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Articles 1 - 8 of 8

Full-Text Articles in Biochemistry, Biophysics, and Structural Biology

Synchronous Opening And Closing Motions Are Essential For Camp-Dependent Protein Kinase A Signaling, Atul K. Srivastava, Leanna R. Mcdonald, Alessandro Cembran, Jonggul Kim, Larry R. Masterson, Christopher L. Mcclendon, Susan S. Taylor, Gianluigi Veglia Nov 2014

Synchronous Opening And Closing Motions Are Essential For Camp-Dependent Protein Kinase A Signaling, Atul K. Srivastava, Leanna R. Mcdonald, Alessandro Cembran, Jonggul Kim, Larry R. Masterson, Christopher L. Mcclendon, Susan S. Taylor, Gianluigi Veglia

Larry Masterson

Conformational fluctuations play a central role in enzymatic catalysis. However, it is not clear how the rates and the coordination of the motions affect the different catalytic steps. Here, we used NMR spectroscopy to analyze the conformational fluctuations of the catalytic subunit of the cAMP-dependent protein kinase (PKA-C), a ubiquitous enzyme involved in a myriad of cell signaling events. We found that the wild-type enzyme undergoes synchronous motions involving several structural elements located in the small lobe of the kinase, which is responsible for nucleotide binding and release. In contrast, a mutation (Y204A) located far from the active site desynchronizes the opening and …


Characterization Of A Recently Purified Thermophilic Dnase From A Novel Thermophilic Fungus, Kyle Landry, Robert Levin Jul 2014

Characterization Of A Recently Purified Thermophilic Dnase From A Novel Thermophilic Fungus, Kyle Landry, Robert Levin

Kyle S Landry

A newly isolated thermophilic fungus was found to produce a partially inducible extracellular DNase. This manuscript focuses on the characterization of this novel thermophilic DNase in terms of optimal enzyme conditions, molecular weight, and certain kinetic properties. The DNase was found to be inactivated by the presence of EDTA demonstrating its dependence on metal cofactors for activity. Maximum activity occurred at pH 6.0 with no activity at pH 2.0 or 10.0. The optimal temperature for the purified DNase was 65 °C. The thermophilic DNase was found to be an exonuclease with an estimated molecular weight of 56 kDa.


Crystal Structure And Functional Assignment Of Yfau, A Metal Ion Dependent Class Ii Aldolase From Escherichia Coli K12, Dean Rea, Rebecca Hovington, John Rakus, John Gerlt, Vilmos Fu¨Lo¨P, Timothy Bugg, David Roper Jun 2014

Crystal Structure And Functional Assignment Of Yfau, A Metal Ion Dependent Class Ii Aldolase From Escherichia Coli K12, Dean Rea, Rebecca Hovington, John Rakus, John Gerlt, Vilmos Fu¨Lo¨P, Timothy Bugg, David Roper

John F. Rakus

One of the major challenges in the postgenomic era is the functional assignment of proteins using sequence- and structure-based predictive methods coupled with experimental validation. We have used these approaches to investigate the structure and function of theEscherichia coli K-12 protein YfaU, annotated as a putative 4-hydroxy-2-ketoheptane-1,7-dioate aldolase (HpcH) in the sequence databases. HpcH is the final enzyme in the degradation pathway of the aromatic compound homoprotocatechuate. We have determined the crystal structure of apo-YfaU and the Mg2+−pyruvate product complex. Despite greater sequence and structural similarity to HpcH, genomic context suggests YfaU is instead a 2-keto-3-deoxy sugar aldolase like the …


Development Of A Novel Affinity Membrane Purification System For Deoxyribonuclease, Kyle Landry, Robert Levin Jan 2014

Development Of A Novel Affinity Membrane Purification System For Deoxyribonuclease, Kyle Landry, Robert Levin

Kyle S Landry

A membrane based affinity purification system was developed for the purification of the DNA specific nuclease, DNase I. Single stranded DNA was bound to unmodified polyvinylidene fluoride (PVDF) membranes which were used to purify DNase I from a solution of bovine serum albumin. Using coated membranes, a 6-fold increase in specific activity was achieved with 80 % enzyme recovery. This method provides a simple yet effective way to purify DNase I and can be very useful for the purification of other DNA specific enzymes.


