Identifying the structural boundaries of independent folding domains in the a subunit of tryptophan synthase, a β/α barrel protein

Abstract

Two equilibrium intermediates have previously been observed in the urea denaturation of the a subunit of tryptophan synthase~aTS! from Escherichia coli, an eight-stranded b0a barrel protein. In the current study, a series of aminoterminal fragments were characterized to probe the elementary folding units that may be in part responsible for this complex behavior. Stop-codon mutagenesis was used to produce eight fragments ranging in size from 105-214 residues and containing incremental elements of secondary structure. Equilibrium studies by circular dichroism indicate that all of these fragments are capable of adopting secondary structure. All except for the shortest fragment fold cooperatively. The addition of the fourth, sixth, and eighth b-strands leads to distinct increases in structure, cooperativity, and0or stability, suggesting that folding involves the modular assembly of bab supersecondary structural elements. Onedimensional NMR titrations at high concentrations of urea, probing the environment around His92, were also performed to test for the presence of residual structure in the fragments. All fragments that contained the first four ba units of structure exhibited a cooperative unfolding transition at high concentrations of urea with significant but reduced stability relative to the full-length protein. These results suggest that the residual structure in aTS requires the participation of hydrophobic residues in multiple b-strands that span the entire sequence.Keywords: autonomous folding unit; circular dichroism; equilibrium intermediates; nuclear magnetic resonance; protein fragments; protein folding; residual structureThe equilibrium unfolding reactions of many proteins exhibit two-state behavior in which only the native and unfolded conformations are populated at significant concentrations. This high cooperativity often hinders a detailed analysis of partially folded forms that could provide clues to the relationship between protein sequence and structure. Protein fragmentation offers one approach toward reducing the cooperativity of the folding reaction and producing stable partially folded forms that can be extensively studied under equilibrium conditions~Kim & Baldwin, 1982; Lecomte & Matthews, 1993!.Protein fragmentation studies, employing proteolysis, chemical cleavage, or protein engineering methods, have provided a variety of insights into protein folding reactions. Fragments corresponding to isolated structural domains in the multidomain proteins phosphoglycerate kinase~Missiakas et al !, streptokinase~Par-rado et al., 1996!, and thermolysin~Corbett & Roche, 1986! have been shown to fold independently, providing evidence for a hierarchical folding mechanism in multidomain proteins~Jaenicke, 1991!. A structural subdomain revealed by proteolysis of the dimeric trp repressor~Tasayco & Carey, 1992! has also been demonstrated to fold autonomously. The folding of a 123-residue C-terminal fragment of dihydrofolate reductase to a presumably non-native structure~Gegg et al., 1997! may provide...

Random-coil behavior and the dimensions of chemically unfolded proteins

“…We find that in 6 M GuHCl this value expands to 48.8 Ϯ 1.0 Å, consistent with random-coil predictions. This observation is also consistent with NMR-based reports that at concentrations above 6 M urea the protein undergoes an additional cooperative transition (36). In contrast, we observe an R G of 46 Ϯ 1.5 Å for creatine kinase in 6 M GuHCl, which is within error of previous reports (34) but significantly less than the 67 Å predicted for a 380-residue random coil.…”

Anatomy of protein structures: Visualizing how a one-dimensional protein chain folds into a three-dimensional shape

“…Our simulations of a 16-residue ␤-hairpin peptide fragment from protein G (40) have shown that it folds rapidly and cooperatively to a conformation with a defined secondary structure and a packed hydrophobic cluster of aromatic side chains. In experiments, this peptide has been observed to be very stable (41).…”

“…By including the two types of terms, with respect to both fragment size and as a function of the fraction of the fragment size to the whole protein, we obtain a balance for all fragment sizes. To validate the success of our fragment-size-independent scoring function will require a set of systematically carried out stability measurements either from experiments (25,41) or from theoretical calculations (42) for protein fragments of different sizes. However, the consistency and the improvement of the HFU assignments via a combinatorial assembly of the assigned building blocks is an indirect evidence for its validity.…”

In vitro selection and characterization of a stable subdomain of phosphoribosylanthranilate isomerase

“…This is in contrast with analogous (ba) 6 fragments of both S. cerevisiae PRAI and the a subunit of tryptophan synthase, which show spectra of a similar shape but of much reduced intensity compared with the fulllength protein [23,33]. In even starker contrast with the (ba) 6 fragment from the yeast enzyme [23], the trPRAIHis near-UV spectrum is also of a similar form and magnitude to that of full-length PRAI.…”

“…In the last five years, a substantial body of evidence has accumulated for the existence of autonomously folding subdomains in (ba) 8 -barrel proteins including triosephosphate isomerase [38,39], the (ba) 8 -barrels of histidine biosynthesis [40][41][42], IGPS [43], and the a subunit of tryptophan synthase [33,44]. Protein folding studies have suggested that PRAI folds through an intermediate consisting of (ba) 1)5 b 6 [34].…”