Newly synthesized proteins must form their native structures in the crowded environment of the cell, while avoiding non-native conformations that can lead to aggregation. Yet remarkably little is known about the progressive folding of polypeptide chains during chain synthesis by the ribosome, or of the influence of this folding environment on productive folding in vivo. P22 tailspike is a homotrimeric protein that is prone to aggregation via misfolding of its central β-helix domain in vitro. We have produced stalled ribosome:tailspike nascent chain complexes of four fixed lengths in vivo, in order to assess co-translational folding of newly synthesized tailspike chains as a function of chain length. Partially synthesized, ribosome-bound nascent tailspike chains populate stable conformations with some native-state structural features even prior to the appearance of the entire β-helix domain, regardless of the presence of the chaperone trigger factor, yet these conformations are distinct from the conformations of released, refolded tailspike truncations. These results suggest that organization of the aggregation-prone β-helix domain occurs co-translationally, prior to chain release, to a conformation that is distinct from the accessible energy minimum conformation for the truncated free chain in solution.As a protein is synthesized by the ribosome, it begins to fold into a three-dimensional shape, and must simultaneously avoid aggregation with other proteins in the crowded cell. In this seemingly hostile environment, where total protein concentrations can exceed 200-300 mg/ mL, de novo protein folding during and after translation is, for many proteins, more efficient than in vitro refolding1. In other words, many proteins that can fold productively in the cell will aggregate severely under in vitro refolding reactions, presumably due to differences between the dominant folding pathway used. For example, experiments with both bacterial and firefly luciferase have demonstrated that these nascent chains adopt conformations cotranslationally that are not populated during refolding from denaturant2,3, and these cotranslational conformations fold to the native state much more efficiently than the conformations populated during refolding from denaturant. However, there continues to be debate as to what extent large, multi-domain proteins can fold co-translationally in prokaryotes4-7.While molecular chaperones certainly play a role in efficient protein folding in the cell, less than 20% of E. coli cytoplasmic proteins require an interaction with one of the three major chaperone systems (trigger factor (TF), DnaK/DnaJ, or GroEL/ES) in order to fold correctly under normal growth conditions8. And remarkably, both chaperone systems responsible for Correspondence should be addressed to P.L.C. (pclark1@nd.edu). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript wil...

Cotranslational protein maturation is often studied in cell-free translation mixtures, using stalled ribosome-nascent chain complexes produced by translating truncated mRNA. This approach has two limitations: (i) it can be technically challenging, and (ii) it only works in vitro, where the concentrations of cellular components differ from concentrations in vivo. We have developed a method to produce stalled ribosomes bearing nascent chains of a specified length by using a 'stall sequence', derived from the Escherichia coli SecM protein, which interacts with residues in the ribosomal exit tunnel to stall SecM translation. When the stall sequence is expressed at the end of nascent chains, stable translation-arrested ribosome complexes accumulate in intact cells or cell-free extracts. SecM-directed stalling is efficient, with negligible effects on viability. This method is straightforward and suitable for producing stalled ribosome complexes in vivo, permitting study of the length-dependent maturation of nascent chains in the cellular milieu.

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While in vitro experiments have contributed much to our understanding of protein folding, we know much less about how proteins fold in the more complex environment of the cell. This review summarizes our current knowledge of the earliest in vivo folding intermediates: the conformations adopted by nascent polypeptides during synthesis by the ribosome. The challenges related to successful folding in the cellular environment, including off-pathway aggregation and macromolecular crowding, are also discussed.

