PapD-like chaperones provide the missing information for folding of pilin proteins.

A fundamental question in molecular biology is how proteins fold into domains that can serve as assembly modules for building up large macromolecular structures. The biogenesis of pili on the surface of Gram-negative bacteria requires the orchestration of a complex process that includes protein synthesis, folding via small chaperones, secretion, and assembly. The results presented here support the hypothesis that pilus subunit folding and biogenesis proceed via mechanisms termed donor strand complementation and donor strand exchange. Here we show that the steric information necessary for pilus subunit folding is not contained in one polypeptide sequence. Rather, the missing information is transiently donated by a strand of a small chaperone to allow folding. Providing the missing information for folding, via a 13-amino acid peptide extension to the C-terminal end of a pilus subunit, resulted in the production of a protein that no longer required the chaperone to fold. This mechanism of small periplasmic chaperone function described here deviates from classical hsp60 chaperone-assisted folding.

Periplasmic chaperone recognition motif of subunits mediates quaternary interactions in the pilus.

The class of proteins collectively known as periplasmic immunoglobulin-like chaperones play an essential role in the assembly of a diverse set of adhesive organelles used by pathogenic strains of Gram-negative bacteria. Herein, we present a combination of genetic and structural data that sheds new light on chaperone-subunit and subunit-subunit interactions in the prototypical P pilus system, and provides new insights into how PapD controls pilus biogenesis. New crystallographic data of PapD with the C-terminal fragment of a subunit suggest a mechanism for how periplasmic chaperones mediate the extraction of pilus subunits from the inner membrane, a prerequisite step for subunit folding. In addition, the conserved N- and C-terminal regions of pilus subunits are shown to participate in the quaternary interactions of the mature pilus following their uncapping by the chaperone. By coupling the folding of subunit proteins to the capping of their nascent assembly surfaces, periplasmic chaperones are thereby able to protect pilus subunits from premature oligomerization until their delivery to the outer membrane assembly site.

The NMR chemical shift pH measurement revisited: analysis of error and modeling of a pH dependent reference.

A standard differential calculus-based propagation of error treatment is applied to the traditional chemical-exchange Henderson-Hasselbalch NMR pH model in which the reference shift is pH independent. It is seen naturally from this analysis that (i) the error minimum in derived pH occurs in the region where pH and indicator pKa are equal and that (ii) the dynamic range, or difference between the limiting chemical shifts of acid and base forms of indicator species, determines the insensitivity of the technique to propagation of errors. To extend the useful pH range and utility of NMR pH determination methodology, a more general model is developed in which the internal reference species is also considered as having a pH-dependent chemical shift. Data from standard solution pH titrations are fitted to both models and parameters are estimated for the normally observed family of ionizable phosphorus metabolites (ATP, inorganic phosphate, phosphoethanolamine and phosphocholine) and the xenometabolite 2-deoxyglucose-6-phosphate with either phosphocreatine, the alpha-phosphate of ATP, or H2O taken as the 31P or 1H chemical shift internal reference species as well as with an external reference.

Tumor 31P NMR pH measurements in vivo: a comparison of inorganic phosphate and intracellular 2-deoxyglucose-6-phosphate as pHnmr indicators in murine radiation-induced fibrosarcoma-1.

Uncertainty regarding the intracellular/extracellular distribution of inorganic phosphate (P(i)) in tumors has raised concerns that pH calculated from the tumor P(i) chemical shift may not accurately represent the intracellular pH (pHin). This issue was addressed in subcutaneously transplanted murine radiation induced fibrosarcoma-1 by directly comparing pH measured via P(i) with pH measured via the in situ generated intracellular xenometabolite 2-deoxyglucose-6-phosphate (2DG6P). In 131 comparative measurements employing eight tumor-bearing mice under both control and hyperglycemic conditions (the latter to extend the range of tumor pH examined), the pH as derived from either 2DG6P or P(i) showed only a small, but statistically significant, difference (0.07 +/- 0.11 SD; P = 0.0001). Scatter in the comparative analysis over the pH range examined (ca. 5.5-7.5) was not uniform. Above pH 6.6, 2DG6P indicated a pH lower than that of P(i) by 0.088 +/- 0.105 SD (n = 107, P = 0.0001); below pH 6.6, 2DG6P indicated a pH essentially identical to and not statistically different from that of P(i) (mean difference 0.003 +/- 0.128 SD (n = 24, P = 0.92)). Evidence is presented in support of this differential arising from a systematic measurement error due to peak overlap between 2DG6P and endogenous phosphomonoester species. These results support the use of P(i) as a tumor 31P NMR pHin indicator, at least in RIF-1 tumors under control and hyperglycemic conditions.

Serial section electron tomography: a method for three-dimensional reconstruction of large structures.

We present a method for combining single axis tomography and serial sectioning techniques to derive a three-dimensional reconstruction of large structures at electron microscopic resolution. This serial-tomography method allows the use of sufficiently thin sections to achieve adequate resolution with electron tomography, yet enables the generation of large reconstructions with considerably fewer sections than would be required using a serial thin section reconstruction technique. Serial thick sections (1-2 microns) are cut through the structure of interest, tomographic volume reconstructions are obtained for each section from a single axis tilt series, and the resulting series of volumes are then aligned and combined to form a single large volume. The serial-tomography method is illustrated with several samples, including red blood cells, the Golgi apparatus, and a spiny dendrite of a cortical pyramidal neuron. In some of these samples, the reconstruction is compared to correlated light microscopic views. The resulting large volume reconstructions appear to represent accurately the size and shape of objects such as red blood cells and spiny dendrites. The continuity of complex, tortuous structures such as the Golgi apparatus is also maintained across serial volumes. These examples demonstrate that it is possible to align and link a series of tomographic volumes accurately and that serial-tomography is a useful method for reconstructing relatively large structures without resorting to large numbers of serial thin sections.