From owner-biophys@hgmp.mrc.ac.uk Fri Nov 24 15:01:57 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 0948217AE8; Fri, 24 Nov 2000 15:01:55 +0000 (GMT) Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 6024) id 9D96417AE7; Fri, 24 Nov 2000 15:01:53 +0000 (GMT) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by mercury.hgmp.mrc.ac.uk (Postfix) with ESMTP id E789D415DE for ; Fri, 24 Nov 2000 15:01:38 +0000 (GMT) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.9.3+Sun/8.8.8) id PAA24500 for biophys-list@hgmp.mrc.ac.uk; Fri, 24 Nov 2000 15:01:38 GMT X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: Frank Fuerst X-Newsgroups: bionet.biophysics Subject: Re: Circular dichroism Date: Fri, 24 Nov 2000 16:01:34 +0100 Lines: 70 Message-ID: <8vlvsd$4u70k$1@fu-berlin.de> References: <3A1E66A6.F374F966@cam.ac.uk> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Trace: fu-berlin.de 975078093 5184532 141.89.201.37 (16 49 955) X-Newsreader: Forte Agent 1.8/32.548 To: biophys@hgmp.mrc.ac.uk Sender: owner-biophys@hgmp.mrc.ac.uk Precedence: bulk Kieran Gillick wrote: >Does anyone know what a 'missing amplitude' is? I'm doing some work on >protein folding pathways, and there's loads of stuff in the literature >about 'missing amplitudes' in CD spectra indicating multi-stage >kinetics/burst phases... Why does a burst phase give a missing >amplitude, and what exactly is a missing amplitude? What you observe when recording any kinetics with a spectroscopic signal is always the difference between the signal of the products and the educts. For a protein folding kinetics, this means the difference of unfolded protein (at the beginning) and native protein (in the end). Let's assume the unfolded species have an ellipticity (CD-signal) of zero, which is sensible, and the native protein has an ellipticity of 1 (just to keep figures simple). Then you would expect to observe a kinetic trace starting at 0 and ending at 1, often with a monoexponential time course [Theta = 1-exp(-t/tau) = 1-exp(-kt)]. But no let's look what happens if there is an intermediate that has a signal between that of the native and that of the unfolded protein, say 0.5 (for far-UV-CD this would mean that the intermediate has about half of the structure formed[1]). Now we would first see a fast rise to nearly 0.5 that smoothly merges into a slower rise to unity. However, if the intermediate is formed very fast, its formation is completed in the dead-time of mixing, so we cannot see the fast rise to 0.5 - we simply start at 0.5 and see the slow rise from 0.5 to 1. And the range from 0 to 0.5 is the 'missing amplitude'. Generally, 'missing amplitude' means that the amplitude of the change of a spectroscopic signal is smaller than expected from the difference in amplitude of the native and denatured signal. To be able to conclude that there is a kinetic intermediate on the folding pathway present, one has to make sure, however, that this missing amplitude is really missing *in*the*folding*reaction*. Two main other sources, or better sinks ;-), of amplitude occur to me: 1. Refolding yield One should always compare the absolute end value of the kinetics with the signal of native protein at the same protein concentration, because if the refolding yield is below 100%, you don't see 100% of the amplitude change, of course. 2. Dependence of the signal of denatured protein on the concentration of denaturant. This is especially important for fluorescence: When one measures the fluorescence of denatured protein at different concentrations of denaturant, all high enough to be sure that it is completely unfolded, one usually gets different values, because the denaturant interacts with the solvent exposed fluorophores. (And the effect is even stronger with heat denaturation). Thus, to calculate the expected amplitude of a refolding reaction, one has to extrapolate these values to the denaturant concentration at which the refolding experiment is carried out. Of course this can also lead to an increase in refolding amplitude. I hope this clarifies the topic a little. Yours, Frank [1] or 3/4 of the secondary structure, but it's in fast equilibrium with unfolded forms and populated only 2/3 of the time, or similar numbers. -- Was W. Richard Stevens angeht: Auf dessen Buecher verweist man nicht einzeln, dessen Buecher sind eigentlich Pflichtlektuere, da schaeme ich mich eigentlich, wenn ich eines nicht auswendig kann. Und ich muss mich da sehr kraeftig schaemen. [Detlef Bosau in dcoun] .