56 research outputs found
Dimeric SecA Couples the Preprotein Translocation in an Asymmetric Manner
The Sec translocase mediates the post-translational translocation of a number of preproteins through the inner membrane in bacteria. In the initiatory translocation step, SecB targets the preprotein to the translocase by specific interaction with its receptor SecA. The latter is the ATPase of Sec translocase which mediates the post-translational translocation of preprotein through the protein-conducting channel SecYEG in the bacterial inner membrane. We examined the structures of Escherichia coli Sec intermediates in solution as visualized by negatively stained electron microscopy in order to probe the oligomeric states of SecA during this process. The symmetric interaction pattern between the SecA dimer and SecB becomes asymmetric in the presence of proOmpA, and one of the SecA protomers predominantly binds to SecB/proOmpA. Our results suggest that during preprotein translocation, the two SecA protomers are different in structure and may play different roles
Investigating Homology between Proteins using Energetic Profiles
Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding) and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved local stability, may provide guidance for a future thermodynamically informed classification of protein homology
Interference control in children with attention deficit/hyperactivity disorder
The view that Attention Deficit/Hyperactivity Disorder (ADHD) is associated with a diminished ability to control interfference is controversial and based exclusively on results of (verbal)-visual interference tasks, primarily the Stroop Color Word task. The present study compares medication-naĂŻve children with ADHD (nâ=â35 and nâ=â51 in Experiments 1 and 2, respectively) with normal controls (nâ=â26 and nâ=â32, respectively) on two interference tasks to assess interference control in both the auditory and the visual modality: an Auditory Stroop task and a Simon task. Both groups showed reliable but equal degrees of interference on both tasks, suggesting that children with ADHD do not differ from normal controls in their ability to control interference in either modality. © 2008 The Author(s)
SecA, a remarkable nanomachine
Biological cells harbor a variety of molecular machines that carry out mechanical work at the nanoscale. One of these nanomachines is the bacterial motor protein SecA which translocates secretory proteins through the protein-conducting membrane channel SecYEG. SecA converts chemically stored energy in the form of ATP into a mechanical force to drive polypeptide transport through SecYEG and across the cytoplasmic membrane. In order to accommodate a translocating polypeptide chain and to release transmembrane segments of membrane proteins into the lipid bilayer, SecYEG needs to open its central channel and the lateral gate. Recent crystal structures provide a detailed insight into the rearrangements required for channel opening. Here, we review our current understanding of the mode of operation of the SecA motor protein in concert with the dynamic SecYEG channel. We conclude with a new model for SecA-mediated protein translocation that unifies previous conflicting data
De novo purification scheme and crystallization conditions yield high-resolution structures of chitinase A and its complex with the inhibitor allosamidin
The purification scheme of chitinase A (ChiA) from S. marcescens has
been extensively revised. The pure enzyme crystallizes readily under new
crystallization conditions. The ChiA crystal structure has been refined
to 1.55 Angstrom resolution and the crystal structure of ChiA
co-crystallized with the inhibitor allosamidin has been refined to 1.9
Angstrom resolution. Allosamidin is located in the deep active-site
tunnel of ChiA and interacts with three important residues: Glu315, the
proton donor of the catalysis, Asp313, which adopts two conformations in
the native structure but is oriented towards Glu315 in the inhibitor
complex, and Tyr390, which lies opposite Glu315 in the active-site
tunnel
High resolution structural analyses of mutant chitinase A complexes with substrates provide new insight into the mechanism of catalysis
Chitinase A (ChiA) from the bacterium Serratia marcescens is a
hydrolytic enzyme, which cleaves beta -1,4-glycosidic bonds of the
natural biopolymer chitin to generate di-N-acetyl-chitobiose. The
refined structure of ChiA at 1.55 Angstrom shows that residue Asp313,
which is located near the catalytic proton donor residue Glu315, is
found in two alternative conformations of equal occupancy. In addition,
the structures of the cocrystallized mutant proteins D313A, E315Q,
Y390F, and D391A with octa- or hexa- N-acetyl-glucosamine have been
refined at high resolution and the interactions with the substrate have
been characterized. The obtained results clearly show that the active
site is a semiclosed tunnel. Upon binding, the enzyme bends and rotates
the substrate in the vicinity of the scissile bond. Furthermore, the
enzyme imposes a critical âchairâ to âboatâ conformational
change on the sugar residue bound to the - 1 subsite. According to our
results, we suggest that residues Asp313 and Tyr390 along with Glu315
play a central role in the catalysis. We propose that after the
protonation of the substrate glycosidic bond, Asp313 that interacts with
Asp311 flips to its alternative position where it interacts with Glu315
thus forcing the substrate acetamido group of - 1 sugar to rotate around
the C2-N2 bond. As a result of these structural changes, the water
molecule that is hydrogen-bonded to Tyr390 and the NH of the acetamido
group is displaced to a position that allows the completion of
hydrolysis. The presented results suggest a mechanism for ChiA that
modifies the earlier proposed âsubstrate assistedâ catalysis
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