Steele - Lecture 2

Question 0

Where are disulfide bonds largely found?

Answer 0

• In active site of enzymes
• In extracellular environment, which is more oxidative(harsher) than the intracellular environment (reductive environment).
• -> Helps stabilize proteins in these environments

Question 1

What are 2 protein motifs found in Src tyrosine-kinase?

Answer 1

1) glycine-rich loop: Binds phosphate of ATP
2) DFG: coordinates Mg binding to ATP
* motifs are functional/structural subunits of specific domains

Question 2

What are 2 physiological examples of disulfide bonds?

Answer 2

1) insulin; one within alpha chain, 2 occurring between alpha and beta chains
2) antibodies: disulfide bonds link heavy to light chains and heavy to heavy chains

Question 3

How are 3D structures identified?

Answer 3

Through X-ray diffraction pattern resulting from Fourier transformations of PROTEIN CRYSTALS

Question 4

What is a method of determining protein structure for smaller proteins (<100K MW)?

Answer 4

NMR- apply magnetic field to proteins in solution, aligning spin states of nuclei. Energy derived from them returning to original state provides structural information.

Advantage- don’t need to crystallize.
Disadvantage- Size limit(<100K MW or 900 AA- can be used for structure of some whole viruses though)

Question 5

How is quaternary structure stabilized?

Answer 5

multiprotein assemblies, stabilized by weak interactions like 2’ and 3’ structures

Question 6

What interactions qualify as 4’ Structure?

Answer 6

All protein interactions COULD be considered 4’ structure, but it depends on the stability of the interaction

Can be homo or heteromeric

Question 7

What is the generic formula for MW of proteins?

Answer 7

AA x 100 = #Da

Question 8

What are two general categories of protein shape?

Answer 8

1) globular (dominant)

2) Structural proteins - elongated (fibrous)

Question 9

What are the sources of charge for proteins?

Answer 9

1) amino (+) and carboxy (-) terminus)
2) side chains of basic and acidic amino acids
3) covalent modifications (eg phosphorylation) - transient
4) metal ions (eg cofactors)

Question 10

How can you determine if an R group or terminus will be protonated?

Answer 10

Look at pKa. If,

pHpKa, deprotonated form predominates (- charge)

Question 11

What is pKa?

Answer 11

pH at which 1/2 of ionizable group is protonated, 1/2 deprotonated

Question 12

Are proteins good buffers? Why or why not?

Answer 12

No. A buffer is a molecule that resists pH change in solution within 1 pH unit above and below the pKa. Requires much more acid/base to change pH within buffer zone.

Proteins only have His residues and amino-terminus with pKa values within the right range (~6.5-7.5), but they are too sparse to have a significant impact.

Question 13

Glycosylation and disulfide bonds are most commonly found on which side for transmembrane domains?

Answer 13

Extracellular domain

Question 14

What modifications are found on the intracellular domain?

Answer 14

1) reduced sulfhydryl groups
2) phosphorylation
3) no complex carbs

Question 15

What secondary structure is found in transmembrane proteins?

Answer 15

Strongly hydrophobic alpha helix (single-pass) or helices (multi-pass)

Question 16

What are the two types of glycosylation? What AA do they modify?

Answer 16

1) N-linked: side chain N on asparagine
2) O-linked: OH group on serine or threonine

(don’t need to memorize)

Question 17

What is Leri-Weill dyschondrosteosis caused by?

Answer 17

mutation in nuclear localization signal in SHOX transcription factor.

Mutations in localization signal can cause disease

Question 18

What are some protein destinations in the cell?

Answer 18

1) ER
2) nucleus (reversible)
3) mitochondrion
4) cytoplasm (default mode)
5) peroxisome

Question 19

What is a common characteristic of nuclear localization signals?

Answer 19

Mostly basic amino acids (arg, lys)

Question 20

What is the difference between monopartite and bipartite localization signals?

Answer 20

Monopartite signals are continuous, bipartite signals have a sequence in the middle of the basic amino acids

(don’t need to memorize)

Question 21

How are proteins isolated?

Answer 21

By

1) charge: ion-exchange chromatography
2) size: gel filtration chromatography
3) hydrophobicity: Absorption chromatography
4) interaction with other molecules: affinity chromatography w/ ligands, substrates, products, binding proteins, antibodies

Question 22

What factors tell you if two different proteins evolved together?

Answer 22

Primary structure, tertiary structure (may be better index than 1’ structure), function

ex: hemoglobin, myoglobin

Question 23

Define ortholog and paralog

Answer 23

ortholog- proteins with similar function and structure in different species

paralog- proteins with similar structure but different function in same species, often arise from gene duplication

Question 24

What is the structure of hemoglobin?

Answer 24

2 alpha, 2 beta chains

Question 25

What is the mutation related to sickle cell disease?

Answer 25

glutamate to valine at position 6 in beta chain. Causes Hb to aggregate due to hydrophobic interactions, result in HbS (sickled appearance)