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Journal Issue - Volume 17 Issue 3 (March 2008)

Abstract Human general transcription factor IIF (TFIIF), a component of the transcription pre‐initiation complex (PIC) associated with RNA polymerase II (Pol II), was characterized by size‐exclusion chromatography (SEC), electrospray ionization mass spectrometry (ESI‐MS), and chemical cross‐linking. Recombinant TFIIF, composed of an equimolar ratio of α and β subunits, was bacterially expressed, purified to homogeneity, and found to...

Abstract A major goal of polymerase research is to determine the mechanism through which a nucleotide complementary to a templating DNA base is selected and delivered to the polymerase active site. Structural evidence suggests a large open‐to‐closed conformational change affecting the fingers subdomain as being crucial to the process. We previously designed a FRET system capable of measuring the rate of fingers subdomain closure in...

Abstract The flavoprotein cholesterol oxidase (CO) from Brevibacterium sterolicum is a monomeric flavoenzyme containing one molecule of FAD cofactor covalently linked to His69. The elimination of the covalent link following the His69Ala substitution was demonstrated to result in a significant decrease in activity, in the midpoint redox potential of the flavin, and in stability with respect to the wild‐type enzyme, but does not modify the...

Abstract The translation of the unspliced and partially spliced viral mRNAs that encode the late, structural proteins of HIV‐1 depends on the viral‐protein Rev. Oligomeric binding of Rev to the Rev response element (RRE) in these mRNAs promotes their export from the nucleus and thus controls their expression. Here, we compared the effects of hydrophobic to hydrophilic mutations within the oligomerization domain of Rev using assays...

Abstract Mammals express two parvalbumins—an α isoform and a β isoform. In rat, the α‐parvalbumin (α‐PV) exhibits superior divalent ion affinity. For example, the standard free energies for Ca2+ binding differ by 5.5 kcal/mol in 0.15 M KCl (pH 7.4). High‐resolution structures of the Ca2+‐bound proteins provide little insight into this disparity, prompting a structural analysis of the apo‐proteins. A recent analysis of rat β‐PV suggested that...

Abstract Electrostatic interactions are important for molecular recognition processes including Ca2+‐binding and cell adhesion. To understand these processes, we have successfully introduced a novel Ca2+‐binding site into the non‐Ca2+‐dependent cell adhesion protein CD2 using our criteria that are specifically tailored to the structural and functional properties of the protein environment and charged adhesion surface. This designed site with ligand...

Abstract The cell wall in Gram‐negative bacteria is surrounded by an outer membrane comprised of charged lipopolysaccharide (LPS) molecules that prevent entry of hydrophobic agents into the cell and protect the bacterium from many antibiotics. The hydrophobic anchor of LPS is lipid A, the biosynthesis of which is essential for bacterial growth and viability. UDP‐3‐O‐(R‐3‐hydroxymyristoyl)‐N‐acetylglucosamine deacetylase (LpxC) is an essential...

Abstract X‐ray crystallography is currently the most successful method for determining the three‐dimensional structure of membrane proteins. Nevertheless, growing the crystals required for this technique presents one of the major bottlenecks in this area of structural biology. This is especially true for the α‐helical type membrane proteins that are of particular interest due to their medical relevance. To address this problem we...

Abstract Protein cysteine thiols can be divided into four groups based on their reactivities: those that form permanent structural disulfide bonds, those that coordinate with metals, those that remain in the reduced state, and those that are susceptible to reversible oxidation. Physicochemical parameters of oxidation‐susceptible protein thiols were organized into a database named the Balanced Oxidation Susceptible Cysteine Thiol...

Abstract We propose a method to extensively characterize the native state ensemble of cyclic cysteine‐rich peptides. The method uses minimal information, namely, amino acid sequence and cyclization, as a topological feature that characterizes the native state. The method does not assume a specific disulfide bond pairing for cysteines and allows the possibility of unpaired cysteines. A detailed view of the conformational space...

Abstract Nucleoporin Nup98, a 98‐kDa protein component of the nuclear pore complex, plays an important role in both protein and RNA transport. During its maturation process, Nup98 undergoes post‐translational autoproteolysis, which is critical for targeting to the NPC. Here we present high‐resolution crystal structures of the C‐terminal autoproteolytic domains of Nup98 (2.3 Å for the wild type and 1.9 Å for the S864A precursor), and...

Abstract A four‐pulse electron paramagnetic resonance experiment was used to measure long‐range inter‐subunit distances in reconstituted KvAP, a voltage‐dependent potassium (Kv) channel. The measurements have allowed us to reach the following five conclusions about the native structure of the voltage sensor of KvAP. First, the S1 helix of the voltage sensor engages in a helix packing interaction with the pore domain. Second, the...

Abstract The ability of proteins to self‐assemble into complex, functional nanoscale structures is expected to become of significant use in the manufacture of artificial nanodevices with a wide range of novel applications. The bacterial protein TRAP has potential uses as a nanoscale component as it is ring‐shaped, with a central, modifiable cavity. Furthermore, it can be engineered to make a ring of 12‐fold symmetry, which is...

Abstract Single‐chain antibodies (scFvs) are comprised of IgG variable light and variable heavy domains tethered together by a peptide linker whose length and sequence can affect antigen binding properties. The ability to modulate antigen binding affinity through the use of environmental triggers would be of great interest for many biotechnological applications. We have characterized the antigen binding properties of an...

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