Biomedical subjects

Erika B Gebel

Publications and source records attributed to Erika B Gebel.

5 recordsLinked to original sources

PubMed2007

Characterization of denatured proteins using residual dipolar couplings.

The conventional view of denatured proteins as random coils is being challenged by nuclear magnetic resonance (NMR) experiments that can measure the relative orientation of backbone segments with respect to each other in a distance-independent manner. Known as the residual dipolar coupling (RDC), this NMR parameter is measurable on proteins that have been weakly aligned inside a NMR tube. The simple observation of dipolar couplings unequivocally establishes that a denatured protein is not a random coil. Structures of denatured states under differing experimental conditions can be compared by a simple scatter plot of the RDCs measured under each condition; direct structural interpretation, however, is much more problematic. Here some of the technical issues for achieving weak alignment of denatured proteins are addressed using three types of media: strained polyacrylamide gels, both neutral or highly charged, and neutral lipid liquid crystalline bicelles. All three alignment media are experimentally robust and compatible with 8 M urea and low pH, conditions frequently used to denature proteins.

Acrylic Resins↗

PubMed2006

Multiple alignment tensors from a denatured protein.

The structural content of the denatured state has yet to be fully characterized. In recent years, large residual dipolar couplings (RDCs) from denatured proteins have been observed under alignment conditions produced by bicelles and strained polyacrylamide gels. In this report, we describe efforts to extend our picture of the residual structure in denatured nuclease by measuring RDCs with multiple alignment tensors. Backbone amide 15N-1H RDCs were collected from 4 M urea for a total of eight RDC data sets. The RDCs were analyzed by singular value decomposition (SVD) to determine the number of independent alignment tensors present in the data. On the basis of the resultant singular values and propagated error estimates, it is clear that there are at least three independent alignment tensors. These three independent RDC datasets can be reconstituted as orthogonal linear combinations, (OLC)-RDC datasets, of the eight actually recorded. The first, second, and third OLC-RDC datasets are highly robust to the removal of any single experimental RDC dataset, establishing the presence of three independent alignment tensors, sampled well above the level of experimental uncertainty. The observation that the RDC data span three or more dimensions of the five-dimensional parameter space demonstrates that the ensemble average structure of denatured nuclease must be asymmetric with respect to these three orthogonal principal axes, which is not inconsistent with earlier work demonstrating that it has a nativelike topology.

Bacteriophage Pf1↗

PubMed2005

Changes in hydrogen-bond strengths explain reduction potentials in 10 rubredoxin variants.

The rubredoxin from Clostridium pasteurianum (CpRd) provides an excellent system for investigating how the protein sequence modulates the reduction potential of the active site in an iron-sulfur protein. (15)N NMR spectroscopy has allowed us to determine with unprecedented accuracy the strengths of all six key hydrogen bonds between protein backbone amides and the sulfur atoms of the four cysteine residues that ligate the iron in the oxidized (Fe(III)) and reduced (Fe(II)) forms of wild-type CpRd and nine mutants (V44G, V44A, V44I, V44L, V8G, V8A, V8I, V8L, and V8G/V44G). The length (or strength) of each hydrogen bond was inferred from the magnitude of electron spin delocalized across the hydrogen bond from the iron atom onto the nitrogen. The aggregate lengths of these six hydrogen bonds are shorter in both oxidation states in variants with higher reduction potential than in those with lower reduction potential. Differences in aggregate hydrogen bonding upon reduction correlate linearly with the published reduction potentials for the 10 CpRd variants, which span 126 mV. Sequence effects on the reduction potential can be explained fully by their influence on hydrogen-bond strengths.

Binding Sites↗

PubMed2004

Crystallographic studies of V44 mutants of Clostridium pasteurianum rubredoxin: effects of side-chain size on reduction potential.

Understanding the structural origins of differences in reduction potentials is crucial to understanding how various electron transfer proteins modulate their reduction potentials and how they evolve for diverse functional roles. Here, the high-resolution structures of several Clostridium pasteurianum rubredoxin (Cp Rd) variants with changes in the vicinity of the redox site are reported in order to increase this understanding. Our crystal structures of [V44L] (at 1.8 A resolution), [V44A] (1.6 A), [V44G] (2.0 A) and [V44A, G45P] (1.5 A) Rd (all in their oxidized states) show that there is a gradual decrease in the distance between Fe and the amide nitrogen of residue 44 upon reduction in the size of the side chain of residue 44; the decrease occurs from leucine to valine, alanine or glycine and is accompanied by a gradual increase in their reduction potentials. Mutation of Cp Rd at position 44 also changes the hydrogen-bond distance between the amide nitrogen of residue 44 and the sulfur of cysteine 42 in a size-dependent manner. Our results suggest that residue 44 is an important determinant of Rd reduction potential in a manner dictated by side-chain size. Along with the electric dipole moment of the 43-44 peptide bond and the 44-42 NH--S type hydrogen bond, a modulation mechanism for solvent accessibility through residue 41 might regulate the redox reaction of the Rds.

Bacterial Proteins↗

PubMed2003

Correlation between hydrogen bond lengths and reduction potentials in Clostridium pasteurianum rubredoxin.

15N NMR hyperfine-shift data were collected for wild-type and site-specific mutant (V44I, V44A, and V44G) Clostridium pasteurianum rubredoxins in the oxidized state. Whereas most of the (15)N NMR signals did not exhibit large systematic changes upon mutation of residue 44, the signal from the backbone nitrogen of residue 44 itself (arrows) shifted by approximately 400 ppm. These shifts were used to determine the lengths of the hydrogen bond between the backbone amide of residue 44 and the side-chain sulfur of cysteine-44, which is covalently ligated to the iron of the metal center. The results, which demonstrated that this hydrogen bond is shorter in mutants with higher reduction potential, point to the importance of hydrogen bonds in modulating the reduction potential of iron-sulfur proteins.

Clostridium↗