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The native serpin fold is characterized by being metastable. This thermodynamic characteristic is manifested in the conversion of the native state to other more stable conformations. Whilst this structural transition is required for proteinase inhibition and regulation of a range of biological phenomena, inappropriate structural changes can result in a number of disease states. Identification of these...
Partially folded and denatured proteins can give important insights into protein folding, misfolding, and aggregation. Such non-native states of proteins are however very difficult to characterise in detail as they are dynamic, heterogeneous systems comprising of ensembles of interconverting conformers. This article describes methods that produce models for non-native proteins in atomic detail. A...
The measurement of amino acid-resolved hydrogen exchange (HX) has provided the most detailed information so far available on the structure and properties of protein folding intermediates. Direct HX measurements can define the structure of tenuous molten globule forms that are generally inaccessible to the usual crystallographic and NMR methods (C. Redfield review in this issue). HX pulse labeling...
Stopped-flow mixing coupled with time-resolved Fourier transform infrared (FT-IR) spectroscopy represents a new experimental approach to explore protein folding events, which has become possible only recently with the development of appropriate techniques. Here, we discuss experimental apparatus that are capable of initiating and monitoring protein folding processes on the millisecond to minute timescale...
All atom molecular dynamics simulations have become a standard method for mapping equilibrium protein dynamics and non-equilibrium events like folding and unfolding. Here, we present detailed methods for performing such simulations. Generic protocols for minimization, solvation, simulation, and analysis derived from previous studies are also presented. As a measure of validation, our water model is...
Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for the study of the structure, dynamics, and folding of proteins in solution. It is particularly powerful when applied to dynamic or flexible systems, such as partially folded molten globule states of proteins, which are not usually amenable to X-ray crystallography. In this article, NMR methods suitable for the detailed characterisation...
Chemically induced dynamic nuclear polarization (CIDNP) is a nuclear magnetic resonance phenomenon that can be used to probe the solvent-accessibility of tryptophan, tyrosine, and histidine residues in proteins by means of laser-induced photochemical reactions, resulting in significant enhancement of NMR signals. CIDNP offers good sensitivity as a surface probe of protein structure and is particularly...
Real-time NMR spectroscopy developed to a generally applicable method to follow protein folding reactions. It combines the access to high resolution data with kinetic experiments allowing very detailed insights into the development of the protein structure during different steps of folding. The present review concentrates mainly on the progress of real-time NMR during the last 5 years. Starting from...
Extensive structural studies using high-pressure NMR spectroscopy have recently been carried out on proteins, which potentially contribute to our understanding of the mechanisms of protein folding. Pressure shifts the conformational equilibrium from higher to lower volume conformers. If the pressure is varied, starting from the folded native structure, in many cases we observe intermediate conformers...
Newly synthesized polypeptides entering the endoplasmic reticulum (ER) encounter a large array of molecular chaperones and folding factors that facilitate proper folding as well as assess folding status, retaining non-native proteins within the ER. Calnexin (CNX), an ER membrane protein, and its soluble homologue, calreticulin (CRT), are two important molecular chaperones that contribute to both processes...
We have developed a partially recombinant, cell-free, protein-synthesis system reconstituted solely from those essential elements of the Escherichia coli translation system, termed protein synthesis using recombinant elements (PURE). It provides higher reaction controllability in comparison to crude cell-free protein-synthesis systems for translation studies and biotechnology applications. The PURE...
THz spectroscopy is combined with MD simulations to study the dynamical properties of water in the solvation shell of proteins. The solvation dynamics is found to be influenced on length-scales of several hydration layers which is significantly more than what is found for static properties. Our experiments show that the properties of this dynamical solvation shell depend on the folding state of the...
Protein folding is a fundamental biological process of great significance for cell function and life-related processes. Surprisingly, very little is presently known about how proteins fold in vivo. The influence of the cellular environment is of paramount importance, as molecular chaperones, the ribosome, and the crowded medium affect both folding pathways and potentially even equilibrium structures...
Inherently hierarchic nature of proteins makes multiscale computational methods especially useful in the studies of folding and other functional dynamics. With the multiscale strategies, one can achieve improved accuracy and efficiency by coupling the atomistic and the coarse grained simulations. Depending on the problems studied, very different implementation protocols can be used to realize the...
We record proton NMR spectra of the protein ubiquitin at 1 atmosphere pressure and at negative pressures (under tension), under conditions where the native and denatured states are nearly equally populated. Analysis of the unique histidine aromatic resonance of ubiquitin shows that negative pressure destabilizes the protein, in accord with a quadratic free energy dependence on pressure and temperature...
Polarization methods, introduced in the 1800s, offered one of the earliest ways to examine protein structure. Since then, many other structure-sensitive probes have been developed, but circular dichroism (CD) remains a powerful technique because of its versatility and the specificity of protein structural information that can be explored. With improvements in time resolution, from millisecond to picosecond...
The studies of the folding of structurally related proteins have proved to be a very important tool for investigating protein folding. Here we review some of the insights that have been gained from such studies. Our highlighted studies show just how such an investigation should be designed and emphasise the importance of the synergy between experiment and theory. We also stress the importance of choosing...
Simulating protein folding has been a challenging problem for decades due to the long timescales involved (compared with what is possible to simulate) and the challenges of gaining insight from the complex nature of the resulting simulation data. Markov State Models (MSMs) present a means to tackle both of these challenges, yielding simulations on experimentally relevant timescales, statistical significance,...
Polyglutamine (polyQ)-expansion proteins cause protein aggregation in the cytosol and nucleus of neuronal cells, leading to neurodegenerative diseases. For example, expansion of the polyQ tract (>40 repeats) in huntingtin (htt) proteins leads to Huntington disease, while polyQ-expanded ataxins cause several types of ataxias. PolyQ-rich inclusions are found in neuronal cells of patients, suggesting...
During the last two decades single-molecule manipulation techniques such as atomic force microscopy (AFM) has risen to prominence through their unique capacity to provide fundamental information on the structure and function of biomolecules. Here we describe the use of single-molecule AFM to track protein unfolding and refolding pathways, enzymatic catalysis and the effects of osmolytes and chaperones...
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