Counter-selectable markers can be used in two-hybrid systems to search libraries for a protein or compound that interferes with a macromolecular interaction or to identify macromolecules from a population that cannot mediate a particular interaction. In this report, we describe the adaptation of the yeast URA3/5-FOA counter-selection system for use in bacterial interaction trap experiments. Two...

Most of the standard methods of solving macromolecular structures involve producing a protein crystal that is derivatized by an anomalous scatterer or heavy atom (MIR, SIRAS, MAD, SAD etc.). The theoretical methodology which underpins the extraction of phase information from such derivatives is widely available in the literature. In addition, there are comprehensive sources of information on th...

Accurate measurement of the amount of specific protein a cell produces is important for investigating basic molecular processes. We have developed a technique that allows for quantitation of protein levels in single cells in vivo. This protein quantitation ratioing (PQR) technique uses a genetic tag that produces a stoichiometric ratio of a fluorescent protein reporter and the protein of intere...

We present a novel type of spectral diffusion experiment in the millikelvin range to characterize the energy landscape of a protein as compared with that of a glass. We measure the time evolution of spectral holes for more than 300 hr after well-defined initial nonequilibrium conditions. We show that the model of noninteracting two-level systems can describe spectral diffusion in the glass, but...

We developed and implemented an ensemble-refinement method to study dynamic biomolecular assemblies with intrinsically disordered segments. Data from small angle X-ray scattering (SAXS) experiments and from coarse-grained molecular simulations were combined by using a maximum-entropy approach. The method was applied to CHMP3 of ESCRT-III, a protein with multiple helical domains separated by fle...

Is highly approximate knowledge of a protein's backbone structure sufficient to successfully identify its family, superfamily, and tertiary fold? To explore this question, backbone dihedral angles were extracted from the known three-dimensional structure of 2,439 proteins and mapped into 36 labeled, 60 degrees x 60 degrees bins, called mesostates. Using this coarse-grained mapping, protein conf...

Multivalent polymers are macromolecules containing multiple chemical moieties designed to bind to complementary moieties on a target; for example, a protein with multiple ligands that have affinity for receptors on a cell surface. Though the individual ligand-receptor bonds are often weak, the combinatorial entropy associated with the different possible ligand-receptor pairs leads to a binding ...

Adaptive Multilevel Splitting (AMS) is a replica-based rare event sampling method that has been used successfully in high-dimensional stochastic simulations to identify trajectories across a high potential barrier separating one metastable state from another, and to estimate the probability of observing such a trajectory. An attractive feature of AMS is that, in the limit of a large number of r...

TNF-resistant lines of L cells can be derived from TNF-sensitive populations by repeated exposure to TNF, and these resistant L cells, in contrast to sensitive L cells and other types of cells, lack demonstrable cell surface receptors for TNF. We have now found that TNF-resistant L cells produce a factor that is cytotoxic for L cells and has the following distinguishing characteristics of mouse...

Naturally occurring proteins in cellular networks often share peptide motifs. These motifs have been known to play a pivotal role in protein interactions among the components of a network. However, it remains unknown how these motifs have contributed to the evolution of the protein network. Here we addressed this issue by a synthetic biology approach. Through the motif programming method, we ha...