Quantification of H/D Isotope Effects on Protein Hydrogen-bonds by h3JNC′ and 1JNC′ Couplings and Peptide Group 15N and 13C′ Chemical Shifts

The effect of hydrogen/deuterium exchange on protein hydrogen bond coupling constants (h3)J(NC') has been investigated in the small globular protein ubiquitin. The couplings across deuterated or protonated hydrogen bonds were measured by a long-range quantitative HA(CACO)NCO experiment. The analysis is combined with a determination of the H(N)/D(N) isotope effect on the amide group (1)J(NC') couplings and the (15)N and (13)C' chemical shifts. On average, H-bond deuteration exchange weakens (h3)J(NC') and strengthens (1)J(NC') couplings. A correlation is found between the size of the (15)N isotope shift, the (15)N chemical shift, and the (h3)J(NC') coupling constants. The data are consistent with a reduction of donor-acceptor overlap as expected from the classical Ubbelohde effect and the common understanding that H(N)/D(N) exchange leads to a shortening of the N-hydron bond length.     

A tetramer-octamer equilibrium in Mycobacterium leprae and Escherichia coli RuvA by analytical ultracentrifugation

In the context of the bacterial RuvABC system, RuvA protein binds to and is involved in the subsequent processing of a four-way DNA structure called Holliday junction that is formed during homologous recombination. Four crystal structures of RuvA from Escherichia coli (EcoRuvA) showed that it was tetrameric, while neutron scattering and two other crystal structures for RuvA from Mycobacterium leprae (MleRuvA) and EcoRuvA showed that it was an octamer. To clarify this discrepancy, sedimentation equilibrium experiments by analytical ultracentrifugation were carried out and the results showed that MleRuvA existed as a tetramer-octamer equilibrium between 0.2-0.5 mg/ml in 0.1 M NaCl with a dissociation constant of 4 muM, and is octameric at higher concentrations. The same experiments in 0.3 M NaCl showed that MleRuvA is a tetramer up to 3.5 mg/ml, indicating that salt bridges are involved in octamer formation. Sedimentation equilibrium experiments with EcoRuvA showed that it was tetrameric at low concentration in both salt buffers but the protein was insoluble at high-protein concentrations in 0.1 M NaCl. It is concluded that free RuvA exists in an equilibrium between tetrameric and octameric forms in the typical concentration range and buffer found in bacterial cells.     

Future directions in folding: The multi-state nature of protein structure

All possible protein folding intermediates exist in equilibrium with the native protein at native as well as non-native conditions, with occupation determined by their free energy level. The study of these forms can illuminate the fundamental principles of protein structure and folding. Hydrogen exchange methods can be used to detect and characterize these partially unfolded forms at native conditions and as a function of mild denaturant and temperature. This information illuminates the requirements that govern the ability of kinetic and equilibrium methods to study folding intermediates.     

Investigation of a side-chain-side-chain hydrogen bond by mutagenesis, thermodynamics, and NMR spectroscopy.

Anomalous NMR behavior of the hydroxyl proton resonance for Ser 31 has been reported for histidine-containing protein (HPr) from two microorganisms: Escherichia coli and Staphylococcus aureus. The unusual slow exchange and chemical shift exhibited by the resonance led to the proposal that the hydroxyl group is involved in a strong hydrogen bond. To test this hypothesis and to characterize the importance of such an interaction, a mutant in which Ser 31 is replaced by an alanine was generated in HPr from Escherichia coli. The activity, stability, and structure of the mutant HPr were assessed using a reconstituted assay system, analysis of solvent denaturation curves, and NMR, respectively. Substitution of Ser 31 yields a fully functional protein that is only slightly less stable (delta delta G(folding) = 0.46 +/- 0.15 kcal mol-1) than the wild type. The NMR results confirm the identity of the hydrogen bond acceptor as Asp 69 and reveal that it exists as the gauche- conformer in wild-type HPr in solution but exhibits conformational averaging in the mutant protein. The side chain of Asp 69 interacts with two main-chain amide proteins in addition to its interaction with the side chain of Ser 31 in the wild-type protein. These results indicate that removal of the serine has led to the loss of all three hydrogen bond interactions involving Asp 69, suggesting a cooperative network of interactions. A complete analysis of the thermodynamics was performed in which differences in side-chain hydrophobicity and conformational entropy between the two proteins are accounted for.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences between the prion protein and its homolog Doppel: a partially structured state with implications for scrapie formation

