Skp is a multivalent chaperone of outer membrane proteins

The trimeric chaperone Skp sequesters outer-membrane proteins (OMPs) within a hydrophobic cage, thereby preventing their aggregation during transport across the periplasm in Gram-negative bacteria. Here, we studied the interaction between Escherichia coli Skp and five OMPs of varying size. Investigations of the kinetics of OMP folding revealed that higher Skp/OMP ratios are required to prevent the folding of 16-stranded OMPs compared with their 8-stranded counterparts. Ion mobility spectrometry–mass spectrometry (IMS–MS) data, computer modeling and molecular dynamics simulations provided evidence that 10- to 16-stranded OMPs are encapsulated within an expanded Skp substrate cage. For OMPs that cannot be fully accommodated in the expanded cavity, sequestration is achieved by binding of an additional Skp trimer. The results suggest a new mechanism for Skp chaperone activity involving the coordination of multiple copies of Skp in protecting a single substrate from aggregation

Effects of Periplasmic Chaperones and Membrane Thickness on BamA-Catalyzed Outer-Membrane Protein Folding

The biogenesis of outer-membrane proteins (OMPs) in gram-negative bacteria involves delivery by periplasmic chaperones to the β-barrel assembly machinery (BAM), which catalyzes OMP insertion into the outer membrane. Here, we examine the effects of membrane thickness, the Escherichia coli periplasmic chaperones Skp and SurA, and BamA, the central subunit of the BAM complex, on the folding kinetics of a model OMP (tOmpA) using fluorescence spectroscopy, native mass spectrometry, and molecular dynamics simulations. We show that prefolded BamA promotes the release of tOmpA from Skp despite the nM affinity of the Skp:tOmpA complex. This activity is located in the BamA β-barrel domain, but is greater when full-length BamA is present, indicating that both the β-barrel and polypeptide transport-associated (POTRA) domains are required for maximal activity. By contrast, SurA is unable to release tOmpA from Skp, providing direct evidence against a sequential chaperone model. By varying lipid acyl chain length in synthetic liposomes we show that BamA has a greater catalytic effect on tOmpA folding in thicker bilayers, suggesting that BAM catalysis involves lowering of the kinetic barrier imposed by the hydrophobic thickness of the membrane. Consistent with this, molecular dynamics simulations reveal that increases in membrane thinning/disorder by the transmembrane domain of BamA is greatest in thicker bilayers. Finally, we demonstrate that cross-linking of the BamA barrel does not affect tOmpA folding kinetics in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes, suggesting that lateral gating of the BamA barrel and/or hybrid barrel formation is not required, at least for the assembly of a small 8-stranded OMP in vitro

Network Databases: Past, Present, and Future

The purpose of this paper is to review the past, present, and future in terms of three "generations" of network databases.  Particular emphasis is placed upon the Internet and the booming explosion of restricted-access subsets of the Internet known as intranets.  The Internet is a collection of millions upon millions of local, regional, national, and global networks.  It commenced in 1969 with four supercomputers across the U. S. networked to the Pentagon.  Very few people used the Internet for the first 20 years.  Explosive growth took place after 1992 when the Netscape Navigator web browser incorporated HTML protocols to read HTML codes which were invented by particle physicists in Switzerland in 1990.  This was the beginning of the "first generation" of network computing on what became known as the world wide web (WWW) or simply the "web."  The first generation was mainly one way flows of information from web server computers to client user computers on the web.  At this same time, the first generation of database interactive computing was confined to local and wide area networks (LANs and WANs).  Although data files could be transmitted across the Internet using FTP and other protocols, databases could not interact on the WWW or the Internet as a whole.  Most web applications are still in the first HTML generation.  The second generation of networking and databases followed quickly when web servers became able to interact in a more formal way with remote clients on the Internet.  With special types of middleware software, database servers could process data transmitted from remote Internet client computers.  For example, customer orders and market surveys could be processed and server-side databases could be updated without human intervention.  Middleware CGI scripting and later ActiveX and Java software enabled web servers, database servers, and remote clients on the Internet to become more interactive.  The second generation is relatively new and growing in popularity at this time.  The third generation is only just emerging and is best described as distributed network computing.  In the second generation, middleware updates "front ends" of database servers on the server side when clients transmit signals.  In the third generation, databases can be distributed globally and can communicate with each other with "back-end" distributed network computing.  There is virtually no difference between having all databases on one computer with one operator versus having databases on 100 computers with 100 operators residing anywhere in the world

Deed from J. L. and F. E. Darby to R. C. Sandlin, Coosa County, Alabama, February 15, 1878

An item from the papers of Barrie Lucien Holt. These papers cover Holt's life as a capitalist, planter, financier and Quartermaster General of the Alabama National Guard. Holt was an organizer and majority shareholder in the Prattville Cotton Mills and Banking Company, as well as a partner in D.M. Snow and Company which owned a hardware store and engaged in money-lending

Deed from William E. and M. C. Moore to R. C. Sandlin, Coosa County, Alabama, March 15, 1877

An item from the papers of Barrie Lucien Holt. These papers cover Holt's life as a capitalist, planter, financier and Quartermaster General of the Alabama National Guard. Holt was an organizer and majority shareholder in the Prattville Cotton Mills and Banking Company, as well as a partner in D.M. Snow and Company which owned a hardware store and engaged in money-lending

Warranty Deed from A. P. and E. R. Harris to R. C. Sandlin, Coosa County, Alabama, October 12, 1885

An item from the papers of Barrie Lucien Holt. These papers cover Holt's life as a capitalist, planter, financier and Quartermaster General of the Alabama National Guard. Holt was an organizer and majority shareholder in the Prattville Cotton Mills and Banking Company, as well as a partner in D.M. Snow and Company which owned a hardware store and engaged in money-lending

Deed from A. M. and E. J. Kendrick to R. C. Sandlin, Coosa County, Alabama, June 27, 1874

An item from the papers of Barrie Lucien Holt. These papers cover Holt's life as a capitalist, planter, financier and Quartermaster General of the Alabama National Guard. Holt was an organizer and majority shareholder in the Prattville Cotton Mills and Banking Company, as well as a partner in D.M. Snow and Company which owned a hardware store and engaged in money-lending

Deed from C. J. and E. J. Girdner to R. C. Sandlin, Coosa County, Alabama, February 8, 1879

An item from the papers of Barrie Lucien Holt. These papers cover Holt's life as a capitalist, planter, financier and Quartermaster General of the Alabama National Guard. Holt was an organizer and majority shareholder in the Prattville Cotton Mills and Banking Company, as well as a partner in D.M. Snow and Company which owned a hardware store and engaged in money-lending