1) A single-?molecule microscopy experiment is performed on a slide containing
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Question
1) A single-?molecule microscopy experiment is performed on a slide containing 1000 molecules each of green, red, cyan, and yellow fluorescent proteins (GFP, RFP, CFP, and YFP, respectively). The experiment measures the number of antibodies bound to each type of protein, finding that 900 molecules of GFP, 10 of RFP, 1 of CFP, and 3 of YFP are bound at 1 nM concentration of antibody.
(a) What is the specificity of the antibody for GFP?
(b) What is the KD of the GFP–antibody interaction?
A tryptophan residue near the periphery of a protein-protein interface is mutated to alanine and changes the KD of binding from 1 nM to 40 ?M at 300K. How much binding energy was contributed by that residue?
A protein-protein interface has a 10 nM affinity at 300K. A series of mutants are made in which each residue at the interface is replaced by alanine. A lysine residue at the center of the interface is mutated, and found to contribute 4 kJ•mol–1 to the binding free energy. What is the new KD?
Explanation / Answer
To solve this kind of problems firstly the major question is that what actually specificity refers to and as per the question it's in case of four different types of florocent protiens in a slide containing 1000 molecules i.e GFP,RFP,YFP,CFP.. Now for the (a) part of questions let us know about specificity
Specificity Definition :--specificity "An antigen - antibody interaction... the specificity of the binding is due to specific chemical constitution of each antibody. In other words we can say it's the property of antibodies which enables them to react with some antigenic determinants and not with others. Specificity is dependent of various factors like chemical composition, physical forces, and molecular structures at the binding site. So according to the data given above the specificity of GFP i.e green flurocent protien to an antibody is maximum. And it can be further confirmed by antibody cross reactivity testing. It's 900 per 1000 molecules of given slide in 1 nM concentration.
b) KD is the ratio of the antibody dissociation rate (koff),how quickly it dissociates from its antigen,to the antibody
Association rate (kon) of the antibody,how quickly it binds to it's antigen. The binding of an antibody to its antigen is a reversible process,and rate of binding reaction is proportional to concentration of reactants. At equilibrium , rate of (antibody ) ( antigen ) complex formation is equal to rate of dissociation into it's components (antibody) +(antigen).so here in question it's 1nM.
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