While some members of the ubiquitous DExD/H box family of proteins have RNA helicase activity in vitro, their roles in vivo remain virtually unknown. Here, we show that the function of an otherwise essential DEAD box protein, Prp28p, can be bypassed by mutations that alter either the protein U1-C or the U1 small nuclear RNA. Further analysis suggests that the conserved L13 residue in the U1-C protein makes specific contact to stabilize the U1 snRNA/5' splice site duplex in the prespliceosome, and that Prp28p functions to counteract the stabilizing effect of the U1-C protein, thereby promoting the dissociation of the U1 small nuclear ribonucleoprotein particle from the 5' splice site. Thus, in addition to unwinding RNA, the DExD/H box proteins may affect RNA-RNA rearrangements by antagonizing specific RNA-stabilizing proteins.

beta-Barrel membrane proteins are found in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. Little is known about how residues in membrane beta-barrels interact preferentially with other residues on adjacent strands. We have developed probabilistic models to quantify propensities of residues for different spatial locations and for interstrand pairwise contact interactions involving strong H-bonds, side-chain interactions, and weak H-bonds. Using the reference state of exhaustive permutation of residues within the same beta-strand, the propensity values and p-values measuring statistical significance are calculated exactly by analytical formulae we have developed. Our findings show that there are characteristic preferences of residues for different membrane locations. Contrary to the "positive-inside" rule for helical membrane proteins, beta-barrel membrane proteins follow a significant albeit weaker "positive-outside" rule, in that the basic residues Arg and Lys are disproportionately favored in the extracellular cap region and disfavored in the periplasmic cap region. We find that different residue pairs prefer strong backbone H-bonded interstrand pairings (e.g. Gly-aromatic) or non-H-bonded pairings (e.g. aromatic-aromatic). In addition, we find that Tyr and Phe participate in aromatic rescue by shielding Gly from polar environments. We also show that these propensities can be used to predict the registration of strand pairs, an important task for the structure prediction of beta-barrel membrane proteins. Our accuracy of 44% is considerably better than random (7%). It also significantly outperforms a comparable registration prediction for soluble beta-sheets under similar conditions. Our results imply several experiments that can help to elucidate the mechanisms of in vitro and in vivo folding of beta-barrel membrane proteins. The propensity scales developed in this study will also be useful for computational structure prediction and for folding simulations.

Although the structures of many ␤-barrel membrane proteins are available, our knowledge of the principles that govern their energetics and oligomerization states is incomplete. Here we describe a computational method to study the transmembrane (TM) domains of ␤-barrel membrane proteins. Our method is based on a physical interaction model, a simplified conformational space for efficient enumeration, and an empirical potential function from a detailed combinatorial analysis. Using this method, we can identify weakly stable regions in the TM domain, which are found to be important structural determinants for ␤-barrel membrane proteins. By calculating the melting temperatures of the TM strands, our method can also assess the stability of ␤-barrel membrane proteins. Predictions on membrane enzyme PagP are consistent with recent experimental NMR and mutant studies. We have also discovered that out-clamps, in-plugs, and oligomerization are 3 general mechanisms for stabilizing weakly stable TM regions. In addition, we have found that extended and contiguous weakly stable regions often signal the existence of an oligomer and that strands located in the interfaces of protein-protein interactions are considerably less stable. Based on these observations, we can predict oligomerization states and can identify the interfaces of protein-protein interactions for ␤-barrel membrane proteins by using either structure or sequence information. In a set of 25 nonhomologous proteins with known structures, our method successfully predicted whether a protein forms a monomer or an oligomer with 91% accuracy; in addition, our method identified with 82% accuracy the protein-protein interaction interfaces by using sequence information only when correct strands are given.in-plug ͉ membrane protein oligomerization ͉ out-clamp ͉ protein-protein interaction ͉ weakly stable TM strand D eveloping a general understanding of how proteins behave in membranes is of fundamental importance. ␤-barrel membrane proteins, one of the 2 major structural classes of membrane proteins, have been studied extensively. Currently, structures of 70 ␤-barrel membrane proteins have been resolved, and much has been learned about their thermodynamic stability (1), folding kinetics (2-4), biogenesis (5), and biological functions (6). These membrane proteins are thought to have very regular structures, with the basic principles of their architectural organization well understood (7). An overwhelming structural feature is the existence of an extensive regular hydrogen bond network between the transmembrane (TM) ␤-strands, which is thought to confer extreme stability on the proteins (8).However, ␤-barrel membrane proteins have diverse structures and often deviate significantly from the standard barrel architecture. For example, there are often small ␣-helices and ␤-strands, called in-plugs, found inside the ␤-barrel (9). Nonbarrel-embedded helices are also found to pack against the TM ␤-strands (10). In addition, some ␤-barrel membrane proteins exist in monomeric form, whe...

Objective: To evaluate the association between early (within 10d) pRBC transfusion and the development of severe ROP. Study Design and Methods: This was a single-center retrospective study. Inclusion criteria were preterm infants born ≤ 32 weeks gestation or weighing ≤ 1500g. Severe ROP was defined as infants requiring retinal laser ablation or bevacizumab injection. Logistic regression was used to identify the association between transfusions and severe ROP. Results: A total of 1635 infants were included in the final analysis. The severe ROP incidence was 8% (126/1635). Ninety-one percent (115/126) of infants who developed severe ROP received a pRBC transfusion in the first 10d. Early transfusion was associated with severe ROP; adjusted odds ratio of 3.8 (95% CI: 1.8-8.1). Conclusion: pRBC transfusions in the first 10 days of life are associated with an almost four-fold increased risk of severe ROP, independent of gestational age at birth or bronchopulmonary dysplasia (BPD) status.

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