Sequence replacements in the central β‐turn of plastocyanin

Abstract

The role of 0-turns in dictating the structure of a @-barrel protein is assessed by probing the tolerance of the central 0-turn of poplar plastocyanin to substitution by arbitrary sequences. Native plastocyanin binds copper and is colored bright blue. However, when the wild-type Pr~,,-Ser,,-Gly,,-Val~~ turn sequence is replaced by arbitrary tetrapeptides, the vast majority (92/98 = 94%) of mutant proteins cannot fold into the native blue structure. Characterization of the colorless mutant proteins demonstrates that the majority of substitutions in this type I1 0-turn disrupt the native structure severely. Gross structural changes are indicated by major differences in the CD spectra of the mutants relative to the wild-type protein, and by the much larger apparent size of mutant proteins in gel filtration experiments. These mutant proteins do not bind copper. Furthermore, Cys,, forms a disulfide bond readily in the colorless mutant proteins, indicating that it has moved away from the buried position it occupies in the native copper binding site and has become exposed. These results indicate that the central 0-turn in plastocyanin is not merely a default structure arising in response to the surrounding context; rather, sequence information in this turn plays an active role in dictating the location of a chain reversal in the &barrel structure. These findings are discussed in terms of their implications for the folding of natural proteins, as well as the design of de novo proteins.

Diabetes Mellitus due to Misfolding of a β-Cell Transcription Factor: Stereospecific Frustration of a Schellman Motif in HNF-1α

“…This perspective is supported by the interrelation between turn sequence and β-sheet registry in a β-barrel. 42 The intertwined 4HB of HNF-1α (Figure 4(c)) provides an example of an α-turn-α motif in a globular domain in which the turn both modulates thermodynamic stability and confers architectural specificity. A clinical mutation in this turn violates its strict requirement for a positive ϕ angle at position 20 and so impairs dimerizationcoupled protein folding.…”

Intestinal fatty acid binding protein: A specific residue in one turn appears to stabilize the native structure and be responsible for slow refolding

“…Regardless of whether the same region of the protein is involved with both the early and late stages of the folding process, it is likely that triple mutants lacking Leu 64 represent an intermediate on the folding path prior to the final stabilization of the molecule. Finally, it is of interest to compare the results with those obtained by Ybe and Hecht (1996). These investigators have made a number of mutations in the central P-turn of plastocyanin.…”

MSH3 Polymorphisms and Protein Levels Affect CAG Repeat Instability in Huntington's Disease Mice

“…In particular, although our homology modeling results did not offer insight into which variants resided in regions critical to overall protein structure nor did the polymorphism reside in known protein-binding domains (Figure 5A and Figure S6), the highly conserved T321I variant occurs within a Type I β-Turn which could be critical for protein folding [71], [72]. Changes in β-Turn sequences modulate protein stability [71], [72], [75], [76], where unfavourable sequence changes can dramatically decrease protein folding rate [77], [78] and in some cases completely ablate protein expression [79]. In addition to potential changes brought by the T321I variant, the CBy strain gains a potential serine phosphosite at amino acid 79 as experimentally determined in the homologous human MSH3 protein (site 116 in hMSH3) [80], which could impact overall protein conformation, its protein-binding capacity and stability [81].…”