Protein structure & function

Question 1

List possible functions of proteins:

Answer 1

• signaling
• transport
• catalysis
• movement
• structure
• regulation

Question 2

The basic structure of an amino acid

Answer 2

Question 3

The bond between amino acids:

Answer 3

the peptide bond

Question 4

List types of amino acids:

Answer 4

Hydrophilic:

• Basic amino acids (Lysine, Arginine, Histidine)
• Acidic amino acids (Aspartic acid, Glutamic acid)
• Polar amino acids with uncarged groups (Serine, Threonine, Asparagine, Glutamine)

Hydrophobic:

• Alanine, Valine, Isoleucine, Methionine, Phenylalanine, Tyrosine, Tryptophan

“Special”:

• Cystine, Glycine, Proline

Question 5

Levels of protein structure:

Answer 5

• Primary (sequence)
• Secondary (local folding)
• Tertiary (long-range folding)
• Quaternary (multimeric organization)
• Supramolecular (large-scale assemblies)

Question 6

What is the secondary level of protein structure?

Answer 6

• the alpha-helix or the beta-sheet - the localized organization of parts of a polypeptide chain

Question 7

What is a motif?

Answer 7

• a regular combination of secondary structures

Question 8

List examples of motifs:

Answer 8

• a coiled coil motif is formed by two or more helices wound around each other
• a Helix-loop-helix motif
• a Zinc-finger motif

Question 9

All molecules of any protein species adopt a single conformation -

Answer 9

• the native state, which is the most stably folded form of the molecule

Question 10

Proteins bind a variety of molecules (ligants). The binding is characterized by two properties:

Answer 10

• affinity
• specificity

Question 11

Mechanisms for the regulation of protein function:

Answer 11

• allosteric transitions
• phosphorylation/dephosphorylation
• proteolytic activation
• compartmentalization

Question 12

Allosteric transitions

Answer 12

Allosteric regulation is the regulation of an enzyme or other protein by binding an effector molecule at the protein’s allosteric site (that is, a site other than the protein’s active site). Effectors that enhance the protein’s activity are referred to as allosteric activators, whereas those that decrease the protein’s activity are called allosteric inhibitors. The term allostery comes from the Greek allos (ἄλλος), “other”, and stereos (στερεὀς), “solid (object)”, in reference to the fact that the regulatory site of an allosteric protein is physically distinct from its active site. Allosteric regulations are a natural example of control loops, such as feedback from downstream products or feedforward from upstream substrates. Long-range allostery is especially important in cell signaling.

Question 13

Phosphorylation

Answer 13

Phosphorylation is the addition of a phosphate (PO43-) group to a protein or other organic molecule. Phosphorylation turns many protein enzymes on and off, thereby altering their function and activity. Protein phosphorylation is one type of post-translational modification.

Question 14

What is proteolytic activation?

Answer 14

Many enzymes, signal proteins, or other protein forms are synthesized as inactive precursors, called zymogens, to be subsequently activated by cleavage or one or more peptide bonds. Examples include proteolytic digestive enzymes, the cascade of proteolytic activation in blood clotting, and the activation of many protein hormones.

Question 15

What is protein compartmentalization?

Answer 15

A regulatory mechanism which basically involves having different proteins in different locales of the cell.

Question 16

How can membrane proteins be isolated from the lipid membrane?

Answer 16

By using non-ionic detergents, which break the membrane apart.

Question 17

A technique for separating molecules based on density and/or mass:

Answer 17

• centrifugation

More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture migrate towards the axis. Chemists and biologists may increase the effective gravitational force on a test tube so as to more rapidly and completely cause the precipitate (“pellet”) to gather on the bottom of the tube. The remaining solution is properly called the “supernate” or “supernatant liquid”.

Question 18

What is Electrophoresis and how does it work?

Answer 18

Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge.

Gel electrophoresis uses a gel as an anticonvective medium and/or sieving medium during electrophoresis, the movement of a charged particle in an electrical field. Gels suppress the thermal convection caused by application of the electric field, and can also act as a sieving medium, retarding the passage of molecules; gels can also simply serve to maintain the finished separation, so that a post electrophoresis stain can be applied.[3] DNA Gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via PCR, but may be used as a preparative technique prior to use of other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting for further characterization.

Question 19

Relationship between pH and electronegativity

Answer 19

• The more electronegative the atom attached to the acidic hydrogen is, the more easily that hydrogen will be lost which of course means higher acidity ie higher pH.

Question 20

What is gel filtration chromatography?

Answer 20

Gel filtration (GF) chromatography separates proteins solely on the basis of molecular size. Separation is achieved using a porous matrix to which the molecules, for steric reasons, have different degrees of access–i.e., smaller molecules have greater access and larger molecules are excluded from the matrix. Hence, proteins are eluted from the GF column in decreasing order of size.

Question 21

What is ion exchange chromatography?

Answer 21

Question 22

What is affinity chromatography?

Answer 22

The separation of proteins by specific binding to another molecule.

Question 23

A technique for determining the amino acid sequence of a protein:

Answer 23

• Edman degradation. In this method, the amino-terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid residues.

Question 24

What is time-of-flight mass spectrometry?

Answer 24

(can be used to measure the mass of proteins and peptides) Time-of-flight mass spectrometry (TOFMS) is a method of mass spectrometry in which an ion’s mass-to-charge ratio is determined via a time measurement. Ions are accelerated by an electric field of known strength.[1] This acceleration results in an ion having the same kinetic energy as any other ion that has the same charge. The velocity of the ion depends on the mass-to-charge ratio. The time that it subsequently takes for the particle to reach a detector at a known distance is measured. This time will depend on the mass-to-charge ratio of the particle (heavier particles reach lower speeds). From this time and the known experimental parameters one can find the mass-to-charge ratio of the ion.

Question 25

A technique used to determine protein structure:

Answer 25

• X-ray christallography

X-ray crystallography is a method used for determining the atomic and molecular structure of a crystal, in which the crystalline atoms cause a beam of X-rays to diffract into many specific directions. By measuring the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional picture of the density of electrons within the crystal. From this electron density, the mean positions of the atoms in the crystal can be determined, as well as their chemical bonds, their disorder and various other information.

Question 26

List common techniques for purifying, detecting, and characterizing proteins:

Answer 26

• membrane protein solubilization
• centrifugation
• electrophoresis
• gell filtration chromatography (by size)
• ion exhange chromatography (by charge)
• affinity chromatography (by specific binding to another molecule)
• highly specific enzymes and antibodies to detect individual proteins
• gene sequencing and chemical methods (e.g. Edman degradation) to determine protein primary structure
• time-of-flight mass spectrometry to determine the mass of proteins
• x-ray crystallography to determine protein structure