Proteins can exist in a trinity of structures: the ordered state, the molten globule, and the random coil. The five following examples suggest that native protein structure can correspond to any of the three states (not just the ordered state) and that protein function can arise from any of the three states and their transitions. (1) In a process that likely mimics infection, fd phage converts from the ordered into the disordered molten globular state. (2) Nucleosome hyperacetylation is crucial to DNA replication and transcription; this chemical modification greatly increases the net negative charge of the nucleosome core particle. We propose that the increased charge imbalance promotes its conversion to a much less rigid form. (3) Clusterin contains an ordered domain and also a native molten globular region. The molten globular domain likely functions as a proteinaceous detergent for cell remodeling and removal of apoptotic debris. (4) In a critical signaling event, a helix in calcineurin becomes bound and surrounded by calmodulin, thereby turning on calcineurin's serine/threonine phosphatase activity. Locating the calcineurin helix within a region of disorder is essential for enabling calmodulin to surround its target upon binding. (5) Calsequestrin regulates calcium levels in the sarcoplasmic reticulum by binding approximately 50 ions/molecule. Disordered polyanion tails at the carboxy terminus bind many of these calcium ions, perhaps without adopting a unique structure. In addition to these examples, we will discuss 16 more proteins with native disorder. These disordered regions include molecular recognition domains, protein folding inhibitors, flexible linkers, entropic springs, entropic clocks, and entropic bristles. Motivated by such examples of intrinsic disorder, we are studying the relationships between amino acid sequence and order/disorder, and from this information we are predicting intrinsic order/disorder from amino acid sequence. The sequence-structure relationships indicate that disorder is an encoded property, and the predictions strongly suggest that proteins in nature are much richer in intrinsic disorder than are those in the Protein Data Bank. Recent predictions on 29 genomes indicate that proteins from eucaryotes apparently have more intrinsic disorder than those from either bacteria or archaea, with typically > 30% of eucaryotic proteins having disordered regions of length > or = 50 consecutive residues.

Intrinsically Disordered Proteins

Even under optimal conditions, many metabolic processes, including the chloroplastic, mitochondrial, and plasma membrane-linked electron transport systems of higher plants, produce active oxygen species (AOS). Furthermore, the imposition of biotic and abiotic stress conditions can give rise to excess concentrations of AOS, resulting in oxidative damage at the cellular level. Therefore, antioxidants and antioxidant enzymes function to interrupt the cascades of uncontrolled oxidation in each organelle. Ascorbate peroxidase (APX) exists as isoenzymes and plays an important role in the metabolism of H(2)O(2) in higher plants. APX is also found in eukaryotic algae. The characterization of APX isoenzymes and the sequence analysis of their clones have led to a number of investigations that have yielded interesting and novel information on these enzymes. Interestingly, APX isoenzymes of chloroplasts in higher plants are encoded by only one gene, and their mRNAs are generated by alternative splicing of the gene's two 3'-terminal exons. Manipulation of the expression of the enzymes involved in the AOS-scavenging systems by gene-transfer technology has provided a powerful tool for increasing the present understanding of the potential of the defence network against oxidative damage caused by environmental stresses. Transgenic plants expressing E. coli catalase to chloroplasts with increased tolerance to oxidative stress indicate that AOS-scavenging enzymes, especially chloroplastic APX isoenzymes are sensitive under oxidative stress conditions. It is clear that a high level of endogenous ascorbate is essential effectively to maintain the antioxidant system that protects plants from oxidative damage due to biotic and abiotic stresses.

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Regulation and function of ascorbate peroxidase isoenzymes

Publisher Summary This chapter discusses the chemical analysis of microbial cells. The preparation of material for analysis is discussed, because changes in the chemical composition of cells may occur as a result of the washing and storage conditions used. Wet- and dry-weight determinations of bacterial samples and assay of total cell numbers are described, because analytical results must refer to one or other of these values. Selection of an analytical procedure is a subjective process, because the number of suitable methods is large and each will have different merits and defects. Primary considerations are sensitivity, specificity, reproducibility, and absolute accuracy. Automatic methods for performing biochemical analyses, already widely accepted in hospitals and in industry, are beginning to make their way into the research laboratory. All automatic analyzers developed so far may be classified as either “continuous-flow” or “discrete” types. All of them use colorimetric methods exclusively and contain some form of automatic colorimeter for final read-out. The first and best-known is the Technicon “AutoAnalyzer,” which is of the continuous-flow type.

