Slow motion analysis of protein folding intermediates within wet silica gels.
Resolving the complete folding pathway of a protein is a major challenge to conventional experimental methods because of the rapidity and complexity of folding. Here, we show that entrapment of the protein cytochrome c in wet, optically transparent, porous silica gel matrices has enabled a dramatic expansion, to days or weeks, of the folding time, allowing direct observation of the entire folding pathway using a combination of three spectroscopic techniques, far-ultraviolet circular dichroism, tryptophan fluorescence, and Soret absorption spectroscopy. During refolding in silica gels, collapse and helix formation occur in a stepwise manner, as observed in aqueous solution. Analysis of kinetics and transient spectra indeed reveals a sequence of four distinct intermediates with progressively increasing degrees of folding, two of which closely resemble those previously characterized in solution, namely, the early collapsed and the molten globule intermediates. The other two are the precollapsed and pre-molten globule intermediates that may escape detection by conventional kinetic methods. Interestingly, varying the strength of the gel network has a dramatic effect on the folding time, but no significant effect on the structural features of each folding intermediate, indicating that the interaction between the protein and gel matrix has no measurable effect on the folding pathway. These results better define the major pathway of cytochrome c folding. In addition, in this paper we present the results of the application of this method to a simple, apparent two-state folder ubiquitin, helping to interpret the results for cytochrome c.
Shibayama
Naoya
N
Department of Physiology, Division of Biophysics, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. shibayam@jichi.ac.jp
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D002942:Circular Dichroism / Q000295:instrumentation / Q000379:methods; D045304:Cytochromes c / Q000737:chemistry*; D005782:Gels; D006736:Horses; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012822:Silicon Dioxide / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods; D013816:Thermodynamics; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D005782:Gels; D012822:Silicon Dioxide; D014364:Tryptophan; D045304:Cytochromes c