A new approach to artificial and modified proteins: theory-based design, synthesis in a cell-free system and fast testing of structural properties by radiolabels.
A novel approach to the creation of artificial and modified proteins has been elaborated. The approach includes a sequence design based on the molecular theory of protein secondary structure and folding patterns, gene expression in a cell-free system and testing of structural properties of the synthesized polypeptides at a nanogram level using radiolabelled chains. The approach has been applied to a new synthetic protein albebetin which has been designed to form a 3-D fold which does not contradict any structural rule but has been never observed up to now in natural proteins. Using size-exclusion chromatography, urea-gradient electrophoresis and limited proteolysis of a radiolabelled chain, it has been shown that the artificial protein is nearly as compact as natural proteins, cooperatively unfolds at high urea concentrations and has some structural features of a definite structure consistent with the designed one. As albebetin has been designed as consisting of two structural repeats, a 'half-albebetin' (one of these repeats) has also been synthesized and studied. It was shown that 'half-albebetin' is also compact.
Chemeris
V V
VV
Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region.
Dolgikh
D A
DA
Fedorov
A N
AN
Finkelstein
A V
AV
Kirpichnikov
M P
MP
Uversky
V N
VN
Ptitsyn
O B
OB
D000595:Amino Acid Sequence; D001483:Base Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005813:Genes, Synthetic; D007553:Isotope Labeling; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis; D014176:Protein Biosynthesis; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D014158:Transcription, Genetic; D014508:Urea / Q000494:pharmacology
D010446:Peptide Fragments; D011506:Proteins; C074146:albebetin; D014508:Urea