Biomedical subjects

Samuel S Cho

Publications and source records attributed to Samuel S Cho.

8 recordsLinked to original sources

PubMed2006

Stabilizing IkappaBalpha by "consensus" design.

IkappaBalpha is the major regulator of transcription factor NF-kappaB function. The ankyrin repeat region of IkappaBalpha mediates specific interactions with NF-kappaB dimers, but ankyrin repeats 1, 5 and 6 display a highly dynamic character when not in complex with NF-kappaB. Using chemical denaturation, we show here that IkappaBalpha displays two folding transitions: a non-cooperative conversion under weak perturbation, and a major cooperative folding phase upon stronger insult. Taking advantage of a native Trp residue in ankyrin repeat (AR) 6 and engineered Trp residues in AR2, AR4 and AR5, we show that the cooperative transition involves AR2 and AR3, while the non-cooperative transition involves AR5 and AR6. The major structural transition can be affected by single amino acid substitutions converging to the "consensus" ankyrin repeat sequence, increasing the native state stability significantly. We further characterized the structural and dynamic properties of the native state ensemble of IkappaBalpha and the stabilized mutants by H/(2)H exchange mass spectrometry and NMR. The solution experiments were complemented with molecular dynamics simulations to elucidate the microscopic origins of the stabilizing effect of the consensus substitutions, which can be traced to the fast conformational dynamics of the folded ensemble.

Amino Acid Sequence↗

PubMed2006

P versus Q: structural reaction coordinates capture protein folding on smooth landscapes.

Minding your p's and q's has become as important to protein-folding theorists as it is for those being instructed in the rules of etiquette. To assess the quality of structural reaction coordinates in predicting the transition-state ensemble (TSE) of protein folding, we benchmarked the accuracy of four structural reaction coordinates against the kinetic measure P(fold), whose value of 0.50 defines the stochastic separatrix for a two-state folding mechanism. For two proteins that fold by a simple two-state mechanism, c-src SH3 and CI-2, the Phi-values of the TSEs predicted by native topology-based reaction coordinates (including Q, the fraction of native contacts) are almost identical to those of the TSE based on P(fold), with correlation coefficients of >0.90. For proteins with complex folding mechanisms that have especially broad, asymmetrical free energy barriers such as the designed 3-ankyrin repeating protein (3ANK) or proteins with distinct intermediates such as cyanovirin-N (CV-N), we show that the ensemble of structures with P(fold) = 0.50 generally does not include the chemically relevant transition states. This weakness of P(fold) limits its usefulness in protein folding studies. For such systems, elucidating the essential features of folding mechanisms requires using multiple reaction coordinates, although the number is still rather small. At the same time, for simple folding mechanisms, there is no indication of superiority for P(fold) over structurally chosen and thermodynamically relevant reaction coordinates that correctly measure the degree of nativeness.

CSK Tyrosine-Protein Kinase↗

PubMed2005

The energy landscape of modular repeat proteins: topology determines folding mechanism in the ankyrin family.

Proteins consisting of repeating amino acid motifs are abundant in all kingdoms of life, especially in higher eukaryotes. Repeat-containing proteins self-organize into elongated non-globular structures. Do the same general underlying principles that dictate the folding of globular domains apply also to these extended topologies? Using a simplified structure-based model capturing a perfectly funneled energy landscape, we surveyed the predicted mechanism of folding for ankyrin repeat containing proteins. The ankyrin family is one of the most extensively studied classes of non-globular folds. The model based only on native contacts reproduces most of the experimental observations on the folding of these proteins, including a folding mechanism that is reminiscent of a nucleation propagation growth. The confluence of simulation and experimental results suggests that the folding of non-globular proteins is accurately described by a funneled energy landscape, in which topology plays a determinant role in the folding mechanism.

Algorithms↗

PubMed2005

Symmetry and frustration in protein energy landscapes: a near degeneracy resolves the Rop dimer-folding mystery.

Protein folding has become one of the best understood biochemical reactions from a kinetic viewpoint. The funneled energy landscape, a consequence of the minimal frustration achieved by evolution in sequences, explains how most proteins fold efficiently and robustly to their functional structure and allows robust prediction of folding kinetics. The folding of Rop (repressor of primer) dimer is exceptional because some of its mutants with a redesigned hydrophobic core both fold and unfold much faster than the WT protein, which seems to conflict with a simple funneled energy landscape for which topology mainly determines the kinetics. We propose that the mystery of Rop folding can be unraveled by assuming a double-funneled energy landscape on which there are two basins that correspond to distinct but related topological structures. Because of the near symmetry of the molecule, mutations can cause a conformational switch to a nearly degenerate yet distinct topology or lead to a mixture of both topologies. The topology predicted to have the lower free-energy barrier height for folding was further found by all-atom modeling to give a better structural fit for those mutants with the extreme folding and unfolding rates. Thus, the non-Hammond effects can be understood within energy-landscape theory if there are in fact two different but nearly degenerate structures for Rop. Mutations in symmetric and regular structures may give rise to frustration and thus result in degeneracy.

