Probing protein structure by limited proteolysis.
Limited proteolysis experiments can be successfully used to probe conformational features of proteins. In a number of studies it has been demonstrated that the sites of limited proteolysis along the polypeptide chain of a protein are characterized by enhanced backbone flexibility, implying that proteolytic probes can pinpoint the sites of local unfolding in a protein chain. Limited proteolysis was used to analyze the partly folded (molten globule) states of several proteins, such as apomyoglobin, alpha-lactalbumin, calcium-binding lysozymes, cytochrome c and human growth hormone. These proteins were induced to acquire the molten globule state under specific solvent conditions, such as low pH. In general, the protein conformational features deduced from limited proteolysis experiments nicely correlate with those deriving from other biophysical and spectroscopic techniques. Limited proteolysis is also most useful for isolating protein fragments that can fold autonomously and thus behave as protein domains. Moreover, the technique can be used to identify and prepare protein fragments that are able to associate into a native-like and often functional protein complex. Overall, our results underscore the utility of the limited proteolysis approach for unravelling molecular features of proteins and appear to prompt its systematic use as a simple first step in the elucidation of structure-dynamics-function relationships of a novel and rare protein, especially if available in minute amounts.
Fontana
Angelo
A
CRIBI Biotechnology Centre, University of Padua, Padua, Italy. angelo.fontana@unipd.it
de Laureto
Patrizia Polverino
PP
Spolaore
Barbara
B
Frare
Erica
E
Picotti
Paola
P
Zambonin
Marcello
M
D000818:Animals; D001059:Apoproteins / Q000737:chemistry; D001671:Biochemistry / Q000379:methods*; D002118:Calcium / Q000737:chemistry; D045304:Cytochromes c / Q000737:chemistry; D019382:Human Growth Hormone / Q000737:chemistry; D006801:Humans; D007768:Lactalbumin / Q000737:chemistry; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry; D009211:Myoglobin / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
D001059:Apoproteins; D009211:Myoglobin; D011506:Proteins; C010857:apomyoglobin; D019382:Human Growth Hormone; D045304:Cytochromes c; D007768:Lactalbumin; D009113:Muramidase; D002118:Calcium