CRM197 is an enzymatically inactive and nontoxic form of diphtheria toxin that contains a single amino acid substitution (G52E). Being naturally nontoxic, CRM197 is an ideal carrier protein for conjugate vaccines against encapsulated bacteria and is currently used to vaccinate children globally against Haemophilus influenzae, pneumococcus, and meningococcus. To understand the molecular basis for lack of toxicity in CRM197, we determined the crystal structures of the full-length nucleotide-free CRM197 and of CRM197 in complex with the NAD hydrolysis product nicotinamide (NCA), both at 2.0-Å resolution. The structures show for the first time that the overall fold of CRM197 and DT are nearly identical and that the striking functional difference between the two proteins can be explained by a flexible active-site loop that covers the NAD binding pocket. We present the molecular basis for the increased flexibility of the active-site loop in CRM197 as unveiled by molecular dynamics simulations. These structural insights, combined with surface plasmon resonance, NAD hydrolysis, and differential scanning fluorimetry data, contribute to a comprehensive characterization of the vaccine carrier protein, CRM197.

The proteasome-dependent protein degradation participates in multiple essential cellular processes. Modulation of proteasomal activities may alter cardiac function and disease phenotypes. However, cardiovascular studies reported thus far have yielded conflicting results. We hypothesized that a contributing factor to the contradicting literature may be caused by existing proteasome heterogeneity in the myocardium. In this investigation, we provide the very first direct demonstration of distinct proteasome subpopulations in murine hearts. The cardiac proteasome subpopulations differ in their molecular compositions and proteolytic activities. Furthermore they were distinguished from proteasome subpopulations identified in murine livers. The study was facilitated by the development of novel protocols for in-solution isoelectric focusing of multiprotein complexes in a laminar flow that support an average resolution of 0.04 pH units. Utilizing these protocols, the majority of cardiac proteasome complexes displayed an isoelectric point of 5.26 with additional subpopulations focusing in the range from pH 5.10 to 5.33. In contrast, the majority of hepatic 20 S proteasomes had a pI of 5.05 and focused from pH 5.01 to 5.29. Importantly proteasome subpopulations degraded specific model peptides with different turnover rates. Among cardiac subpopulations, proteasomes with an approximate pI of 5.21 showed 40% higher trypsin-like activity than those with pI 5.28. Distinct proteasome assembly may be a contributing factor to variations in proteolytic activities because proteasomes with pI 5.21 contained 58% less of the inducible subunit ␤2i compared with those with pI 5.28. In addition, dephosphorylation of 20 S proteasomes demonstrated that besides molecular composition posttranslational modifications largely contribute to their pI values. These data suggest the possibility of mixed 20 S proteasome assembly, a departure from the currently hypothesized two subpopulations: constitutive and immuno forms. The identification of multiple distinct proteasome subpopulations in heart provides key mechanistic insights for achieving selective and targeted regulation of this essential protein degradation machinery.

The analysis of complex cellular proteomes by means of two-dimensional gel electrophoresis (2-DE) is significantly limited by the power of resolution of this technique. Although subcellular fractionation can be a fundamental first step to increase resolution, it frequently leads to preparations contaminated with other cellular structures. Here, we chose mitochondria of Saccharomyces cerevisiae to demonstrate that an integrated zone-electrophoretic purification step (ZE), with a free-flow electrophoresis device (FFE), can assist in overcoming this problem, while significantly improving their degree of purity. Whereas mitochondrial preparations isolated by means of differential centrifugation include a considerable degree of non-mitochondrial proteins (16%), this contamination could be effectually removed by the inclusion of a ZE-FFE purification step (2%). This higher degree of purity led to the identification of many more proteins from ZE-FFE purified mitochondrial protein extracts (n = 129), compared to mitochondrial protein extracts isolated by differential centrifugation (n = 80). Moreover, a marked decrease of degraded proteins was found in the ZE-FFE purified mitochondrial protein extracts. It is noteworthy that even at a low 2-DE resolution level, a four-fold higher number (17 versus 4) of presumably low abundance proteins could be identified in the ZE-FFE purified mitochondrial protein extracts. Therefore these results represent a feasible approach for an in-depth proteome analysis of mitochondria and possibly other organelles.

Using antibody-related methods, we recently found that human thyroid cells express various proteins differently depending on whether they are cultured under normal gravity (1g) or simulated microgravity (s-mg). In this study, we performed proteome analysis in order to identify more gravity-sensitive thyroid proteins. Cells cultured under 1g or s-mg conditions were sonicated. Proteins released into the supernatant and those remaining in the cell fragments were fractionated by free-flow IEF. The fractions obtained were further separated by SDS-gel electrophoresis. Selected gel pieces were excised and their proteins were determined by MS. A total of 235 different proteins were found. Out of 235 proteins, 37 appeared to be first identifications in human thyroid cells. Comparing SDS gel lanes of equally numbered free-flow IEF fractions revealed similar patterns with a number of identical bands if proteins of a distinct cell line had been applied, irrespective of whether the cells had been cultured under 1g or s-mg. Most of the identical band pairs contained identical proteins. However, the concentrations of some types of proteins were different within the two pieces of gel. Proteins that concentrated differently in such pieces of gel are considered as candidates for further investigations of gravitational sensitivity.

The cytochrome p450 enzyme, CYP2D6, metabolises approximately 20% of marketed drugs. CYP2D6 multiple variants are associated with altered enzyme activities. Genotyping 1018 Caucasians for CYP2D6 polymorphisms (G1846A, delT1707, delA2549 and A2935C), known to result in the recessive CYP2D6 poor drug metaboliser (PM) phenotype, identified 41 individuals with predicted PM phenotype. These 41 individuals were classified as 'cases'. Single nucleotide polymorphisms (SNPs) mapping within an 880 kb region flanking CYP2D6, were identified to evaluate potential association between genetic variation and the CYP2D6 PM phenotype. The 41 PM cases and 977 controls were genotyped and analysed for 27 SNPs. Associations were observed across a 390 kb region between 14 SNPs and the PM phenotype (P values from 6.20 ϫ 10 Ϫ4 to 4.54 ϫ 10 Ϫ35 ). Haplotype analysis revealed more significant levels of association (P ϭ 3.54 ϫ 10 Ϫ56 ). Strong (DЈ Ͼ 0.7) linkage disequilibrium (LD) between SNPs was observed across the same 390 kb region associated with the CYP2D6 phenotype. The observed phenotype:genotype association reached genome-wide levels of significance, and supports the strategy for potential application of LD mapping and whole genome association scans to pharmacogenetic studies.