Protein engineering techniques: gateways to synthetic protein universe

This brief provides a broad overview of protein-engineering research, offering a glimpse of the most common experimental methods. It also presents various computational programs with applications that are widely used in directed evolution, computational and de novo protein design. Further, it sheds light on the advantages and pitfalls of existing methodologies and future perspectives of protein engineering techniques

Structural Characterization of the CXCL5 dimer.

<p>(A) ¹⁵N-¹H HSQC 800 MHz NMR spectrum of the CXCL5 dimer at pH 6.0 and 25°C. The spectrum shows excellent chemical shift dispersion indicating a well-folded single species with no evidence of heterogeneity. The NH2 resonances of asparagines and glutamines are boxed. (B) Sections of the ¹⁵N-¹H HSQC spectra at pH 7.5 and 25°C at 5 μM (red) and 80 μM (blue). A new set of peaks corresponding to the monomer is evident in the 5 μM spectrum. The equilibrium constant was calculated to be ∼0.3 μM on the basis of monomer and dimer intensities.</p

Stability features from amide exchange.

<p>HSQC spectra of 100 μM CXCL5 in 50 mM sodium phosphate pH 6.0 after initiating exchange with D₂O after ∼8 min and after ∼24 hrs. The panel C shows the plot of ΔG_HX calculated from the amide exchange data.</p

Electrostatic representation of CXCR2-activating chemokines.

<p>The upper panels show the helical surface that highlight distribution of the GAG-binding residues and the lower panels show the opposite β-sheet surface after a 180° flip.</p

Backbone dynamics.

<p>A plot of the backbone {¹H}-¹⁵N NOE values as a function of residue number. The data show that the terminal residues are flexible and also the N-loop and the turn residues are more dynamic compared to the helical and strand residues.</p

Structural comparison between CXCL5 and other CXCR2-activating chemokines.

<p>(A) Superposition of CXCL5 [8–78] (red) on CXCL1, CXCL2, CXCL7, and CXCL8 (blue) in two different orientations. The superposition was optimized using residues 8 to 78 of CXCL5, residues 8 to 73 of CXCL1, residues 6 to 68 of CXCL2, residues 7 to 68 of CXCL7, and residues 8 to 72 of CXCL8.</p

A schematic of dimer-interface interactions.

<p>(A) β1/β1′ and (B) α1/α1′ residues that stabilize the dimer interface are highlighted. The interface residues in the monomer units are represented in blue and yellow, respectively.</p

NMR Solution Structure of CXCL5 dimer.

<p>Panels A and B show the superposition of the backbone atoms N, Cα and CO of residues 10 to 78 for the calculated twenty structures in two orientations. The polypeptide backbone is colored in blue and magenta for the two monomers. (C) Ribbon representation of the CXCL5 [10–78] structure. The protein dimer comprises of a six-stranded antiparallel β-sheet and a pair of α-helices. (D) Ribbon representation of CXCL5 monomer. The monomer consists of three antiparallel beta strands and an alpha helix; the disulfide bonds are shown in yellow.</p

Sequence alignment of CXCR2-activating chemokines.

<p>The conserved ELR, cysteine, and GAG-binding residues are highlighted in magenta, red, and blue, respectively. In the 30 s loop region, GP motif and residues that are aligned with I35 and K41 are highlighted in yellow and green, respectively.</p