Arteriovenous carboxyhemoglobin difference in critical illness: fiction or fact?

It is still unclear whether the paradoxical arteriovenous carboxyhemoglobin (COHb) difference found in critical illness is due to increased COHb production by the lung, or whether this gradient is caused by technical artifacts using spectrophotometry. In healthy and matched endotoxemic sheep, blood gases were analyzed with a standard ABL 625 and the updated version, an ABL 725. The latter one was accurately calibrated for COHb wavelengths (SAT 100) to eliminate the FCOHb dependency on oxygen tension. All endotoxemic sheep exhibited a hypotensive-hyperdynamic circulation and a pulmonary hypertension. Interestingly, arteriovenous COHb difference occurred in both healthy and endotoxemic sheep (P<0.001 each). Arterial and central venous COHb concentrations determined with the ABL 625 were significantly lower than those measured with the ABL 725 (P<0.001 each). We conclude that (a) arteriovenous COHb difference per se does not reflect critical illness and (b) measurements with an ABL 625 underestimate COHb concentrations.     

E2 conjugating enzymes must disengage from their E1 enzymes before E3-dependent ubiquitin and ubiquitin-like transfer

During ubiquitin ligation, an E2 conjugating enzyme receives ubiquitin from an E1 enzyme and then interacts with an E3 ligase to modify substrates. Competitive binding experiments with three human E2-E3 protein pairs show that the binding of E1s and of E3s to E2s are mutually exclusive. These results imply that polyubiquitination requires recycling of E2 for addition of successive ubiquitins to substrate.     

Temperature-induced transitions of function and structure in sarcoplasmic reticulum membranes

A transition in the temperature dependences of Ca²⁺ accumulation and ATPase activity occurs at 20 ° C in Sarcoplasmic reticulum membranes. The transition is characterized by an abrupt change in the activation energies for the cation transport process and the associated enzyme activities. The difference in activation energies below and above 20 °C appears to be due to changes in the entropy of activation rather than in the free energy of activation. Also, the temperature dependences of spectral parameters of lipophilic spin-labeled probes and protein-bound spin labels exhibit different behaviors on either side of this temperature. Above 20 °C the lipid matrix probed by the labels exhibits a large increase in molecular motion and a decrease in the apparent ordering of lipid alkyl chains. In addition, labels covalently bound to enzymic reactive sites indicate that the motion of protein side-chains is sensitive to this transition. The results are consistent with an order-disorder transition involving the lipid alkyl chains of the Sarcoplasmic membrane, and with a model in which molecular motion, Ca²⁺ transport and enzyme activity are limited by local viscosity of hydrophobic regions at temperatures below the transition.Another modification of the Sarcoplasmic reticulum membrane occurs between 37 and 40 °C. It appears that at this temperature the processes governing Ca²⁺ accumulation and ATPase activity are uncoupled, and Ca²⁺ accumulation is inhibited, while ATPase activity and passive Ca²⁺ efflux proceed at rapid rates. Parallel transitions of spectroscopic parameters originating from spin labels, covalently bound to the Sarcoplasmic reticulum ATPase, indicate that the uncoupling is due to a thermally-induced protein conformational change.     

Computational design of second‐site suppressor mutations at protein–protein interfaces

The importance of a protein–protein interaction to a signaling pathway can be established by showing that amino acid mutations that weaken the interaction disrupt signaling, and that additional mutations that rescue the interaction recover signaling. Identifying rescue mutations, often referred to as second‐site suppressor mutations, controls against scenarios in which the initial deleterious mutation inactivates the protein or disrupts alternative protein–protein interactions. Here, we test a structure‐based protocol for identifying second‐site suppressor mutations that is based on a strategy previously described by Kortemme and Baker. The molecular modeling software Rosetta is used to scan an interface for point mutations that are predicted to weaken binding but can be rescued by mutations on the partner protein. The protocol typically identifies three types of specificity switches: knob‐in‐to‐hole redesigns, switching hydrophobic interactions to hydrogen bond interactions, and replacing polar interactions with nonpolar interactions. Computational predictions were tested with two separate protein complexes; the G‐protein Gα_i1 bound to the RGS14 GoLoco motif, and UbcH7 bound to the ubiquitin ligase E6AP. Eight designs were experimentally tested. Swapping a buried hydrophobic residue with a polar residue dramatically weakened binding affinities. In none of these cases were we able to identify compensating mutations that returned binding to wild‐type affinity, highlighting the challenges inherent in designing buried hydrogen bond networks. The strongest specificity switches were a knob‐in‐to‐hole design (20‐fold) and the replacement of a charge–charge interaction with nonpolar interactions (55‐fold). In two cases, specificity was further tuned by including mutations distant from the initial design. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

A spin-label study of the role of phospholipids in the regulation of membrane-bound microsomal enzymes

