Selective precipitation of proteins can be used as a bulk method to recover the majority of proteins from a crude lysate, as a selective method to fractionate a subset of proteins from a protein solution, or as a very specific method to recover a single protein of interest from a purification step. This unit describes a number of methods suitable for selective precipitation. In each of the prot...

One of the general paradigms for ab initio protein structure prediction involves sampling the conformational space such that a large set of decoy (candidate) structures are generated and then selecting native-like conformations from those decoys using various scoring functions. In this study, based on a physical/geometric approach first suggested by Banavar and colleagues, we formulate a knowle...

The mechanism of import-competent precursor protein-induced inactivation of a 50-pS anion channel of the chloroplast envelope is investigated using single-channel recordings. The inactivation by precursor protein is the result of the induction of a long-lived closed state of the channel. The mean duration of this state does not depend on precursor concentration. From this it can be concluded th...

Photoswitchable distance constraints in the form of photoisomerizable chemical cross-links offer a general approach to the design of reversibly photocontrolled proteins. To apply these effectively, however, one must have guidelines for the choice of cross-linker structure and cross-linker attachment sites. Here we investigate the effects of varying cross-linker structure on the photocontrol of ...

We investigate the effect of temperature and pressure on polypeptide conformational stability using a two-dimensional square lattice model in which water is represented explicitly. The model captures many aspects of water thermodynamics, including the existence of density anomalies, and we consider here the simplest representation of a protein: a hydrophobic homopolymer. We show that an explici...

We perform theoretical studies of stretching of 20 proteins with knots within a coarse-grained model. The knot's ends are found to jump to well defined sequential locations that are associated with sharp turns, whereas in homopolymers they diffuse around and eventually slide off. The waiting times of the jumps are increasingly stochastic as the temperature is raised. Knots typically do not retu...

All proteins sample a range of conformations at physiologic temperatures and this inherent flexibility enables them to carry out their prescribed functions. A comprehensive understanding of protein function therefore entails a characterization of protein flexibility. Here we describe a novel approach for quantifying a protein's flexibility in solution using small-angle X-ray scattering (SAXS) d...

Quantifying metal-binding by force: A quantitative single-molecule force-spectroscopy-based assay is developed to measure the binding affinity of metal ions to proteins. The method uses the unfolding force of a protein as a direct probe to distinguish the apo and metal-ion-bound forms of that protein and quantify the partitioning between the two forms (see figure).