tertiary structure. Multiunit complex.
Proteins exhibit a wide range of functions depending on their properties. Discuss the properties
which make the proteins are having a wide range of functions
(large answer give more emphasis)
1. Proteins are linear polymers built of monomer units called amino acids which are linked end to end.
The sequence of amino acid is called primary structure. Proteins fold up into three-dimensional structures such as secondary or quaternary structure. Protein function depend on this structure.
- Proteins contain wide range of functional groups such as thiols, thioesters, carboxylic acids, carboxamides that are chemically active. When combined in various sequences it accounts for broad spectrum of protein function. Functions such as growth, act as messenger and provides structure.
- Proteins can interact with one another and with other biological macromolecules to form complex assemblies.
- Some proteins are quite rigid and others have flexibilty.
Those that are rigid function as structural elements in cytoskeleton or connective tissue. Flexible proteins can undergo conformational changes to make regulated assembly of large protein complexes and transmission of information between or within cell.
Discuss the functional groups of proteins which provides specific chemical nature to the proteins? Provide the examples for each type of functional groups?
Alcohols Thiols Thioesters Carboxylic acids Carboxamides
Example:
Differentiate the structural features of the secondary structure of protein
Major elements are alpha helix and beta strand.
Alpha Helix:
The polypeptide chain twists into a tightly packed rod like a spiral. Within the helix, the carboxyl group of each amino acid is hydrogen bonded to amine group of the amino acid four residues father along polypeptide chain.
Beta strand:
Two or more beta strands connected by NH to CO hydrogen bonds come together to form beta sheets in a extended structure not spiral. These sheets can be antiparallel, parallel or mixed
The Amino Acid Sequence of a Protein Determines Its Three-Dimensional Structure. Explain the
above statement
(WRITE IT DOWN PROPERLY)Primary structure with bonds. Mention the bonds.
Primary structure with bonding form secondary structure.
DNA encoding RNA
Amino acid peptide bonds
Folding for stability
Outer Layer hydrophilic, inside hydrophobic
Modifications and thermostability. stability.
What are the common techniques used to elucidate the structure of protein
- X-ray crystallography
- NMR spectroscopy
What are the reasons for crystalizing the protein prior to the structure identification.
The main requirement for protein structure determination by X-ray crystallography is the attainment of protein crystals diffracting at high resolution. During crystallization some solvents and impurities are being precipitated and removed. Hence, protein concentration is high. Another reason for crystallization is to remove intermolecular forces. These forces or interactions can change the electron density map affecting structure identification.
Briefly explain, how you would achieve the protein crystallization
The proteins should be dissolved in a suitable solvent from which it must be precipitated in crystalline form. Solvent used is water buffer solution
Super-saturation conditions can be obtained by addition of preceipitating agents such as salts or polyethylene glycol polymers or by the modification of pH, temp, protein concentration. The super-saturated state of solution evolves towards a thermodynamically stable state in which protein precipitate to large crystals.
What are the main steps used for the structure identification using protein x-ray crystallography? Briefly explain each step
- Determination of protein degree of purity.
If protein is not 90-95% pure, further purification should be carried out to achieve crystallization. - The proteins should be dissolved in a suitable solvent from which it must be precipitated in crystalline form. Solvent used is water buffer solution
- Solution is bought to super saturation.
Small aggregates are formed which are the nuclei for crystal growth. Once nuclei have been formed, actual crystal growth begins. - Crystals are exposed to X-ray beam which results in diffraction pattern (crystal size and pattern and intensities)
- Based on diffraction pattern, and form electron density map
- Using electron density map, protein structure is formed after refining electron density map.
Discuss the importance of NMR spectroscopy on structure determination?
Based on chemical shift, Used to derive a three-dimensional model of a protein in a solution. Detailed structural information based on chemical shift.
Identify isotopes and chemical groups
Use NMR while product is intact and crystallisation is not required.
Can maintain NMR libraries and refer back to library for reference.
Speedy measurement unlike X-ray crystallography.
There are many models to display the protein structures. Differentiate three models depending on its structural difference
- Space filling Model
Each atom is shown as a sphere with a size corresponding to the van der Waals radius of the atom. (Most realistic representation) - Ball and stick model
Show bonding arrangment, reveal complex structure than space filling model but not as relalistic as atoms occupy more space. - Ribbon Diagram
Highly schematic, used to visualize aspected protein structure such as helix, beta strand and loops to provide clean views of the folding pattern of proteins.
This cannot be done using the other two models, the space filling and ball and stick depict protein strucutres at atomic level, large number of atoms makes it difficult to to distinguish relevant structural features.
A new drug is developed which selectively cleaves covalent bonds between two sulfur atoms of non-adjacent amino acids in a polypeptide chain. Which level of protein structure in affected
molecules would be most directly affected by the drug?
Discuss the reason for selecting the answer
Tertiary structure because of the disulfide bond involved between the two sulfur atoms of non- adjacent structure.
A thiol is a functional group that is not commonly focused upon during this course but is present amongst some of the amino acids. What element is found in thiols to distinguish them from other functional groups? How many amino acids contain thiols in their side chains? Name each of them.
Thiols: sulfur takes the place of oxygen in the hydroxyl group of an alcohol
Amino acid Cysteine has thiol
Only one amino acid has cysteine.
