A ß-sheet structure interacting peptide for intracellular protein delivery into human pluripotent stem cells and their derivatives.

The advance in stem cell research relies largely on the efficiency and biocompatibility of technologies used to manipulate stem cells. In our previous study, we had designed an amphipathic peptide RV24 that can deliver proteins into cancer cell lines efficiently without significant side effects. Encouraged by this observation, we moved forward to test whether RV24 could be used to deliver proteins into human embryonic stem cells and human induced pluripotent stem cells. RV24 successfully mediated protein delivery into these pluripotent stem cells, as well as their derivatives including neural stem cells and dendritic cells. Based on NMR studies and particle surface charge measurements, we proposed that hydrophobic domain of RV24 interacts with ß-sheet structures of the proteins, followed by formation of "peptide cage" to facilitate delivery across cellular membrane. These findings suggest the feasibility of using amphipathic peptide to deliver functional proteins intracellularly for stem cell research.

Structural, stability, dynamic and binding properties of the ALS-causing T46I mutant of the hVAPB MSP domain as revealed by NMR and MD simulations.

T46I is the second mutation on the hVAPB MSP domain which was recently identified from non-Brazilian kindred to cause a familial amyotrophic lateral sclerosis (ALS). Here using CD, NMR and molecular dynamics (MD) simulations, we characterized the structure, stability, dynamics and binding capacity of the T46I-MSP domain. The results reveal: 1) unlike P56S which we previously showed to completely eliminate the native MSP structure, T46I leads to no significant disruption of the native secondary and tertiary structures, as evidenced from its far-UV CD spectrum, as well as Cα and Cß NMR chemical shifts. 2) Nevertheless, T46I does result in a reduced thermodynamic stability and loss of the cooperative urea-unfolding transition. As such, the T46I-MSP domain is more prone to aggregation than WT at high protein concentrations and temperatures in vitro, which may become more severe in the crowded cellular environments. 3) T46I only causes a 3-fold affinity reduction to the Nir2 peptide, but a significant elimination of its binding to EphA4. 4) EphA4 and Nir2 peptide appear to have overlapped binding interfaces on the MSP domain, which strongly implies that two signaling networks may have a functional interplay in vivo. 5) As explored by both H/D exchange and MD simulations, the MSP domain is very dynamic, with most loop residues and many residues on secondary structures highly fluctuated or/and exposed to bulk solvent. Although T46I does not alter overall dynamics, it does trigger increased dynamics of several local regions of the MSP domain which are implicated in binding to EphA4 and Nir2 peptide. Our study provides the structural and dynamic understanding of the T46I-causing ALS; and strongly highlights the possibility that the interplay of two signaling networks mediated by the FFAT-containing proteins and Eph receptors may play a key role in ALS pathogenesis.

Dynamically-driven inactivation of the catalytic machinery of the SARS 3C-like protease by the N214A mutation on the extra domain.

Despite utilizing the same chymotrypsin fold to host the catalytic machinery, coronavirus 3C-like proteases (3CLpro) noticeably differ from picornavirus 3C proteases in acquiring an extra helical domain in evolution. Previously, the extra domain was demonstrated to regulate the catalysis of the SARS-CoV 3CLpro by controlling its dimerization. Here, we studied N214A, another mutant with only a doubled dissociation constant but significantly abolished activity. Unexpectedly, N214A still adopts the dimeric structure almost identical to that of the wild-type (WT) enzyme. Thus, we conducted 30-ns molecular dynamics (MD) simulations for N214A, WT, and R298A which we previously characterized to be a monomer with the collapsed catalytic machinery. Remarkably, three proteases display distinctive dynamical behaviors. While in WT, the catalytic machinery stably retains in the activated state; in R298A it remains largely collapsed in the inactivated state, thus implying that two states are not only structurally very distinguishable but also dynamically well separated. Surprisingly, in N214A the catalytic dyad becomes dynamically unstable and many residues constituting the catalytic machinery jump to sample the conformations highly resembling those of R298A. Therefore, the N214A mutation appears to trigger the dramatic change of the enzyme dynamics in the context of the dimeric form which ultimately inactivates the catalytic machinery. The present MD simulations represent the longest reported so far for the SARS-CoV 3CLpro, unveiling that its catalysis is critically dependent on the dynamics, which can be amazingly modulated by the extra domain. Consequently, mediating the dynamics may offer a potential avenue to inhibit the SARS-CoV 3CLpro.

Elimination of the native structure and solubility of the hVAPB MSP domain by the Pro56Ser mutation that causes amyotrophic lateral sclerosis.

The Pro56Ser mutation in the human VAPB MSP domain causes a familial amyotrophic lateral sclerosis. Here we present the first structural investigation of both wild-type and Pro56Ser mutant MSP domains. The results reveal that the wild-type MSP domain is well-folded at neutral pH but can undergo acid-induced unfolding reversibly. It has a thermodynamic stability energy (DeltaG degrees (N-U)) of 7.40 kcal/mol and is also active in binding to a Nir2 peptide with a K(D) of 0.65 muM. Further determination of its crystal structure reveals that it adopts a seven-strand immunoglobulin-like beta sandwich in which Pro56 adopts the unusual cis-peptide bond conformation that appears to be critical in maintaining the characteristic S-shaped loop. Markedly, the Pro56Ser mutation renders the MSP domain insoluble in buffer. Nevertheless, as facilitated by our recent discovery that "insoluble proteins" can be solubilized in salt-free water, we have successfully characterized the residue-specific conformation of the Pro56Ser mutant by CD and heteronuclear NMR spectroscopy. The Pro56Ser mutant remains lacking of the native tight packing and secondary structures under various conditions and was further characterized as having a non-native helical conformation weakly populated at pH 3.5. Intriguingly, Pro12 located in another S-shaped loop also adopts the cis-peptide bond conformation, and its mutation to Ser is able to make the MSP domain highly insoluble and unfolded like the Pro56Ser mutant. Our study thus implies that the Pro56Ser mutation might lead to ALS by eliminating the native MSP structure, which consequently leads to aggregation and loss of functions under physiological conditions.

Identification, recombinant production and structural characterization of four silk proteins from the Asiatic honeybee Apis cerana.

Unlike silkworm and spider silks assembled from very large and repetitive fibrous proteins, the bee and ant silks were recently demonstrated to consist of four small and non-repetitive coiled-coil proteins. The design principle for this silk family remains largely unknown and so far no structural study is available on them in solution. The present study aimed to identify, express and characterize the Asiatic honeybee silk proteins using DLS, CD and NMR spectroscopy. Consequently, (1) four silk proteins are identified, with approximately 6, 10, 9 and 8% variations, respectively, from their European honeybee homologs. Strikingly, their recombinant forms can be produced in Escherichia coil with yields of 10-60 mg/l. (2) Despite containing approximately 65% coiled-coil sequences, four proteins have very low alpha-helix (9-27%) but unusually high random coil (45-56%) contents. Surprisingly, beta-sheet is also detected in four silk proteins (26-35%), implying the possible presence of beta-sheet in the bee and ant silks. (3) Four proteins lacking of the tight tertiary packing appear capable of interacting with each other weakly but this interaction triggers no significant formation of the tight tertiary packing. The study not only implies the promising potential to produce recombinant honeybee silk proteins for the development of various biomaterials; but also provides the first structural insight into the molecular mechanism underlying the formation of the coiled-coil silks.