Crystal structure of the ribosomal protein S6 from Thermus thermophilus.

To explore the plasticity and structural constraints of the proteinfolding nucleus we have constructed through circular permutation four topological variants of the ribosomal protein S6. In effect, these topological variants represent entropy mutants with maintained spatial contacts. The proteins were characterized at two complementary levels of detail: by -value analysis estimating the extent of contact formation in the transition-state ensemble and by Hammond analysis measuring the site-specific growth of the folding nucleus. The results show that, although the loop-entropy alterations markedly influence the appearance and structural location of the folding nucleus, it retains a common motif of one helix docking against two strands. This nucleation motif is built around a shared subset of side chains in the center of the hydrophobic core but extends in different directions of the S6 structure following the permutant-specific differences in local loop entropies. The adjustment of the critical folding nucleus to alterations in loop entropies is reflected by a direct correlation between the -value change and the accompanying change in local sequence separation.O ur current understanding of the protein-folding nucleus is based on a synthesis of results from simulation (1) and experimental mapping of site-specific contacts in the transitionstate ensemble by protein engineering (2, 3). From these results it is apparent that the free-energy landscape controlling the folding process is highly evolved, with few traps and a characteristic bias toward native contacts (4, 5). Consistent with the general insensitivity of the transition-state structure to point mutation and the remarkable success of reproducing experimental data with simplistic Go models, the prediction from such biased landscapes is that the sequence of folding events is largely determined by the topology of the native structure (1, 4, 6, 7). One intriguing possibility is that folding follows a trajectory of the lowest successive loop-entropy cost (8, 9). To directly test this idea we have analyzed the folding behavior of four S6 variants in which the loop-entropy cost of forming pairwise contacts has been systematically altered through circular permutation (10-12). The results show that the -value distribution defining the S6 nucleus is plastic and responds to circular permutation in a systematic manner. Contacts are recruited in directions with decreased sequence separation, and contacts are lost at the entropically penalized regions of the backbone incisions. Even so, the critical nuclei of the permuted proteins share a minimal two-strand-helix motif with variable but overlapping composition of secondary-structure elements. Moreover, it is apparent that a specific number of side-chain contacts are required to turn the folding free energy profile downhill, and that the dimension of this cluster matches the size of the smallest cooperatively folding proteins. Results and DiscussionPronounced Changes of the -Value Distribution upon Circular Permutation. Ch...

Section: Methodsmentioning

confidence: 99%

Identification of the minimal protein-folding nucleus through loop-entropy perturbations

Lindberg

Haglund

Hubner

et al. 2006

“…When position 41 is polar or hydrophobic, there is a charged residue in position 42 or 43 that could act as a substitute gatekeeper. In the case of Arg-46, however, a strong functional conservation obscures the picture, because this residue is also involved in RNA binding (31).…”

Section: A Link Between Tetramerization Transient Aggregation and Pmentioning

confidence: 99%

“…The major protein component of the Alzheimer plaque is the 39-to 43-aa ␤-peptide (␤-AP), an aberrant proteolytic product of a 695-to 770-aa membranebound precursor protein (3). ␤-AP consists of a hydrophilic N-terminal part (amino acids 1-28) and a hydrophobic Cterminal sequence (amino acids [29][30][31][32][33][34][35][36][37][38][39][40][41][42][43], which is believed to be buried in the membrane in the precursor protein. Although ␤-AP readily forms fibrils in vitro, the fibrils' insolubility has been an obstacle to all attempts to solve their atomic structure.…”

mentioning

confidence: 99%

Designed protein tetramer zipped together with a hydrophobic Alzheimer homology: A structural clue to amyloid assembly

Otzen

Kristensen

Oliveberg

2000

177

147

Limited solubility and precipitation of amyloidogenic sequences such as the Alzheimer peptide (␤-AP) are major obstacles to a molecular understanding of protein fibrillation and deposition processes. Here we have circumvented the solubility problem by stepwise engineering a ␤-AP homology into a soluble scaffold, the monomeric protein S6. The S6 construct with the highest ␤-AP homology crystallizes as a tetramer that is linked by the ␤-AP residues forming intermolecular antiparallel ␤-sheets. This construct also shows increased coil aggregation during refolding, and a 14-mer peptide encompassing the engineered sequence forms fibrils. Mutational analysis shows that intermolecular association is linked to the overall hydrophobicity of the sticky sequence and implies the existence of ''structural gatekeepers'' in the wild-type protein, that is, charged side chains that prevent aggregation by interrupting contiguous stretches of hydrophobic residues in the primary sequence.

“…The split ␤-␣-␤ ribosomal protein S6 from Thermus thermophilus consists of 97 residues in a four-stranded ␤-sheet packed against two ␣-helices with a hydrophobic core (9). Functionally, S6 binds to both RNA and its protein partner S18 in a cooperative manner during the intermediate stage of 30S ribosomal subunit formation (10).…”

mentioning

confidence: 99%

Simulation, experiment, and evolution: Understanding nucleation in protein S6 folding

Hubner

Oliveberg

Shakhnovich

2004

In this study, we explore nucleation and the transition state ensemble of the ribosomal protein S6 using a Monte Carlo (MC) Go model in conjunction with restraints from experiment. The results are analyzed in the context of extensive experimental and evolutionary data. The roles of individual residues in the folding nucleus are identified, and the order of events in the S6 folding mechanism is explored in detail. Interpretation of our results agrees with, and extends the utility of, experiments that shift -values by modulating denaturant concentration and presents strong evidence for the realism of the mechanistic details in our MC Go model and the structural interpretation of experimental -values. We also observe plasticity in the contacts of the hydrophobic core that support the specific nucleus. For S6, which binds to RNA and protein after folding, this plasticity may result from the conformational flexibility required to achieve biological function. These results present a theoretical and conceptual picture that is relevant in understanding the mechanism of nucleation in protein folding.U nderstanding the transition state (TS) is among the major technical and intellectual milestones toward understanding the protein folding reaction (1). Several recent studies (2-6) have attempted to construct TS ensemble (TSE) structures by using -values as structural restraints in unfolding simulations. Through extensive studies of the experimentally and computationally benchmarked protein G (7), we have shown that experimental -values ( exp ) may be used in simulation to construct a putative TSE, but that measurement of a conformation's transmission coefficient (''probability to fold,'' p fold ) is the only means by which a structure may be classified as a member of the TSE. However, one must also be cautious in choosing which exp to use because the point mutations on which they are based may alter protein stability or structural features of the TSE, making normalization to the wildtype data ambiguous (8). Given that our method for studying the structure of the TSE has been validated in the complicated case of protein G folding (7), we are now able to carry out such analysis for other proteins on a comparative basis to aid in the distinction between experimental inconsistencies, noise, or artifacts and to determine the common denominators of the critical nucleus in protein folding.The split ␤-␣-␤ ribosomal protein S6 from Thermus thermophilus consists of 97 residues in a four-stranded ␤-sheet packed against two ␣-helices with a hydrophobic core (9). Functionally, S6 binds to both RNA and its protein partner S18 in a cooperative manner during the intermediate stage of 30S ribosomal subunit formation (10). S6 is an ideal candidate for computational study, due to the large body of high-quality experimental data available, including extensive kinetic and -value data at varying denaturant concentrations (11), circular-permutant studies that reflect rearrangements in the TSE (12), and studies of salt-induced off-pathway intermed...