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MCDB 126B/226B -- Winter 2015Homework 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. Thisarticle 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 nAChreceptor.

We plan to organize this week’s section meeting in a discussion format, so please be preparedto discuss the article. Reading journal articles takes practice, and one of the goals of this classis to develop those skills. You aren’t expected to understand all of the details of the article, butdo your best. (For additional information on methods and background see the Dougherty labwebsite: 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 arebeing studied?

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

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

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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 incorporatingunnatural amino acids into a protein?

6. In carrying out this study, the authors needed to overcome several technical hurdles inorder to express the nAChR proteins in Xenopus (frog) oocytes. For one of thesehurdles, they used the property of “inward rectification”.

As background information, “rectification” refers to asymmetry in the ability of an ionchannel to carry ions across the membrane, and “inward rectification” means that thechannel can carry ions into the cell better than out of the cell. Whether a channelrectifies or not can be determined by measuring the ratio of the current through thechannel at positive voltage (outward current) compared to current at negative voltages(inward current), and a low ratio (

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MCDB 126B/226B -- Winter 2015Homework 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 practicesetting 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 ligandconcentrations, the equilibrium concentrations of free ligand and receptor-ligand complex weredetermined as shown below.

a) Plot [LR] vs. [L].b) What would you estimate for the K d for the binding of this ligand to the receptor?c) What do you estimate for the total concentration of receptors, [R t]?

[L] [LR]10 M 50 nM20 M 81 nM30 M 99 nM40 M 115 nM50 M 125 nM

where [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 againestimate Kd and [R t].

3) (2 pts) Now plot the same data with a double reciprocal plot, and again determine K d and[Rt].

4) (2 pts) Plot again using a Scatchard plot, and determine K d and the total concentration ofreceptors, [R t].

5) (2 pts) Compare the estimates of R t and K d obtained for problems 1-4. Are they the same?Which method(s) gives the better estimate for this data set?