Chemical rescue of a site-modified ligand to a [4Fe-4S] cluster in PsaC, a bacterial-like dicluster ferredoxin bound to Photosystem I

Chemical rescue of site-modified amino acids using externally supplied organic molecules represents a powerful method to investigate structure-function relationships in proteins. Here we provide definitive evidence that aryl and alkyl thiolates, reagents typically used for in vitro iron-sulfur cluster reconstitutions, serve as rescue ligands to a site-specifically modified [4Fe-4S](1+,2+) cluster in PsaC, a bacterial dicluster ferredoxin-like subunit of Photosystem I. PsaC binds two low-potential [4Fe-4S](1+,2+) clusters termed F(A) and F(B). In the C13G/C33S variant of PsaC, glycine has replaced cysteine at position 13 creating a protein that is missing one of the ligating amino acids to iron-sulfur cluster F(B). Using a variety of analytical techniques, including non-heme iron and acid-labile sulfur assays, and EPR, resonance Raman, and M?ssbauer spectroscopies, we showed that the C13G/C33S variant of PsaC binds two [4Fe-4S](1+,2+) clusters, despite the absence of one of the biological ligands. (19)F NMR spectroscopy indicated that the external thiolate replaces cysteine 13 as a substitute ligand to the F(B) cluster. The finding that site-modified [4Fe-4S](1+,2+) clusters can be chemically rescued with external thiolates opens new opportunities for modulating their properties in proteins. In particular, it provides a mechanism to attach an additional electron transfer cofactor to the protein via a bound, external ligand.     

Robust Concentration and Frequency Control in Oscillatory Homeostats

Homeostatic and adaptive control mechanisms are essential for keeping organisms structurally and functionally stable. Integral feedback is a control theoretic concept which has long been known to keep a controlled variable robustly (i.e. perturbation-independent) at a given set-point by feeding the integrated error back into the process that generates . The classical concept of homeostasis as robust regulation within narrow limits is often considered as unsatisfactory and even incompatible with many biological systems which show sustained oscillations, such as circadian rhythms and oscillatory calcium signaling. Nevertheless, there are many similarities between the biological processes which participate in oscillatory mechanisms and classical homeostatic (non-oscillatory) mechanisms. We have investigated whether biological oscillators can show robust homeostatic and adaptive behaviors, and this paper is an attempt to extend the homeostatic concept to include oscillatory conditions. Based on our previously published kinetic conditions on how to generate biochemical models with robust homeostasis we found two properties, which appear to be of general interest concerning oscillatory and homeostatic controlled biological systems. The first one is the ability of these oscillators (“oscillatory homeostats”) to keep the average level of a controlled variable at a defined set-point by involving compensatory changes in frequency and/or amplitude. The second property is the ability to keep the period/frequency of the oscillator tuned within a certain well-defined range. In this paper we highlight mechanisms that lead to these two properties. The biological applications of these findings are discussed using three examples, the homeostatic aspects during oscillatory calcium and p53 signaling, and the involvement of circadian rhythms in homeostatic regulation.     

Structure of GlnK1 with bound effectors indicates regulatory mechanism for ammonia uptake

A binary complex of the ammonia channel Amt1 from Methanococcus jannaschii and its cognate P(II) signalling protein GlnK1 has been produced and characterized. Complex formation is prevented specifically by the effector molecules Mg-ATP and 2-ketoglutarate. Single-particle electron microscopy of the complex shows that GlnK1 binds on the cytoplasmic side of Amt1. Three high-resolution X-ray structures of GlnK1 indicate that the functionally important T-loop has an extended, flexible conformation in the absence of Mg-ATP, but assumes a compact, tightly folded conformation upon Mg-ATP binding, which in turn creates a 2-ketoglutarate-binding site. We propose a regulatory mechanism by which nitrogen uptake is controlled by the binding of both effector molecules to GlnK1. At normal effector levels, a 2-ketoglutarate molecule binding at the apex of the compact T-loop would prevent complex formation, ensuring uninhibited ammonia uptake. At low levels of Mg-ATP, the extended loops would seal the ammonia channels in the complex. Binding of both effector molecules to P(II) signalling proteins may thus represent an effective feedback mechanism for regulating ammonium uptake through the membrane.     

Native-like conformations are sampled by partially folded and disordered variants of bovine pancreatic trypsin inhibitor

Partially folded conformational ensembles of bovine pancreatic trypsin inhibitor (BPTI) are accessed by replacing Cys 5, 30, 51, and 55 by alpha-amino-n-butyric acid (Abu) while retaining the disulfide between Cys 14 and 38; the resultant variant is termed [14-38](Abu). Two new analogues with modifications in the beta-turn, P26D27[14-38](Abu) and N26G27K28[14-38](Abu), are compared to partially folded [14-38](Abu), as well as to [R](Abu), the unfolded protein with all six Cys residues replaced by Abu. Structural features of the new analogues of [14-38](Abu) have been determined by circular dichroism (CD), one-dimensional (1)H NMR, and 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence experiments. Both analogues are more disordered than the parent [14-38](Abu), but while P26D27[14-38](Abu) has a small population of native-like conformations observed by NMR, no ordered structure is detected for N26G27K28[14-38](Abu). Trypsin inhibition assays were carried out using a modified rat trypsin, C191A/C220A, that minimizes cleavage of unfolded peptides. Both [14-38](Abu) and P26D27[14-38](Abu) significantly inhibit modified trypsin. N26G27K28[14-38](Abu) has low but measurable inhibitor activity, while [R](Abu) has no activity even when in very high molar excess relative to trypsin. ANS fluorescence is enhanced by [14-38](Abu) and by both variants but not by [R](Abu). We conclude that partially folded ensembles of BPTI, even those with little or no CD- or NMR-detectable structure, contain minor populations of native-like conformations. Partially folded [14-38](Abu) and both variants, as well as [R](Abu), have enhanced negative ellipticity in CD spectra acquired in the presence of the osmolyte trimethylamine N-oxide (TMAO). TMAO-induced structure is formed cooperatively, as indicated by thermal unfolding curves. Inhibitor activity as a function of TMAO concentration implies that the osmolyte-induced structure is native-like for [14-38](Abu) and P26D27[14-38](Abu) and is probably native-like for N26G27K28[14-38](Abu). [R](Abu) also shows increased CD-detected structure in the presence of TMAO, but such structure is likely to be collapsed and non-native.