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Chemical synthesis allows the incorporation of nonnatural amino acids into proteins that may provide previously untried probes of their folding pathway and thermodynamic stability. We have used a flexible thioether linker as a loop mimetic in the human yes kinase-associated protein (YAP 65) WW domain, a three-stranded, 44-residue, ␤-sheet protein. This linkage avoids problems of incorporating sequences that constrain loops to the extent that they significantly change the nature of the denatured state with concomitant effects on the folding kinetics. An NMR solution structure shows that the thioether linker had little effect on the global fold of the domain, although the loop is apparently more dynamic. The thioether variants are destabilized by up to 1.4 kcal͞mol (1 cal ‫؍‬ 4.18 J). Preliminary ⌽-value analysis showed that the first loop is highly structured in the folding transition state, and the second loop is essentially unstructured. These data are consistent with results from simulated unfolding and detailed protein-engineering studies of structurally homologous WW domains. Previously, ⌽-value analysis was limited to studying side-chain interactions. The linkers used here extend the protein engineering method directly to secondary-structure interactions. U nderstanding how proteins spontaneously fold to a stable three-dimensional structure is exceptionally difficult. This difficulty applies especially to ␤-sheet proteins, whose secondary structure is stabilized more by long-range sequence-distant interactions than is that in ␣-helical proteins. Small ␤-sheet proteins are useful paradigms for understanding the search problem. There is increasing evidence of the importance of ␤-hairpins and loops in driving ␤-sheet folding (1-6). The stability of these loops may also influence the overall stability of ␤-sheet proteins in two ways. One way is by contributing to the overall thermodynamic stability of the system (7-9). The other way is in the effect on the folding pathway (2, 3), such that folding may or may not be facilitated by loop or hairpin formation, with concomitant effects on the folding rate and, thus, on stability.The WW domains are a family of small, triple-stranded, antiparallel ␤-sheet proteins (typically, of 34-44 amino acids) with a small hydrophobic core involving residues from both the N and C termini (10). The loop structures and overall bend of the ␤-sheet are well conserved in the different members of this family (11-13). A number of these domains are well suited for biophysical studies of ␤-sheet folding and loop formation as they reversibly denature, are amenable to mutagenesis, and are easily synthesized by using both recombinant and organic chemistry techniques (12,14,15). Thus, these domains can be synthesized with both natural and nonnatural amino acids in their sequences, allowing for sophisticated experiments that cannot easily be performed with recombinant proteins. The effects of substitutions on the stabilities of WW domains have been studied in the context of a designed WW-domain...

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Comprehensive structure–function analyses are possible with an array comprising more than 800 synthetic variants of a protein domain composed of 44 amino acids. This array was produced by SPOT synthesis on a cellulose membrane (see schematic representation) and successfully employed for a parallel ligand‐binding assay.

Comprehensive structure–function analyses are possible with an array comprising more than 800 synthetic variants of a protein domain composed of 44 amino acids. This array was produced by SPOT synthesis on a cellulose membrane (see schematic representation) and successfully employed for a parallel ligand‐binding assay.

The cover picture shows an array of several hundred synthetically produced variants of the 44 amino acids comprising the hyAP‐WW protein domain. The array was produced by a stepwise SPOT synthesis on a cellulose membrane. At each synthesis site (spot) a WW domain is bound to the membrane through a C‐terminal ester bond. The secondary structure of the WW domain is shown in green as a ribbon. The domains of the single spots differ only by a single amino acid. All the domains were tested simultaneously for their ability to bind to a peptide motif (red) common to many proteins. The binding was evident when visualized by chemoluminescence, with the Spots having various intensities. The systematic analysis undertaken here enabled molecular biology to be carried out that would have otherwise have required great effort, or not been done at all. This chemical technique also allows the construction of many non‐genetically coded building blocks. Through the use of modern synthetic techniques for the coupling of peptides this method should also allow the synthesis of proteins. The combination of molecular biological and chemical methods opens up opportunities for the preparation of protein chips for diagnostics and drug discovery. More about this can be found in the article by Schneider‐Mergener et al. on p. 897ff.

The cover picture shows an array of several hundred synthetically produced variants of the 44 amino acids comprising the hyAP-WW protein domain. The array was produced by a stepwise SPOT synthesis on a cellulose membrane. At each synthesis site (spot) a WW domain is bound to the membrane through a C-terminal ester bond. The secondary structure of the WW domain is shown in green as a ribbon. The domains of the single spots differ only by a single amino acid. All the domains were tested simultaneously for their ability to bind to a peptide motif (red) common to many proteins. The binding was evident when visualized by chemoluminescence, with the Spots having various intensities. The systematic analysis undertaken here enabled molecular biology to be carried out that would have otherwise have required great effort, or not been done at all. This chemical technique also allows the construction of many non-genetically coded building blocks. Through the use of modern synthetic techniques for the coupling of peptides this method should also allow the synthesis of proteins. The combination of molecular biological and chemical methods opens up opportunities for the preparation of protein chips for diagnostics and drug discovery. More about this can be found in the article by Schneider-Mergener et al. on p. 897ff.