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There is growing interest in understanding how the cellular environment affects protein folding mechanisms, but most spectroscopic methods for monitoring folding in vitro are unsuitable for experiments in vivo or in other complex mixtures. Monoclonal antibody binding represents a sensitive structural probe that can be detected against the background of other cellular components. A panel of antibodies has been raised against Salmonella typhimurium phage P22 tailspike. In this report, nine alpha-tailspike antibody binding epitopes were characterized by measuring the binding of these monoclonal antibodies to tailspike variants bearing surface point mutations. These results reveal that the antibody epitopes are distributed throughout the tailspike structure, with several clustered in the central parallel beta-helix domain. The ability of each antibody to distinguish between tailspike conformational states was assessed by measuring antibody binding to tailspike in vitro refolding intermediates. Interestingly, the binding of all but one of the nine antibodies is sensitive to the tailspike conformational state. Whereas several antibodies bind preferentially to the tailspike native structure, the structural features that comprise the binding epitopes form with different rates. In addition, two antibodies preferentially recognize early refolding intermediates. Combined with the epitope mapping, these results indicate portions of the beta-helix form early during refolding, perhaps serving as a scaffold for the formation of additional structure. Finally, three of the antibodies show enhanced binding to non-native, potentially aggregation-prone tailspike conformations. The refolding results indicate these non-native conformations form early during the refolding reaction, long before the appearance of native tailspike.

Two banded, heavy snowstorms that occurred over the northern mid-Atlantic region are compared and contrasted. On 6-7 January 2002, a narrow, intense band of heavy snow was observed, along with several other weaker bands, embedded within a large area of moderate snow. On 19-20 January 2002, a single, broader band of heavy snow was observed, embedded within a broken area of light snow.The synoptic-scale settings associated with these two storms were strikingly dissimilar. In the first case, strong quasigeostrophic (QG) forcing for ascent was present just to the south of the heavy snowfall area. A highly amplified longwave trough was located over the Mississippi River valley, while a compact shortwave trough moved northward, up the east side of the longwave trough. The result was robust cyclogenesis off of the midAtlantic coast. In the second case, the relatively weaker QG forcing for ascent was located much farther southwest of the snowband. The flow aloft was much less amplified, with weaker cyclogenesis occurring off of the midAtlantic coast.Analysis of the frontal scale environments for both cases indicated that the snowbands were each associated with the collocation of midtropospheric frontogenesis and reduced stability. In the first case, evidence is shown that a layer of potential symmetric instability (PSI) was located just above a deep, sloping zone of frontogenesis, in the presence of deep near-saturated conditions. In the second case, evidence is shown that a layer of potential instability (PI), associated with rapidly decreasing relative humidity with height, was located just above a shallow, sloping zone of frontogenesis. In addition, it is shown that a particularly favorable thermal environment for snowflake growth and accumulation became collocated with the heavy snowband. It is hypothesized that the differences in the intensity and horizontal extent of the bands observed with these two events resulted from differing atmospheric responses associated with the areal extent of large-scale and frontogenetical forcing, moisture availability, degree of instability, and specific thermal profiles.

In June 2006, significant flooding and flash flooding impacted much of the mid-Atlantic region as a continuous supply of deep tropical moisture moved north from the subtropical Atlantic ahead of a slowmoving cold front. A 3-day period of heavy rain resulted in nearly 38.1 cm (15 in) of rain across portions of the northern mid-Atlantic with record flooding along the mainstem Susquehanna and Delaware Rivers. In September 2011, moisture associated with the remnants of Tropical Storm Lee resulted in a 24-h period of heavy rain over which rainfall totals approached 30.3 cm (12 in) across portions of central New York and northern Pennsylvania. Numerous river-stage records that were set in the June 2006 event were shattered along the mainstem Susquehanna River during the September 2011 flood. Damage estimates resulting from the flooding in both events were >2 billion dollars, and 22 lives were lost. Multiple counties across the northern mid-Atlantic were declared disaster areas. Both flood events were investigated to identify the similar meteorological features and patterns responsible for extreme rainfall. Several crucial similarities were identified that likely combined to produce historic socioeconomic and environmental impacts. One of the similarities was that each event had a well-established atmospheric river in place that provided the uninterrupted supply of deep tropical moisture. Additionally, although these events displayed many of the large-scale characteristics identified in previous flash flood classification schemes, both events were associated with the presence of coastal fronts that appeared to make these cases different from many otherwise similar and previously documented flood cases.