Visualization of Early Events in Acetic Acid Denaturation of HIV-1 Protease: A Molecular Dynamics Study

Protein denaturation plays a crucial role in cellular processes. In this study, denaturation of HIV-1 Protease (PR) was investigated by all-atom MD simulations in explicit solvent. The PR dimer and monomer were simulated separately in 9 M acetic acid (9 M AcOH) solution and water to study the denaturation process of PR in acetic acid environment. Direct visualization of the denaturation dynamics that is readily available from such simulations has been presented. Our simulations in 9 M AcOH reveal that the PR denaturation begins by separation of dimer into intact monomers and it is only after this separation that the monomer units start denaturing. The denaturation of the monomers is flagged off by the loss of crucial interactions between the α-helix at C-terminal and surrounding β-strands. This causes the structure to transit from the equilibrium dynamics to random non-equilibrating dynamics. Residence time calculations indicate that denaturation occurs via direct interaction of the acetic acid molecules with certain regions of the protein in 9 M AcOH. All these observations have helped to decipher a picture of the early events in acetic acid denaturation of PR and have illustrated that the α-helix and the β-sheet at the C-terminus of a native and functional PR dimer should maintain both the stability and the function of the enzyme and thus present newer targets for blocking PR function

Dynamic association of PfEMP1 and KAHRP in knobs mediates cytoadherence during Plasmodium invasion

Plasmodium falciparum infected erythrocytes display membrane knobs that are essential for their adherence to vascular endothelia and for prevention of clearance by the spleen. The knob associated histidine rich protein (KAHRP) is indispensable to knob formation and has been implicated in the recruitment and tethering of P. falciparum erythrocyte membrane protein-1 (PfEMP1) by binding to its cytoplasmic domain termed VARC. However, the precise mechanism of interaction between KAHRP and VARC is not very well understood. Here we report that both the proteins co-localize to membrane knobs of P. falciparum infected erythrocytes and have identified four positively charged linear sequence motifs of high intrinsic mobility on KAHRP that interact electrostatically with VARC in solution to form a fuzzy complex. The current study provides molecular insight into interaction between KAHRP and VARC in solution that takes place at membrane knobs

Molecular Insights into Mammalian End-binding Protein Heterodimerization

Microtubule plus-end tracking proteins (+TIPs) are involved in many microtubule-based processes. End binding (EB) proteins constitute a highly conserved family of +TIPs. They play a pivotal role in regulating microtubule dynamics and in the recruitment of diverse +TIPs to growing microtubule plus ends. Here we used a combination of methods to investigate the dimerization properties of the three human EB proteins EB1, EB2, and EB3. Based on Forster resonance energy transfer, we demonstrate that the C-terminal dimerization domains of EBs (EBc) can readily exchange their chains in solution. We further document that EB1c and EB3c preferentially form heterodimers, whereas EB2c does not participate significantly in the formation of heterotypic complexes. Measurements of the reaction thermodynamics and kinetics, homology modeling, and mutagenesis provide details of the molecular determinants of homo-versus heterodimer formation of EBc domains. Fluorescence spectroscopy and nuclear magnetic resonance studies in the presence of the cytoskeleton-associated protein-glycine-rich domains of either CLIP-170 or p150(glued) or of a fragment derived from the adenomatous polyposis coli tumor suppressor protein show that chain exchange of EBc domains can be controlled by binding partners. Extension of these studies of the EBc domains to full-length EBs demonstrate that heterodimer formation between EB1 and EB3, but not between EB2 and the other two EBs, occurs both in vitro and in cells as revealed by live cell imaging. Together, our data provide molecular insights for rationalizing the dominant negative control by C-terminal EB domains and form a basis for understanding the functional role of heterotypic chain exchange by EBs in cells

The extremely slow-exchanging core and acid-denatured state of green fluorescent protein

Green fluorescent protein (GFP) is a large protein with a complex eleven-stranded beta-barrel structure. Previous studies have shown that it has a complex energy landscape for folding on which there are several intermediate states and a denatured state with significant residual structure. Here, we use two different types of H/D exchange measurement and nuclear magnetic resonance (NMR) techniques to probe the energy landscape for folding of GFP in further detail. H/D exchange experiments were performed over a wide range of conditions including different concentrations of denaturant. Results show that the penetration model dominates the exchange mechanism, consistent with the known stability and slow unfolding kinetics of GFP. H/D exchange experiments at high pH establish that there is an extremely slow-exchanging superstable core of amide protons in GFP that are clustered and located in beta-strands 1, 2, 4, 5, and 6. These residues form part of a mini-beta-sheet which we propose constitutes a folding nucleus. Using a pulsed-labeling strategy, the acid-denatured state has been investigated and the residual structure observed in earlier studies shown to locate to beta-strands 1 and 3. There is some evidence that this residual structure is stabilized by a localized hydrophobic collapse of the polypeptide chain. ©2008 HFSP Publishin