Chapter III Chemical Analysis of Microbial Cells

Current single-molecule detection techniques require labeling the target molecule. We report a highly specific and sensitive optical sensor based on an ultrahigh quality (Q) factor (Q > 10^8) whispering-gallery microcavity. The silica surface is functionalized to bind the target molecule; binding is detected by a resonant wavelength shift. Single-molecule detection is confirmed by observation of single-molecule binding events that shift the resonant frequency, as well as by the statistics for these shifts over many binding events. These shifts result from a thermo-optic mechanism. Additionally, label-free, single-molecule detection of interleukin-2 was demonstrated in serum. These experiments demonstrate a dynamic range of 10^(12) in concentration, establishing the microcavity as a sensitive and versatile detector.

/pdf/label-free-single-molecule-detection-with-optical-13uzbeuo9v.pdf

Label-Free, Single-Molecule Detection with Optical Microcavities

Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

/pdf/structures-of-cas9-endonucleases-reveal-rna-mediated-e5eo54au8k.pdf

Structures of Cas9 Endonucleases Reveal RNA- Mediated Conformational Activation

THE JOURNAL OF BIOLOGICAL CHEMISTRY: The Biochemical Behavior of LeadL I. Influence of Calcium, Phosphorus, and Vitamin D on Lead in Blood and Bone

A simple technique for the determination of calcium and magnesium in small amounts of serum

A clinical method for the quantitative determination of potassium in small amounts of serum

At the meeting of the American Pediatric Society held in June, 1920, one of us, in discussing a paper by Park and Howland 1 on the effect of cod liver oil on rachitic bones as demonstrated by the roentgenogram, reported that we had found the concentration of the inorganic phosphorus of the serum to be low in rickets. The amount was definitely increased following the administration of cod liver oil. The present communication represents the work on which these statements were based. Rickets is a disease characterized by a deficient deposition of calcium salts in the bones themselves and in the intercellular cartilage of the epiphyses. Various views have been expressed and various theories advanced to explain the failure of deposition. We have approached the question from the standpoint of the inorganic chemical composition of the blood serum. Inasmuch as the salts of the bones consist preponderantly of calcium phosphate,

Calcium and phosphorus in the serum in relation to rickets

THE object of these experiments was to determine the conditions under which calcification occurs in vitro, and also what factors interfere with calcification. If it can be shown that the position and the character of the calcification, in other words the histological picture, are exactly the same as those occurring in vivo then it would seem justifiable to draw certain deductions regarding in vivo calcification from in vitro experiments. Per contra, if certain factors prevent calcification in vitro, and if it can be shown that these same factors are operative in diseases such as rickets, in which there is a failure of calcification, then it would appear justifiable to explain the failure of calcification by the influence of these factors. The basis for these experiments was the observation of Shipley [1924] that, when the cartilage and bone of a rachitic rat are placed in the serum of rachitic rats and incubated for a number of hours, no calcification occurs. On the other hand, when a piece of cartilage and bone from the same animal is placed in the serum of normal rats and incubated for the same length of time, calcification, similar to that taking place in an animal undergoing cure, can be demonstrated. It is obvious that this method is applicable to the study of various aspects of calcification and for this it has been employed. In previous studies of rickets from this department it has been shown that, in uncomplicated rickets, there is regularly a low concentration of inorganic phosphorus in the serum, 1-5 to 3.5 mg. per 100 cc., as opposed to the normal of 5 to 6 mg. Studies in conjunction with the School of Hygiene have demonstrated that rats can be rendered rachitic by feeding them a diet very low in calcium and relatively high in phosphorus or high in calcium and low in phosphorus (McCollum's 3143 diet). The low phosphorus diet is the most satisfactory one to use, for it produces a very severe rickets with great regularity. The low phosphorus in the diet is reflected in the low inorganic phosphorus of the serum and, instead of the normal 8 mg. of inorganic phosphorus in the serum, the rachitic rat has usually 2-5 to 3 mg. per 100 cc. It has further

Benjamin Kramer

Papers

THE JOURNAL OF BIOLOGICAL CHEMISTRY: The Biochemical Behavior of LeadL I. Influence of Calcium, Phosphorus, and Vitamin D on Lead in Blood and Bone

A simple technique for the determination of calcium and magnesium in small amounts of serum

A clinical method for the quantitative determination of potassium in small amounts of serum

Calcium and phosphorus in the serum in relation to rickets

Studies upon Calcification in vitro.