The in vitro refolding of hen egg-white lysozyme is studied in the presence of various osmolytes. Proline is found to prevent aggregation during protein refolding. However, other osmolytes used in this study fail to exhibit a similar property. Experimental evidence suggests that proline inhibits protein aggregation by binding to folding intermediate~s! and trapping the folding intermediate~s! into enzymatically inactive, "aggregation-insensitive" state~s!. However, elimination of proline from the refolded protein mixture results in significant recovery of the bacteriolytic activity. At higher concentrations~Ͼ1.5 M!, proline is shown to form loose, higher-order molecular aggregate~s!. The supramolecular assembly of proline is found to possess an amphipathic character. Formation of higher-order aggregates is believed to be crucial for proline to function as a protein folding aid. In addition to its role in osmoregulation under water stress conditions, the results of this study hint at the possibility of proline behaving as a protein folding chaperone.
The aim of thiscom pre hen sivereviewisto critically evaluate the progress inresearchin the area of protein fold ing. In the first sec tion, we dis cuss the var i ous mod els pro posed to ex plain the pro tein fold ingpar a dox. In the sue ceed ing sec tion of the re view, a de tailed ac count of the de vel op ments in our un derstand ing of the fold ing path ways of ß-sheet pro teins is pro vided.The foundation for protein folding kinetics was laid about thirty five years ago. Christain Anfinsen showed that pro tein can fold reversibly and the native structures of proteins are ther mo dy nam i cally sta ble states rep re sent ing the global min ima of their ac ces si ble free en er gies. 1 ' 2 For most pro teins, the in for ma tion for fold ing is con tained in the amino acid se quence. 3 " 6 How ever, a pro tein se quence needs to meet two cri te ria dur ing fold ing -one ther mo dy namic and the other kinetic. 7 " 14 The ther mo dy namic re quire ment is that the pro tein mol e cule needs to adopt aunique folded con forma tion (na tive state),whichisstableunderphysiologicalconditions. 15 " 23 The ki netic re quire ment is that the pro tein should fold back from its un folded state to its na tive con for ma tion within a rea sonable time frame. 24 " 34 In prin ci pie, a polypeptide chain (in its unfolded state) can adopt numerous conformations. 35 " 37 Hence, if the pro tein needs to sam pie out all the pos si ble con fer ma tions to de eide on its global min i mum, then for a pro tein con sist ing of 100 amino ac ids, it would take the age of earth (about 10 29 years if one assumes that only 10" 1 's is re quired to con vert from one con for ma tion to the other) to fold into its nativeconformation. 38 " 40 This puz zle is the crux of the pro tein fold ing prob lern and it is pop u larly called the Levinthal par adox. 4 ' Levinthal pos tu lated that the ther mo dy namic and ki netic con trols are the two mu tu ally ex clu sive op tions for a folding molecule. Thermodynamic control meant that the fold ing mol e cule reaches its global min i mum in en ergy and thereby fold ing is path way in de pend ent. Due to the ex ten sive search in volved in this pro cess, it takes a long time for a pro tein to fold un der ther mo dy namic con trol. The ki netic con trol meant that fold ing oc curs on a phy s i o log i cal time scale be cause it is path way de pend ent. 42 " 45 Un der the ki netic con trol, the fi nal struc ture of the pro tein could differ de pend ing on the denaturantconditionsfromwhichfoldingisinitiated 46 " 54 Formu la tion of the Levinthal par a dox led to the search for fold ing pathways.The search for fold ing in ter me di ates be gan in 1971. T anf ord and co work ers re ported sta ble in ter me di ates along the equilibrium-unfolding pathway of hen egg white lysozyme. 55 " 60 Identificationofequilibriumintermediatesinbovine pan ere atic ribonuclease con sol i dated the no tion that pro tein-folding pro ceeds through def i nite struc tural in ter me di ätes. 61 " 64 These re ports trigerred the larg...
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