A design approach to the structural analysis of interleukin‐2

Abstract

A semi-synthetic protein design approach has been employed for the structural investigation of a putative helical region at the C-terminus of Interleukin-2. With crystallographic or NMR derived conformational data as yet unavailable, we have relied only on primary sequence information and computer-assisted modelling to direct the analysis. By employing both chemical peptide synthesis and recombinant DNA methods, the C-terminus of IL-2 was modified according to a strategy designed to stabilize helical secondary structure. A semi-synthetic protein incorporating 12 simultaneous amino acid replacements was constructed, which possessed potentiated biological activity and displayed a far UV circular dichroism spectrum comparable to a hybrid protein with the authentic sequence. By comparison, another hybrid protein containing a C-terminal region designed to contain helix breaking residues was totally devoid of bioactivity. These findings provide evidence that the modelling method correctly identified a helix necessary for the formation of a bioactive tertiary fold. Moreover, by employing semi-synthesis it was possible to circumvent the difficulties associated with the preparation, purification and analysis of multiple recombinant proteins, and also to avoid the unreliability of total chemical synthesis for proteins greater than 100 residues.

Alzheimer disease hyperphosphorylated tau aggregates hydrophobically

“…The CD spectra as a function of temperature were conducted as previously described in Ruben et al (1991) using the method of Ciardelli et al (1988) and an Instruments SA, Jobin Yvon Mark V circular dichro-graph (Longjumeau, France). All of the samples were filtered through a 0.45 µm Millipore centrifuge filter before their spectra were recorded so that the 185 nm signal could be observed and the 195-245 nm signal could be smoothed.…”

Generation of low-toxicity interleukin-2 fusion proteins devoid of vasopermeability activity

“…These IL-2 analogs containing specific amino acid substitutions have added greatly to our basic understanding of the structure of IL-2 and to the importance of specific regions of the molecule that interact with receptors expressed on T lymphocytes and other hematopoietic cells. [11][12][13][14][15][16][17][18][19][20] Among other findings, these studies have shown that point mutations outside the region of the receptor binding domains can also affect IL-2 function by distorting the tertiary structure of the molecule. Although early studies focused on the influence of point mutations on IL-2 function such as T-cell proliferation and receptor binding, more recent studies have examined whether the toxic properties of the molecule can be altered without affecting cytokine activity.…”