Hi, I'm working with proteins at IISc (Bangalore). I'm characterising a 226-residue long protein of an unknown fold. I'm trying to use CD spectroscopy to calculate the ΔG for protein unfolding. I have used both GuHCl (0M - 3M) and Urea (0M - 4M) chemical gradients. Here are the graphs: http://oi41.tinypic.com/2rtpk.jpg Please note that I drew the red lines on MS paint. No curve-fitting here. Ideally, I should get a nice, smooth sigmoidal curve. However, in both cases, I'm getting an initial 'dip' in CD values. This would indicate that the protein is folding BETTER under slight denaturing conditions. These results are reproducible. I've run 3 replicates for each graph. Have you seen chemical denaturation curves like these before? If so, how do you interpret such data? Please post links. Other details: -Protein is pretty soluble (purified to 5-10 mg/ml). -Shows alpha-beta CD spectrum (190-250 nm range). -It's Tm is 45C (this graph is a smooth sigmoid). Thanks a lot.