Problem Set 2

Advanced Chemical BiologyBPS 4129

April 9, 2010

Problem 1

A) State in your own words why the particular types of small molecule libraries were used given that the mutant enzyme involves the elimination of one of the amino acid side chains that binds zinc. (/5)

• Must rescue the altered enzyme by mimicking altered structural motifs

• Bulky hydrophobic group to replace Phe• Aromatic nitrogen containing compounds to replace His for

Zn(II) binding• Chose small molecules containing these two motifs• Qunazolines, isoquinolines, benzimidazole etc.

Problem 1 continued

B) Name an experiment that could be used to independently confirm if small molecule “hits” actually recover the zinc finger’s ability to bind zinc. (/3)

• Reporter assay (Gal, Luc, GFP)

C) In point form, outline how it was determined the transcription factor function is recovered in the presence of small molecule. (/2)

• Reporter assay (GFP)• GFP reporter downstream of Zn finger recognition sequence• Zn finger mutant genes also in vector• Compared fluorescence with and without small molecule

Problem 2

A) Describe the bump-and-hole approach for studying kinase function. (/5)

• Scheme• >500 kinases similar in structure/function • Mutate kinase of interest by introducing a “hole” • Mutated kinase must retain its function• Mutate gatekeeper residue to a smaller amino acid• Screen natural substrate analogs with “bumps” for match• Inhibitor should affect only the mutated kinase

Problem 2 continued

B) What metabolite binding site is modified? (/2)

ATP

C) What is the gatekeeper residue and why is it important? (/3)

• Ile 338• Regulates access of ATP to kinase• Can be modified without disrupting affinity for ATP• Usually mutated to Gly or Ala• Can be targeted in a kinase of interest through bump-&-hole

Problem 3

A) Propose a chemical mechanism for the following transformation: (/3)

• Similar to derivatization of carboxylic acids with diazomethane

Methods Enzymol. 1988, 172, 288-301Angew. Chem. Int. Ed. 2005, 44, 1328-1332Nature Genet. 2001, 28, 317-325

Problem 3 continued

B) Describe how the modified D/RNA in “A” serves as a photochemical switch (/2)

C) What is the chemical basis for photoswitching using azobenzene chromophores? (/2)

• Association with sugar phosphate backbone disrupts structure• Sterics• Perturbed electrostatic interactions

• Photolabile group (as seen before in class) can switch transcription/translation on/off

• UV light induces isomerization to cis isomer• Thermal relaxation to trans isomer

Problem 3 continued

D) An example where azobenzene is used as a chemical switch. (/3)

• Examples from class/literature

Problem 4

A) What does FRET stand for? (/2)

Fluorescence/Förster Resonance Energy Transfer

B) Describe in your own words how FRET works. (/4)

• Two chromophores in close proximity and properly oriented• Excitation of one by light of specific wavelength• Nonradiative transfer of energy from one to other followed by

emission of lower energy light• Emission spectrum of first must overlap with absorption

spectrum of second for FRET

Problem 4 continued

C) Give an example of the use of FRET in chemical biology. (/4)

• I.e. Calmodulin experiment from lecture

Problem 5

A) Give an example of the use of a molecular imaging technique in high throughput screening. (/5)

Describe a technique from class and how it’s been applied to high throughput screens i.e. AFM,

B) Describe the advantages of the method over others. (/5)

From notes

Problem 6

A) Describe chemical force microscopy and the chemistry used to functionalize the tip. (/5)

B) Discuss the use of AFM tips to perform high throughput screening. What are the drawback/limitations of this technique? (/5)

• Chemical interactions between surface and functionalized tip can be measured• Hydrogen bond strength, electrostatic interactions, pH• Force required on cantilever to disrupt interactions between surface and tip are

quantitated• Coat with reactive silanes• Coat with thin layer of gold, then organic thiols• Further functionalization with organic molecules

• Discuss DPN, DNA/DNA interactions, Protein/DNA interactions, ligand/receptor interactions

• Drawbacks: tip functionalization, resolution, surface imaging only, sample height, scanning area limitations, slow scan rate

Problem 7

A) Identify all the functional features of the MRI probe below. (/3)

• cNGR• Biotin• Diethylenetriaminepent

aacetic acid (DTPA)

Problem 7 continuedB) Describe the function of each element of the probe. (/3)

• cNGR - Asparagine, gylcine, arginine cyclic peptide specific for CD13 aminopeptidase, overexpressed in angiogenic endothelial cells. Site specificity

• Biotin – Affinity for avidin, tetramer provides contrast • Diethylenetriaminepentaacetic acid (DTPA) – Chelates

gadolinium which acts as a contrast agent for MRI

C) Describe an alternative approach to add multivalency to a molecular probe. (/4)

Superparamagnetic iron oxideNanotubes (gadolinium based)+ brief description of approach