19 research outputs found
Interrogating and Predicting Tolerated Sequence Diversity in Protein Folds: Application to E. elaterium Trypsin Inhibitor-II Cystine-Knot Miniprotein
Cystine-knot miniproteins (knottins) are promising molecular scaffolds for protein engineering applications. Members of the knottin family have multiple loops capable of displaying conformationally constrained polypeptides for molecular recognition. While previous studies have illustrated the potential of engineering knottins with modified loop sequences, a thorough exploration into the tolerated loop lengths and sequence space of a knottin scaffold has not been performed. In this work, we used the Ecballium elaterium trypsin inhibitor II (EETI) as a model member of the knottin family and constructed libraries of EETI loop-substituted variants with diversity in both amino acid sequence and loop length. Using yeast surface display, we isolated properly folded EETI loop-substituted clones and applied sequence analysis tools to assess the tolerated diversity of both amino acid sequence and loop length. In addition, we used covariance analysis to study the relationships between individual positions in the substituted loops, based on the expectation that correlated amino acid substitutions will occur between interacting residue pairs. We then used the results of our sequence and covariance analyses to successfully predict loop sequences that facilitated proper folding of the knottin when substituted into EETI loop 3. The sequence trends we observed in properly folded EETI loop-substituted clones will be useful for guiding future protein engineering efforts with this knottin scaffold. Furthermore, our findings demonstrate that the combination of directed evolution with sequence and covariance analyses can be a powerful tool for rational protein engineering
The T cell antigen receptor: the Swiss army knife of the immune system
The mammalian T cell receptor (TCR) orchestrates immunity by responding
to many billions of different ligands that it has never encountered before
and cannot adapt to at the protein sequence level. This remarkable receptor
exists in two main heterodimeric isoforms: ab TCR and gd TCR. The ab
TCR is expressed on the majority of peripheral T cells. Most ab T cells
recognize peptides, derived from degraded proteins, presented at the cell
surface in molecular cradles called major histocompatibility complex (MHC)
molecules. Recent reports have described other ab T cell subsets. These
βunconventionalβ T cells bear TCRs that are capable of recognizing lipid
ligands presented in the context of the MHC-like CD1 protein family or
bacterial metabolites bound to the MHC-related protein 1 (MR1). gd T cells
constitute a minority of the T cell pool in human blood, but can represent
up to half of total T cells in tissues such as the gut and skin. The identity
of the preferred ligands for gd T cells remains obscure, but it is now
known that this receptor can also functionally engage CD1-lipid, or
immunoglobulin (Ig) superfamily proteins called butyrophilins in the
presence of pyrophosphate intermediates of bacterial lipid biosynthesis.
Interactions between TCRs and these ligands allow the host to discriminate
between self and non-self and co-ordinate an attack on the latter. Here, we
describe how cells of the T lymphocyte lineage and their antigen receptors
are generated and discuss the various modes of antigen recognition by these
extraordinarily versatile receptors