Understanding GFP posttranslational chemistry: structures of designed variants that achieve backbone fragmentation, hydrolysis, and decarboxylation

The green fluorescent protein (GFP) creates a fluorophore out of three sequential amino acids by promoting spontaneous posttranslational modifications. Here, we use high-resolution crystallography to characterize GFP variants that not only undergo peptide backbone cyclization but additional denaturation-induced peptide backbone fragmentation, native peptide hydrolysis, and decarboxylation reactions. Our analyses indicate that architectural features that favor GFP peptide cyclization also drive peptide hydrolysis. These results are relevant for the maturation pathways of GFP homologues, such as the kindling fluorescent protein and the Kaede protein, which use backbone cleavage to red-shift the spectral properties of their chromophores. We further propose a photochemical mechanism for the decarboxylation reaction, supporting a role for the GFP protein environment in facilitating radical formation and one-electron chemistry, which may be important in activating oxygen for the oxidation step of chromophore biosynthesis. Together, our results characterize GFP posttranslational modification chemistry with implications for the energetic landscape of backbone cyclization and subsequent reactions, and for the rational design of predetermined spontaneous backbone cyclization and cleavage reactions.     

Rational design of a beta-lactamase inhibitor achieved via stabilization of the trans-enamine intermediate: 1.28 A crystal structure of wt SHV-1 complex with a penam sulfone

beta-Lactamases are one of the major causes of antibiotic resistance in Gram negative bacteria. The continuing evolution of beta-lactamases that are capable of hydrolyzing our most potent beta-lactams presents a vexing clinical problem, in particular since a number of them are resistant to inhibitors. The efficient inhibition of these enzymes is therefore of great clinical importance. Building upon our previous structural studies that examined tazobactam trapped as a trans-enamine intermediate in a deacylation deficient SHV variant, we designed a novel penam sulfone derivative that forms a more stable trans-enamine intermediate. We report here the 1.28 A resolution crystal structure of wt SHV-1 in complex with a rationally designed penam sulfone, SA2-13. The compound is covalently bound to the active site of wt SHV-1 similar to tazobactam yet forms an additional salt-bridge with K234 and hydrogen bonds with S130 and T235 to stabilize the trans-enamine intermediate. Kinetic measurements show that SA2-13, once reacted with SHV-1 beta-lactamase, is about 10-fold slower at being released from the enzyme compared to tazobactam. Stabilizing the trans-enamine intermediate represents a novel strategy for the rational design of mechanism-based class A beta-lactamase inhibitors.     

Optimal control landscapes for quantum observables

The optimal control of quantum systems provides the means to achieve the best outcome from redirecting dynamical behavior. Quantum systems for optimal control are characterized by an evolving density matrix and a Hermitian operator associated with the observable of interest. The optimal control landscape is the observable as a functional of the control field. The features of interest over this control landscape consist of the extremum values and their topological character. For controllable finite dimensional quantum systems with no constraints placed on the controls, it is shown that there is only a finite number of distinct values for the extrema, dependent on the spectral degeneracy of the initial and target density matrices. The consequences of these findings for the practical discovery of effective quantum controls in the laboratory is discussed.     

Determination of heavy metals by inductively coupled plasma-optical emission spectrometry in fish from the Piracicaba River in Southern Brazil

A total of 202 fish, representing 16 species, were collected during 2008 (March–October) in the Tanquan region of the Piracicaba River using nets. Flesh samples were collected and analyzed, using inductively coupled plasma-optical emission spectroscopy for Al, As, Cd, Co Cr, Cu, Mn, Mo, Ni, Pb, Se, Sn, Sr, and Zn. The results showed that the flesh of these fish all contained extremely high levels of Al and Sr, and moderately high levels of Cr, As, Zn, Ni, Mn and Pb. The metals were higher in these fish during rainy season, with fish collected during the months of March and October being the highest. In addition, the accumulation of metals was species-dependent. Cascudos (Hypostomus punctatus) and piranhas (Serrasalmus spilopleura) exhibited high levels of almost all of the metals, while curimbata (Prochilodus lineatus) had moderate levels. A few species, including pacu (Piaractus mesopotamicus) and dourado (Salminus maxillosus), had very low levels of most metals. The results show that the Piracicaba River Basin is widely contaminated with high levels of many toxic heavy metals, and that human consumption of some fish species is a human health concern.     

Iterative Denoising

One problem in many fields is knowledge discovery in heterogeneous, high-dimensional data. As an example, in text mining an analyst often wishes to identify meaningful, implicit, and previously unknown information in an unstructured corpus. Lack of metadata and the complexities of document space make this task difficult. We describe Iterative Denoising, a methodology for knowledge discovery in large heterogeneous datasets that allows a user to visualize and to discover potentially meaningful relationships and structures. In addition, we demonstrate the features of this methodology in the analysis of a heterogeneous Science News corpus.     

Efficient, optimal stochastic-action selection when limited by an action budget

The problem that we consider here is a basic operations research problem, but it also a special case of the Stochastic Shortest Path with Recourse Problem and the Canadian Travellers Problem in the probabilistic path planning literature, and it is also a special case of maximizing a submodular set function subject to a matroid constraint. Specifically, suppose an agent has a task and suppose that there is a set of actions, any of which the agent might perform, with respective probabilities of the actions successfully accomplishing the task and respective rewards for the agent if the actions are successful; the agent is to select a sequence of some of these actions that will be performed sequentially, until the task is accomplished or the selected actions are exhausted, but there is a budget on the number of actions that can be performed. We provide an efficient algorithm that chooses a sequence of actions that, under the budget, maximize the agent’s expected reward. An example illustrates how, when conditioning on partial selection of actions, there can be changes to the order of the remaining actions’ adjusted utilities. However, we prove and exploit a nesting result involving solutions.     

Spectral flow and Dixmier traces

We obtain general theorems which enable the calculation of the Dixmier trace in terms of the asymptotics of the zeta function and of the heat operator in a general semi-finite von Neumann algebra. Our results have several applications. We deduce a formula for the Chern character of an odd -summable Breuer-Fredholm module in terms of a Hochschild 1-cycle. We explain how to derive a Wodzicki residue for pseudo-differential operators along the orbits of an ergodic action on a compact space X. Finally, we give a short proof of an index theorem of Lesch for generalised Toeplitz operators.