Metabolic challenge protocols, such as the oral glucose tolerance test, can uncover early alterations in metabolism preceding chronic diseases. Nevertheless, most metabolomics data accessible today reflect the fasting state. To analyze the dynamics of the human metabolome in response to environmental stimuli, we submitted 15 young healthy male volunteers to a highly controlled 4 d challenge protocol, including 36 h fasting, oral glucose and lipid tests, liquid test meals, physical exercise, and cold stress. Blood, urine, exhaled air, and breath condensate samples were analyzed on up to 56 time points by MS- and NMR-based methods, yielding 275 metabolic traits with a focus on lipids and amino acids. Here, we show that physiological challenges increased interindividual variation even in phenotypically similar volunteers, revealing metabotypes not observable in baseline metabolite profiles; volunteer-specific metabolite concentrations were consistently reflected in various biofluids; and readouts from a systematic model of β-oxidation (e.g., acetylcarnitine/palmitylcarnitine ratio) showed significant and stronger associations with physiological parameters (e.g., fat mass) than absolute metabolite concentrations, indicating that systematic models may aid in understanding individual challenge responses. Due to the multitude of analytical methods, challenges and sample types, our freely available metabolomics data set provides a unique reference for future metabolomics studies and for verification of systems biology models.

Cold shock proteins (Csps) comprise a family of small proteins that are structurally highly conserved and bind to single-stranded nucleic acids via their nucleic acid binding motifs RNP1 and RNP2. Bacterial Csps are mainly induced after a rapid temperature downshift to regulate the adaptation to cold stress, but are also present under normal conditions to regulate other biological functions. The structural unit characteristic for Csps occurs also as a cold shock domain (CSD) in other proteins and can be found in wide variety of organisms from bacteria to vertebrates. Important examples are the Y-box proteins that are known to be involved in regulation of several transcription and translation processes. This review describes the role of Csps in protein expression during cold shock with special emphasis on structural aspects of Csps.

Folding and function of proteins are two aspects of proteins which are usually considered as basically unrelated phenomena that are optimized by evolution independently. From the funnel model of folding/unfolding and the associated energy landscape, we infer the paradigm that the minimum number of folding intermediates is determined by the number of all functional states of a protein ("essential" folding intermediates). Here, we demonstrate the supposed fundamental link using the Ras protein complexed with the GTP analogue GppNHp that occurs in two structural states coexisting in solution. State 2 was shown earlier to represent the effector interacting state, and the function of state 1 was hitherto unknown. By (31)P NMR spectroscopy, we demonstrate that state 1 represents the conformation interacting with guanine nucleotide exchange factors (GEFs). Denaturation experiments of the protein with a chaotropic reagent show that both functional states coexist during folding and unfolding. Application of high pressure represents another perturbation of the energy landscape, leading to an increased population of the state 1 as observed by NMR spectroscopy. The specific volume difference between the two states DeltaV(12) is 17.2 +/- 0.5 mL mol(-1), indicating that state 1 represents a more open conformation of the protein. The free energies of stabilization for state 1 and state 2 at 278 K can be determined as 8.3 and 9.8 kJ mol(-1), respectively.

Cold-shock proteins (Csps) are a subgroup of the coldinduced proteins preferentially expressed in bacteria and other organisms on reduction of the growth temperature below the physiological temperature. They are related to the cold-shock domain found in eukaryotes and are some of the most conserved proteins known. Their exact function is still not known, but translational regulation, possibly via RNA chaperoning, has been discussed.Here we present the structure of a hyperthermophilic member of the Csp family. The NMR solution structure of TmCsp from Thermotoga maritima, the hyperthermophilic member of this class of proteins, was solved on the basis of 1015 conformational constraints. It contains five b strands combined in two antiparallel b sheets making up a b barrel structure, in which b strands 1±4 are arranged in a Greekkey topology. The side chain of R2, which is exclusively found in thermophilic members of the Csp family, probably participates in a peripheral ion cluster involving residues D20, R2, E47 and K63, suggesting that the thermostability of TmCsp is based on the peripheral ion cluster around the side chain of R2.Keywords: cold-shock protein; hyperthermophiles; NMR spectroscopy; structure determination; Thermotoga maritima.The cold-shock response in micro-organisms is a transient phenomenon affecting the growth rate of the cell and the saturation of fatty acids as well as the rate of synthesis of DNA, RNA, and protein at temperatures significantly lower than the normal physiological temperature [1]. For most proteins, the expression under cold shock is dramatically decreased, whereas for a few it is increased. Of the latter, one group of small acidic proteins shows an extremely high induction level and high affinity to single stranded nucleic acids [2]. Because of their high sequence homology, they are classified together in the family of cold-shock proteins (Csps) (reviewed in [3±5]). The biological function is still not known, but translational regulation, possibly via RNA chaperoning, is the possibility currently favored [6].The first hyperthermophilic member of this family has been cloned from Thermotoga maritima (TmCsp); its sequence and physicochemical characteristics were recently reported [7]. TmCsp is a small globular protein of 66 amino acids with a molecular mass of 7474 Da. It exhibits extreme intrinsic stability making it the most thermostable Csp known at present [7].The Csps with known 3D structure are Greek-key b barrel proteins and belong to the`OB-fold family' . For EcCspA, the binding specificity has not yet been determined; for ssDNA the sequences CCAAT and ATTGG seem to be preferred while ssRNA is bound unspecifically. A thorough analysis of the interaction of BsCspB with ssDNA templates revealed that BsCspB preferentially binds to polypyrimidine but not polypurine ssDNA templates [2]. Thymine-based ssDNA templates bind with high-affinity and salt-independently, whereas binding of cytosine-based ssDNA templates is strongly salt-dependent, indicating that a large electrostatic c...

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