Nonexponential Kinetics of Loop Formation in Proteins and Peptides: A Signature of Rugged Free Energy Landscapes?

The kinetics of loop formation, i.e., the occurrence of contact between two atoms of a polypeptide, remains the focus of continuing interest. One of the reasons is that contact formation is the elementary event underlying processes such as folding and binding. More importantly, it is experimentally measurable and can be predicted theoretically for ideal polymers. Deviations from single exponential kinetics have sometimes been interpreted as a signature of rugged, protein-like, free energy landscapes. Here we present simulations, with different atomistic models, of short peptides with varied structural propensity, and of a structured protein. Results show exponential contact formation kinetics (or relaxation) at long times, and a power law relaxation at very short times. At intermediate times, a deviation from either power law or simple exponential kinetics is observed that appears to be characteristic of polypeptides with either specific or nonspecific attractive interactions but disappears if attractive interactions are absent. Our results agree with recent experimental measurements on peptides and proteins and offer a comprehensive interpretation for them.

The structure of the PanD/PanZ protein complex reveals negative feedback regulation of pantothenate biosynthesis by coenzyme A.

Coenzyme A (CoA) is an ubiquitous and essential cofactor, synthesized from the precursor pantothenate. Vitamin biosynthetic pathways are normally tightly regulated, including the pathway from pantothenate to CoA. However, no regulation of pantothenate biosynthesis has been identified. We have recently described an additional component in the pantothenate biosynthetic pathway, PanZ, which promotes the activation of the zymogen, PanD, to form aspartate α-decarboxylase (ADC) in a CoA-dependent manner. Here we report the structure of PanZ in complex with PanD, which reveals the structural basis for the CoA dependence of this interaction and activation. In addition, we show that PanZ acts as a CoA-dependent inhibitor of ADC catalysis. This inhibitory effect can effectively regulate the biosynthetic pathway to pantothenate, and thereby also regulate CoA biosynthesis. This represents a previously unobserved mode of metabolic regulation whereby a cofactor-utilizing protein negatively regulates the biosynthesis of the same cofactor.

Effect of external pulling forces on the length distribution of peptides.

BACKGROUND: The distribution of the length of a polypeptide, or that of the distance between any two of its atoms, is an important property as it can be analytically or numerically estimated for a number of polymer models. Importantly, it is directly measurable through a number of different experimental techniques. Length distributions can be straightforwardly assessed from molecular dynamics simulation; however, true convergence through full accurate coverage of the length range is difficult to achieve. METHODS: The application of external constant force combined with the weighted-histogram analysis method (WHAM) is used to enhance sampling of unlikely 'long' or 'short' conformations and obtain the potential of mean force, while also collecting dynamic properties of the chain under variable tension. RESULTS: We demonstrate the utility of constant force to enhance the sampling efficiency and obtain experimentally measurable quantities on a series of short peptides, including charge-rich sequences that are known to be highly helical but whose properties are distinct from those of helical peptides undergoing helix-coil transitions. CONCLUSIONS: Force-enhanced sampling enhances the range and accuracy of the length-based potential of mean force of the peptide, in particular those sequences that contain increased numbers of charged residues. GENERAL SIGNIFICANCE: This approach allows users to simultaneously probe the force-dependent behaviour of peptides directly, enhance the range and accuracy of the length-based PMF of the peptide and also test the convergence of simulations by comparing the overlap of PMF profiles from different constant forces. This article is part of a special issue entitled Recent developments of molecular dynamics.

TROSY NMR with a 52 kDa sugar transport protein and the binding of a small-molecule inhibitor.

Using the sugar transport protein, GalP, from Escherichia coli, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [(15)N-(1)H]- and [(13)C-(1)H]-methyl-TROSY NMR spectra with a 52 kDa membrane protein that putatively has 12 transmembrane-spanning α-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48 h at a temperature of 25 °C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan (15)N backbone positions and also resolved signals for (15)N side-chain positions. For improved sensitivity isoleucine, leucine and valine (ILV) methyl-labelled protein was prepared, which produced unexpectedly well resolved [(13)C-(1)H]-methyl-TROSY spectra showing clear signals for the majority of methyl groups. The GalP/GLUT inhibitor forskolin was added to the ILV-labelled sample inducing a pronounced chemical shift change in one Ile residue and more subtle changes in other methyl groups. This work demonstrates that high-resolution TROSY NMR spectra can be achieved with large complex α-helical membrane proteins without the use of elevated temperatures. This is a prerequisite to applying further labelling strategies and NMR experiments for measurement of dynamics, structure elucidation and use of the spectra to screen ligand binding.

Revealing low-dose radiation damage using single-crystal spectroscopy.

The structural information and functional insight obtained from X-ray crystallography can be enhanced by the use of complementary spectroscopies. Here the information that can be obtained from spectroscopic methods commonly used in conjunction with X-ray crystallography and best-practice single-crystal UV-Vis absorption data collection are briefly reviewed. Using data collected with the in situ system at the Swiss Light Source, the time and dose scales of low-dose X-ray-induced radiation damage and solvated electron generation in metalloproteins at 100 K are investigated. The effect of dose rate on these scales is also discussed.