Biophysical Phenomena↗

PubMed2005

A survey of flexible protein binding mechanisms and their transition states using native topology based energy landscapes.

Many cellular functions rely on interactions between protein pairs and higher oligomers. We have recently shown that binding mechanisms are robust and owing to the minimal frustration principle, just as for protein folding, are governed primarily by the protein's native topology, which is characterized by the network of non-covalent residue-residue interactions. The detailed binding mechanisms of nine dimers, a trimer, and a tetramer, each involving different degrees of flexibility and plasticity during assembly, are surveyed here using a model that is based solely on the protein topology, having a perfectly funneled energy landscape. The importance of flexibility in binding reactions is manifested by the fly-casting effect, which is diminished in magnitude when protein flexibility is removed. Many of the grosser and finer structural aspects of the various binding mechanisms (including binding of pre-folded monomers, binding of intrinsically unfolded monomers, and binding by domain-swapping) predicted by the native topology based landscape model are consistent with the mechanisms found in the laboratory. An asymmetric binding mechanism is often observed for the formation of the symmetric homodimers where one monomer is more structured at the binding transition state and serves as a template for the folding of the other monomer. Phi values were calculated to show how the structure of the binding transition state ensemble would be manifested in protein engineering studies. For most systems, the simulated Phi values are reasonably correlated with the available experimental values. This agreement suggests that the overall binding mechanism and the nature of the binding transition state ensemble can be understood from the network of interactions that stabilize the native fold. The Phi values for the formation of an antibody-antigen complex indicate a possible role for solvation of the interface in biomolecular association of large rigid proteins.

Antigen-Antibody Complex↗

PubMed2005

Overcoming residual frustration in domain-swapping: the roles of disulfide bonds in dimerization and aggregation.

The prevalence of domain-swapping in nature is a manifestation of the principle of minimal frustration in that the interactions designed by evolution to stabilize the protein are also involved in this mode of binding. We previously demonstrated that the Symmetrized-Go potential accurately predicts the experimentally observed domain-swapped structure of Eps8 based solely on the structure of the monomer. There can be, however, multiple modes of domain-swapping, reflecting a higher level of frustration, which is a consequence of symmetry. The human prion and cyanovirin-N are too frustrated to form unique domain-swapped structures on the basis of the Symmetrized-Go potential. However, supplementing the completely symmetric model with intermolecular and intramolecular disulfide bonds in the prion and cyanovirin-N proteins, respectively, yielded unique domain-swapped structures with a remarkable similarity to the experimentally observed ones. These results suggest that the disulfide bonds may sometimes be critical in overcoming the intrinsic frustration of the symmetrized energy landscapes for domain-swapping. We also discuss the implications of intermolecular disulfide bonds in the formation of mammalian prion aggregates.

Amino Acid Sequence↗

PubMed2004

Molecular recognition of aldehydes by aldehyde dehydrogenase and mechanism of nucleophile activation.

Experimental structural data on the state of substrates bound to class 3 Aldehyde Dehydrogenases (ALDH3A1) is currently unknown. We have utilized molecular mechanics (MM) simulations, in conjunction with new force field parameters for aldehydes, to study the atomic details of benzaldehyde binding to ALDH3A1. Our results indicate that while the nucleophilic Cys243 must be in the neutral state to form what are commonly called near-attack conformers (NACs), these structures do not correlate with increased complexation energy calculated with the MM-Generalized Born Molecular Volume (GBMV) method. The negatively charged Cys243 (thiolate form) of ALDH3A1 also binds benzaldehyde in a stable conformation but in this complex the sulfur of Cys243 is oriented away from benzaldehyde yet yields the most favorable MM-GBMV complexation energy. The identity of the general base, Glu209 or Glu333, in ALDHs remains uncertain. The MM simulations reveal structural and possible functional roles for both Glu209 and Glu333. Structures from the MM simulations that would support either glutamate residue as the general base were further examined with Hybrid Quantum Mechanical (QM)/MM simulations. These simulations show that, with the PM3/OPLS potential, Glu209 must go through a step-wise mechanism to activate Cys243 through an intervening water molecule while Glu333 can go through a more favorable concerted mechanism for the same activation process.

Aldehyde Dehydrogenase↗

PubMed2004

Domain swapping is a consequence of minimal frustration.

The same energy landscape principles associated with the folding of proteins into their monomeric conformations should also describe how these proteins oligomerize into domain-swapped conformations. We tested this hypothesis by using a simplified model for the epidermal growth factor receptor pathway substrate 8 src homology 3 domain protein, both of whose monomeric and domain-swapped structures have been solved. The model, which we call the symmetrized Gō-type model, incorporates only information regarding the monomeric conformation in an energy function for the dimer to predict the domain-swapped conformation. A striking preference for the correct domain-swapped structure was observed, indicating that overall monomer topology is a main determinant of the structure of domain-swapped dimers. Furthermore, we explore the free energy surface for domain swapping by using our model to characterize the mechanism of oligomerization.

Binding Sites↗