The activities of two hepatic microsomal enzymes, glucose 6-phosphatase and UDP-glucuronyltransferase, were determined at assay temperatures in the range between 5 °C and 40 °C. Arrhenius plots of the activities of both enzymes display abrupt changes at about 19 °C. An additional discontinuity at 32 °C is observed in the case of UDP-glucuronyltransferase. Only the latter discontinuity is detected in microsomes subjected to partial treatment with phospholipase A.Lipophilic nitroxide radicals were introduced into samples of the same microsomal preparations and the corresponding electron spin resonance spectra were recorded over the same temperature range. Temperature dependence of an empirical spectral parameter, related to the fluidity of the matrix solubilizing the moleoular probes, reveals apparent breaks at 19 °C and 32 °C in intact microsomes. Only the break at 19 °C was observed in microsomes subjected to sonic disruption. No breaks were detected in plots of data measured in microsomes partially treated with phospholipase A.The correlation between the enzymatic data and the data obtained from lipophilic spin-probes is indicative of the dependence of tightly bound membrane enzymes on the physical state of membrane lipids. The relevance of the data to further studies of the protein-lipid interactions is discussed.     

High-resolution structural and thermodynamic analysis of extreme stabilization of human procarboxypeptidase by computational protein design

Recent efforts to design de novo or redesign the sequence and structure of proteins using computational techniques have met with significant success. Most, if not all, of these computational methodologies attempt to model atomic-level interactions, and hence high-resolution structural characterization of the designed proteins is critical for evaluating the atomic-level accuracy of the underlying design force-fields. We previously used our computational protein design protocol RosettaDesign to completely redesign the sequence of the activation domain of human procarboxypeptidase A2. With 68% of the wild-type sequence changed, the designed protein, AYEdesign, is over 10 kcal/mol more stable than the wild-type protein. Here, we describe the high-resolution crystal structure and solution NMR structure of AYEdesign, which show that the experimentally determined backbone and side-chains conformations are effectively superimposable with the computational model at atomic resolution. To isolate the origins of the remarkable stabilization, we have designed and characterized a new series of procarboxypeptidase mutants that gain significant thermodynamic stability with a minimal number of mutations; one mutant gains more than 5 kcal/mol of stability over the wild-type protein with only four amino acid changes. We explore the relationship between force-field smoothing and conformational sampling by comparing the experimentally determined free energies of the overall design and these focused subsets of mutations to those predicted using modified force-fields, and both fixed and flexible backbone sampling protocols.     

Mis-translation of a computationally designed protein yields an exceptionally stable homodimer: implications for protein engineering and evolution

We recently used computational protein design to create an extremely stable, globular protein, Top7, with a sequence and fold not observed previously in nature. Since Top7 was created in the absence of genetic selection, it provides a rare opportunity to investigate aspects of the cellular protein production and surveillance machinery that are subject to natural selection. Here we show that a portion of the Top7 protein corresponding to the final 49 C-terminal residues is efficiently mis-translated and accumulates at high levels in Escherichia coli. We used circular dichroism, size-exclusion chromatography, small-angle X-ray scattering, analytical ultra-centrifugation, and NMR spectroscopy to show that the resulting C-terminal fragment (CFr) protein adopts a compact, extremely stable, homo-dimeric structure. Based on the solution structure, we engineered an even more stable variant of CFr by disulfide-induced covalent circularisation that should be an excellent platform for design of novel functions. The accumulation of high levels of CFr exposes the high error rate of the protein translation machinery. The rarity of correspondingly stable fragments in natural proteins coupled with the observation that high quality ribosome binding sites are found to occur within E. coli protein-coding regions significantly less often than expected by random chance implies a stringent evolutionary pressure against protein sub-fragments that can independently fold into stable structures. The symmetric self-association between two identical mis-translated CFr sub-domains to generate an extremely stable structure parallels a mechanism for natural protein-fold evolution by modular recombination of protein sub-structures.     

Effects of grazing on plant species diversity and carbon partitioning in semiarid rangelands of northeastern China

Grasslands are one of the most widespread landscapes worldwide, covering approximately one-fifth of the world’s land surface, where grazing is a common practice. How carbon storage responds to grazing in steppes remains poorly understood. We quantified the effects of grazing on community composition and species diversity, and carbon storage in two typical grasslands of northeastern China, one in Horqin and the other one in Hulunbeier. In both grasslands, grazing did not influence plant species diversity. However, it substantially decreased aboveground carbon by 31% and 54% in Horqin and Hulunbeier, respectively. Fenced and grazing treatments showed a similar belowground carbon at both locations. The predominant carbon pool in the study grassland ecosystem was found in the upper 100 cm soil depth, from 98.2 to 99.1% of the total carbon storage. There were no significant effects of grazing on soil carbon neither in the whole profile nor in the uppermost 20 cm soil depth in the two study grasslands. Studies on the effects of varying rangeland management, such as region disparity and grazing systems, may have important consequences on species diversity and carbon partitioning, and thus on rangeland stability and ecosystem functioning.