The key event in the pathogenesis of prion diseases is a conformational change in the prion protein (PrP). Models for conversion of PrP(C) into PrP(Sc) typically implicate an, as yet, unidentified intermediate. In an attempt to identify such an intermediate, we used native-state hydrogen exchange monitored with NMR. Although we were unable to detect an intermediate directly, we observed substantial protection above that expected based upon measurements of the global stability of PrP (>2 kcal mol(-1) super protection). This super protection implicates either structure in the denatured state or the presence of an intermediate. Similar experiments with Doppel, a homolog of PrP that does not form infectious prions, failed to demonstrate such super protection. This suggests that the partially structured state of PrP encompassing portions of the B and C helices, may be a significant factor in the ability of PrP to convert from PrP(C) to PrP(Sc).     

Structural Characterization of Bardet-Biedl Syndrome 9 Protein (BBS9)

The Bardet-Biedl syndrome protein complex (BBSome) is an octameric complex that transports membrane proteins into the primary cilium signaling organelle in eukaryotes and is implicated in human disease. Here we have analyzed the 99-kDa human BBS9 protein, one of the eight BBSome components. The protein is composed of four structured domains, including a β-stranded N-terminal domain. The 1.8 Å crystal structure of the 46-kDa N-terminal domain reveals a seven-bladed β-propeller. A structure-based homology search suggests that it functions in protein-protein interactions. We show that the Bardet-Biedl syndrome-causing G141R mutation in BBS9 likely results in misfolding of the β-propeller. Although the C-terminal half of BBS9 dimerizes in solution, the N-terminal domain only does so in the crystal lattice. This C-terminal dimerization interface might be important for the assembly of the BBSome.     

Structural Characterization of the Extracellular Domain of CASPR2 and Insights into Its Association with the Novel Ligand Contactin1

Contactin-associated protein-like 2 (CNTNAP2) encodes for CASPR2, a multidomain single transmembrane protein belonging to the neurexin superfamily that has been implicated in a broad range of human phenotypes including autism and language impairment. Using a combination of biophysical techniques, including small angle x-ray scattering, single particle electron microscopy, analytical ultracentrifugation, and bio-layer interferometry, we present novel structural and functional data that relate the architecture of the extracellular domain of CASPR2 to a previously unknown ligand, Contactin1 (CNTN1). Structurally, CASPR2 is highly glycosylated and has an overall compact architecture. Functionally, we show that CASPR2 associates with micromolar affinity with CNTN1 but, under the same conditions, it does not interact with any of the other members of the contactin family. Moreover, by using dissociated hippocampal neurons we show that microbeads loaded with CASPR2, but not with a deletion mutant, co-localize with transfected CNTN1, suggesting that CNTN1 is an endogenous ligand for CASPR2. These data provide novel insights into the structure and function of CASPR2, suggesting a complex role of CASPR2 in the nervous system.     

Comparison of the folding processes of T. thermophilus and E. coli ribonucleases H

In order to examine how the stabilization of thermophilic proteins affects their folding, we have characterized the folding process of Thermus thermophilus ribonuclease H using circular dichroism, fluorescence, and pulse-labeling hydrogen exchange. Like its homolog from Escherichia coli, this thermophilic protein populates a partially folded kinetic intermediate within the first few milliseconds of folding. The structure of this intermediate is similar to that of E.coli RNase H and corresponds remarkably well to a partially folded form that is populated at low levels in the native state of the protein. Proline isomerization appears to partly limit the folding of the thermophilic but not the mesophilic protein. Lastly, unlike other thermophilic proteins, which unfold much more slowly than their mesophilic counterparts, T.thermophilus RNase H folds and unfolds with overall rates similar to those of E.coli RNase H.