Inactivation of Aβ42 Protomer-dimer Dissociation Reaction via Increasing Protomer Size

Amyloid fibril growth is supposed to be common pathogenic causes for neurodegenerative diseases, triggered by sufficient amounts of growth nuclei species. Since the molecular entity of growth nuclei is regarded as fibril-like aggregates, clarifying the minimum size of thermodynamically stable fibril-like aggregates has been a long standing problem to understand molecular mechanisms of amyloid fibril growth. We studied this problem by examining relationship between the size of fibril-like amyloid-β(1-42) (Aβ42) aggregates and their thermodynamic stability. Seven different protomer dimers were examined as Aβ42 fibril-like aggregate models with employing atomistic molecular dynamics simulations. This study has found that increase of protomer size suppresses conformational fluctuation of these aggregates and inactivates protomer-protomer dissociation reactions by making timescales much longer than mean lifetime of human beings at the point of pentamer dimer formation. This observation shows apparent contribution of protomer size to stabilization of fibril-like aggregates, thus implying that dimer formation of relatively small protomers is a turning point toward growth nuclei formation. Meanwhile, Aβ42 monomer dissociation from the edges of protomers can occur within timescales ranging from microsecond to second and could work for Aβ42 protomer decomposition. This observation implies that suppressing the decomposition route leads to stable Aβ42 growth nuclei formation.

Active-Site Conformational Fluctuations Promote the Enzymatic Activity of NDM-1

ABSTRACT β-Lactam antibiotics are the mainstay for the treatment of bacterial infections. However, elevated resistance to these antibiotics mediated by metallo-β-lactamases (MBLs) has become a global concern. New Delhi metallo-β-lactamase-1 (NDM-1), a newly added member of the MBL family that can hydrolyze almost all β-lactam antibiotics, has rapidly spread all over the world and poses serious clinical threats. Broad-spectrum and mechanism-based inhibitors against all MBLs are highly desired, but the differential mechanisms of MBLs toward different antibiotics pose a great challenge. To facilitate the design of mechanism-based inhibitors, we investigated the active-site conformational changes of NDM-1 through the determination of a series of 15 high-resolution crystal structures in native form and in complex with products and by using biochemical and biophysical studies, site-directed mutagenesis, and molecular dynamics computation. The structural studies reveal the consistency of the active-site conformations in NDM-1/product complexes and the fluctuation in native NDM-1 structures. The enzymatic measurements indicate a correlation between enzymatic activity and the active-site fluctuation, with more fluctuation favoring higher activity. This correlation is further validated by structural and enzymatic studies of the Q123G mutant. Our combinational studies suggest that active-site conformational fluctuation promotes the enzymatic activity of NDM-1, which may guide further mechanism studies and inhibitor design.

Startling temperature effect on proteins when confined: single molecular level behaviour of human serum albumin in a reverse micelle

The present work reports the effect of confinement, and temperature therein, on the conformational fluctuation dynamics of domain-I of human serum albumin (HSA) by fluorescence correlation spectroscopy (FCS).

Interrogating the activities of conformational deformed enzyme by single-molecule fluorescence-magnetic tweezers microscopy

Characterizing the impact of fluctuating enzyme conformation on enzymatic activity is critical in understanding the structure–function relationship and enzymatic reaction dynamics. Different from studying enzyme conformations under a denaturing condition, it is highly informative to manipulate the conformation of an enzyme under an enzymatic reaction condition while monitoring the real-time enzymatic activity changes simultaneously. By perturbing conformation of horseradish peroxidase (HRP) molecules using our home-developed single-molecule total internal reflection magnetic tweezers, we successfully manipulated the enzymatic conformation and probed the enzymatic activity changes of HRP in a catalyzed H2O2–amplex red reaction. We also observed a significant tolerance of the enzyme activity to the enzyme conformational perturbation. Our results provide a further understanding of the relation between enzyme behavior and enzymatic conformational fluctuation, enzyme–substrate interactions, enzyme–substrate active complex formation, and protein folding–binding interactions.

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.