Evolution Of A Plant-Specific Copper Chaperone Family For Chloroplast Copper Homeostasis., José Argüello, Crysten Blaby-Haas, Teresita Padilla-Benavides, Roland Stube, Sabeeha Merchant Dec 2013

Evolution Of A Plant-Specific Copper Chaperone Family For Chloroplast Copper Homeostasis., José Argüello, Crysten Blaby-Haas, Teresita Padilla-Benavides, Roland Stube, Sabeeha Merchant

José M. Argüello

Metallochaperones traffic copper (Cu(+)) from its point of entry at the plasma membrane to its destination. In plants, one destination is the chloroplast, which houses plastocyanin, a Cu-dependent electron transfer protein involved in photosynthesis. We present a previously unidentified Cu(+) chaperone that evolved early in the plant lineage by an alternative-splicing event of the pre-mRNA encoding the chloroplast P-type ATPase in Arabidopsis 1 (PAA1). In several land plants, recent duplication events created a separate chaperone-encoding gene coincident with loss of alternative splicing. The plant-specific Cu(+) chaperone delivers Cu(+) with specificity for PAA1, which is flipped in the envelope relative to …


Insights Into The Cation Permeation Pathway Of Channelrhodopsin-2, Robert Dempski, Ryan Richards Dec 2013

Insights Into The Cation Permeation Pathway Of Channelrhodopsin-2, Robert Dempski, Ryan Richards

Robert E. Dempski

Channelrhodopsin-2 (ChR2) is a light-activated, non-selective cation channel endogenous to the green algae Chlamydomonas reinhardtii. The unique properties of ChR2 have made it a useful tool in the field of optogenetics. However, the mechanism of ion conductance is not well resolved. Elucidation of the crystal structure of the channelrhodopsin chimera C1C2 has provided structural insight on the putative ChR2 ion conductance pathway. However, it is not clear how the chimeric structure correlates to ChR2 function.


Purification Of An Inducible Dnase From A Thermophilic Fungus, Kyle Landry, Andrea Vu, Robert Levin Dec 2013

Purification Of An Inducible Dnase From A Thermophilic Fungus, Kyle Landry, Andrea Vu, Robert Levin

Kyle S Landry

The ability to induce an extracellular DNase from a novel thermophilic fungus was studied and the DNAse purified using both traditional and innovative purification techniques. The isolate produced sterile hyphae under all attempted growing conditions, with an average diameter of 2 μm and was found to have an optimal temperature of 45 °C and a maximum of 65 °C. Sequencing of the internal transcribed region resulted in a 91% match with Chaetomium sp., suggesting a new species, but further clarification on this point is needed. The optimal temperature for DNase production was found to be 55 °C and was induced by the …


Purification And Characterization Of Iso-Ribonucleases From A Novel Thermophilic Fungus, Kyle Landry, Robert Levin Dec 2013

Purification And Characterization Of Iso-Ribonucleases From A Novel Thermophilic Fungus, Kyle Landry, Robert Levin

Kyle S Landry

A thermophilic fungus previously isolated from composted horse manure was found to produce extracellular iso-RNases that were purified 127.6-fold using a combination of size exclusion chromatography and a novel affinity membrane purification system. The extent of purification was determined electrophoretically using 4%–15% gradient polyacrylamide gels. RNase activity was dependent on the presence of a metal co-factor with significantly more activity with Zn2+ or Mn2+ than Mg2+. The RNases exhibited maximum activity at both pH 3.0 and pH 7.0 with no activity at pH 2.0 or 10.0. The optimal temperature for the iso-RNase was 70 °C. The …