Rescuing Abandoned Proteins with Water: CD and NMR Studies of Protein Fragments Solubilized in Salt-Free Water
Minfen Li 1, Jingxian Liu 1, Xiaoyuan Ran 1, Miaoqing Fang 1, Jiahai Shi 1, Haina Qin 1, June-Mui Goh 1 and Jianxing Song 1*
1 National University of Singapore
* To whom correspondence should be addressed. E-mail: bchsj{at}nus.edu.sg.
Submitted on July 12, 2006
Revised on August 11, 2006
Accepted on 28 August 2006
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Abstract |
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Many proteins expressed in E. coli cells form inclusion bodies which are neither refoldable nor soluble in buffers. Very surprisingly, we recently discovered that all 11 buffer-insoluble protein fragments/domains we have, with a great diversity of cellular function, location and molecular size, could be easily solubilized in salt-free water. The CD and NMR characterization led to classification of these proteins into three groups: group 1 with no secondary structure by CD and with narrowly-dispersed but sharp HSQC peaks; group 2 with secondary structure by CD but with HSQC peaks broadened and consequently only a small set of peaks detectable; and group 3 with secondary structure by CD and also well-separated HSQC peaks. Intriguingly, we failed to find any protein with a tight tertiary packing. Therefore we propose that buffer-insoluble proteins may lack intrinsic ability to reach or/and to maintain a well-packed conformation and thus are trapped in partially-folded states with many hydrophobic side chains exposed to the bulk solvent. As such, a very low ionic strength is sufficient to screen out intrinsic repulsive interactions and consequently the hydrophobic clustering/aggregation will be occurring. Marvelously enough, it appears that in the pure water proteins have the potential to manifest their full spectrum of structural states by utilizing intrinsic repulsive interactions to suppress the attractive hydrophobic clustering. Our discovery not only opens up a novel avenue to insight into the properties of insoluble proteins, but also sheds the first light on previously-unknown regimes associated with proteins.
Key Words:
CD spectroscopy, NMR spectroscopy, ionic strength, protein folding, protein solubility, the pure water
