MCDB 126B/226B -- Winter 2015

Homework 1A (10 pts)--Due in section (either Wednesday Jan 14 or Friday Jan 16)
Please read the article on the class website from the Dougherty group at Caltech:
Xiu et al. (2009) Nicotine binding to brain receptors requires a strong cation-
interaction. Nature 458, 534.
Two summary pages are also included with the article to help with your understanding. This
article describes exciting work that explores the remarkable specificty of nicotine for the brain,
and explains at the molecular level why nicotine binds to brain nACh, but not muscle nACh
receptor.
We plan to organize this weeks section meeting in a discussion format, so please be prepared
to discuss the article. Reading journal articles takes practice, and one of the goals of this class
is to develop those skills. You arent expected to understand all of the details of the article, but
do your best. (For additional information on methods and background see the Dougherty lab
website: http://www.its.caltech.edu/~dadgrp/research/ Unnatural Amino Acid Mutagenesis).
Also, bring any questions you have to discuss with members of your section and your TA.
Your written homework assignment (below) will be due at the beginning of discussion section:
1. What is a cation- interaction? In this paper, what are the cation and the that are
being studied?

2. What do the authors conclude is the reason that nicotine can bind to brain nACh
receptors but not to muscle nACh R?

3. What is the key experimental evidence that supports their conclusion?

4. What are unnatural amino acids, and why are they employed in the study?

5. What is nonsense suppression? How is nonsense suppression helpful for incorporating

unnatural amino acids into a protein?

6. In carrying out this study, the authors needed to overcome several technical hurdles in
order to express the nAChR proteins in Xenopus (frog) oocytes. For one of these
hurdles, they used the property of inward rectification.
As background information, rectification refers to asymmetry in the ability of an ion
channel to carry ions across the membrane, and inward rectification means that the
channel can carry ions into the cell better than out of the cell. Whether a channel
rectifies or not can be determined by measuring the ratio of the current through the
channel at positive voltage (outward current) compared to current at negative voltages
(inward current), and a low ratio (<0.1) was used as an indicator of inward rectification.
In this paper, why did the authors measure inward rectification and what did it tell them
about the ACh receptor that they expressed?

MCDB 126B/226B -- Winter 2015

Homework Problems 1B (10 pts) --Due in lecture Friday Jan 16
Please make the Double reciprocal plot (#3) by hand using graph paper in order to practice
setting up your axes; other questions can be done by computer if desired.
1) (2 pts) A binding experiment is made with an isolated receptor. Using a variety of ligand
concentrations, the equilibrium concentrations of free ligand and receptor-ligand complex were
determined as shown below.
a) Plot [LR] vs. [L].
b) What would you estimate for the Kd for the binding of this ligand to the receptor?
c) What do you estimate for the total concentration of receptors, [Rt]?
[L]
10 M
20 M
30 M
40 M
50 M
where

[LR]
50 nM
81 nM
99 nM
115 nM
125 nM
[L]= concentration of ligand
[LR]= concentration of receptor-ligand complex
[Rt]= [R] + [LR] = total concentration of receptor, including bound & free receptor
Note: M = 10-6 M; nM= 10-9 M. Your graphing will be most successful if you convert
[L] and [LR] to the same units before analysis and plotting.

2) (2 pts) Plot the data above using a semi-logarithmic plot of [LR] vs. log[L], and again
estimate Kd and [Rt].
3) (2 pts) Now plot the same data with a double reciprocal plot, and again determine Kd and
[Rt].
4) (2 pts) Plot again using a Scatchard plot, and determine Kd and the total concentration of
receptors, [Rt].
5) (2 pts) Compare the estimates of Rt and Kd obtained for problems 1-4. Are they the same?
Which method(s) gives the better estimate for this data set?