lecture 27 - structure and function of globular proteins

Question 1

how do we study structure and funciton of globular proteins? (3)

Answer 1

(1) sequences:
- look at homologues
- look for conserved / non conserved regions (strict conservation implies importance for function either directly or indirectly (structure))

(2) structural studies:
- determination of 3D structure
- get atomic-level detail of 3D structures using x-ray crystallography, NMR or cyro-EM

(3) functional studies:
- all biological function involves interactions between molecules
- e.g. ligand binding (hormones/receptors, antibodies/antigens)
- e.g. enzyme catalysis

Question 2

what do conservative substitutes like: \_\_\_ suggest?
leu - ile
ser - thr
asp - glu
arg - lys

Answer 2

important for function

Question 3

a lack of conservation at certain areas often implies what?

Answer 3

loops connecting secondary structural elements

Question 4

what can we use sequence conservations for?

Answer 4

to guide experiments such as site-mutagenesis (changing a spec aa for another)

Question 5

explain (generally) ligand binding

what does it occur between?

Answer 5

• the reversible binding of one molecule to another (non-covalent interactions)
• can be small molecule binding to protein, 2 proteins or protein and nucleic acid

Question 6

what is the result of ligand binding?

Answer 6

• results in a change in function
• often involves a conformational change
• may affect the protein interaction with other molecules or its catalytic function
• can result in “signalling”

Question 7

explain (generally) the characteristics of ligand binding interactions

Answer 7

• tend to be specific
• range of affinities
• depend on shape and physiochemical properties (polarity, charge, etc.)

Question 8

how do you describe affinity?

Answer 8

• protien (P) + ligand (L) <=> PL
• Ka = [PL] / [P][L] (association constant)
• Kd = [P][L] / [PL] (dissociation constant)
• experiments measure [PL] and [P] vs [L]
• θ = fraction bound
• θ = [PL] / [PL]+[P]
• θ = [L] / [L] + Kd
• graphically, if you were to plot θ vs [L], Kd is where θ=0.5 intercepts with the trendline
• if you were to compare the Kds of two ligands, a larger Kd value corresponds to a greater affinity

Question 9

range of Kd values observed:

Answer 9

10^-3 M = weak binding —> 10^-12 M (1 pM) = extremely tight binding