Stain-Free technology as a normalization tool in Western blot analysis.

Western blots are used to specifically measure the relative quantities of proteins of interest in complex biological samples. Quantitative measurements can be subject to error due to process inconsistencies such as uneven protein transfer to the membrane. These non-sample-related variations need to be compensated for by an approach known as normalization. Two approaches to data normalization are commonly employed: housekeeping protein (HKP) normalization and total protein normalization (TPN). In this study, we evaluated the performance of Stain-Free technology as a novel TPN tool for Western blotting experiments in comparison with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a representative of the HKP normalization strategy. The target protein (TP) used for this study was MCM7, a DNA licensing replication factor, which was shown previously to be down-regulated by 20% in irradiated lymphoblastoid cell lines (LCLs). We studied the regulation of MCM7 with a multiplex Western blotting approach based on fluorescently labeled secondary antibodies and found that Stain-Free technology appears to be more reliable, more robust, and more sensitive to small effects of protein regulation when compared with HKP normalization with GAPDH. Stain-Free technology offers the additional advantages of providing checkpoints throughout the Western blotting process by allowing rapid visualization of gel separation and protein transfer.

Evidence of functional protein dynamics from X-ray crystallographic ensembles.

It is widely recognized that representing a protein as a single static conformation is inadequate to describe the dynamics essential to the performance of its biological function. We contrast the amino acid displacements below and above the protein dynamical transition temperature, T(D)∼215K, of hen egg white lysozyme using X-ray crystallography ensembles that are analyzed by molecular dynamics simulations as a function of temperature. We show that measuring structural variations across an ensemble of X-ray derived models captures the activation of conformational states that are of functional importance just above T(D), and they remain virtually identical to structural motions measured at 300K. Our results highlight the ability to observe functional structural variations across an ensemble of X-ray crystallographic data, and that residue fluctuations measured in MD simulations at room temperature are in quantitative agreement with the experimental observable.

The influence of protein dynamics on the success of computational enzyme design.

We characterize the molecular dynamics of a previously described computational de novo designed enzyme optimized to perform a multistep retrol-aldol reaction when engineered into a TIM barrel protein scaffold. The molecular dynamics simulations show that the protein dynamics under physiological conditions of temperature and aqueous environment distorts the designed geometric factors of the substrate-enzyme reaction intermediates, such that catalysis is limited by the primary retrol-aldol step of proton abstraction from the covalently bound substrate and its interactions with a histidine-aspartate dyad. These results emphasize that computational enzyme designs will benefit from considerations of dynamical fluctuations when optimizing active site geometries.

Non-sequence-specific interactions can account for the compaction of proteins unfolded under "native" conditions.

Proteins unfolded by high concentrations of chemical denaturants adopt expanded, largely structure-free ensembles of conformations that are well approximated as random coils. In contrast, globular proteins unfolded under less denaturing conditions (via mutations, or transiently unfolded after a rapid jump to native conditions) and molten globules (arising due to mutations or cosolvents) are often compact. Here we explore the origins of this compaction using a truncated equilibrium-unfolded variant of the 57-residue FynSH3 domain. As monitored by far-UV circular dichroism, NMR spectroscopy, and hydrogen-exchange kinetics, CDelta4 (a 4-residue carboxy-terminal deletion variant of FynSH3) appears to be largely unfolded even in the absence of denaturant. Nevertheless, CDelta4 is quite compact under these conditions, with a hydrodynamic radius only slightly larger than that of the native protein. In order to understand the origins of this molten-globule-like compaction, we have characterized a random sequence polypeptide of identical amino acid composition to CDelta4. Notably, we find that the hydrodynamic radius of this random sequence polypeptide also approaches that of the native protein. Thus, while native-like interactions may contribute to the formation of compact "unfolded" states, it appears that non-sequence-specific monomer-monomer interactions can also account for the dramatic compaction observed for molten globules and the "physiological" unfolded state.

The folding process of acylphosphatase from Escherichia coli is remarkably accelerated by the presence of a disulfide bond.

The acylphosphatase from Escherichia coli (EcoAcP) is the first AcP so far studied with a disulfide bond. A mutational variant of the enzyme lacking the disulfide bond has been produced by substituting the two cysteine residues with alanine (EcoAcP mutational variant C5A/C49A, mutEcoAcP). The native states of the two protein variants are similar, as shown by far-UV and near-UV circular dichroism and dynamic light-scattering measurements. From unfolding experiments at equilibrium using intrinsic fluorescence and far-UV circular dichroism as probes, EcoAcP shows an increased conformational stability as compared with mutEcoAcP. The wild-type protein folds according to a two-state model with a very fast rate constant (k(F)(H2O)=72,600 s(-1)), while mutEcoAcP folds ca 1500-fold slower, via the accumulation of a partially folded species. The correlation between the hydrophobicity of the polypeptide chain and the folding rate, found previously in the AcP-like structural family, is maintained only when considering the mutant but not the wild-type protein, which folds much faster than expected from this correlation. Similarly, the correlation between the relative contact order and the folding rate holds only for mutEcoAcP. The correlation also holds for EcoAcP, provided the relative contact order value is recalculated by considering the disulfide bridge as an alternate path for the backbone to determine the shortest sequence separation between contacting residues. These results indicate that the presence of a disulfide bond in a protein is an important determinant of the folding rate and allows its contribution to be determined in quantitative terms.

Is there or isn't there? The case for (and against) residual structure in chemically denatured proteins.

First raised some 60 years ago, the question of whether chemically denatured proteins are fully unfolded has, in recent years, seen significantly renewed interest. This increased attention has been spurred, in large part, by new spectroscopic and computational approaches that suggest even the most highly denatured polypeptides contain significant residual structure. In contrast, the most recent scattering results uphold the long-standing view that chemically denatured proteins adopt random coil configurations. Here we review the evidence both for and against residual structure in chemically denatured proteins, and attempt to reconcile these seemingly contradictory observations.

Engineering a signal transduction mechanism for protein-based biosensors.

Hybridization-induced conformational changes have been successfully used in biosensors for the transduction of DNA-binding events into readily observable optical or electronic signals. Similar signal transduction has not, however, proven of equal utility in protein-based biosensors. The discrepancy arises because, unlike ssDNA, most proteins do not undergo significant conformational changes upon ligand binding. Here, we describe a solution to this problem. We show that an arbitrarily selected, normally well folded protein can be rationally engineered such that it undergoes ligand-induced folding. The engineered protein responds rapidly (milliseconds) and selectively to its target, and it couples recognition with the largest possible conformational change: folding. These traits suggest that ligand-induced folding could serve as an ideal signal-transduction mechanism. Consistent with this claim, we demonstrate a label-free optical biosensor based on the effect that is sufficiently selective to detect its target even in complex, contaminant-ridden samples such as blood serum.

Random-coil behavior and the dimensions of chemically unfolded proteins.

Spectroscopic studies have identified a number of proteins that appear to retain significant residual structure under even strongly denaturing conditions. Intrinsic viscosity, hydrodynamic radii, and small-angle x-ray scattering studies, in contrast, indicate that the dimensions of most chemically denatured proteins scale with polypeptide length by means of the power-law relationship expected for random-coil behavior. Here we further explore this discrepancy by expanding the length range of characterized denatured-state radii of gyration (R(G)) and by reexamining proteins that reportedly do not fit the expected dimensional scaling. We find that only 2 of 28 crosslink-free, prosthetic-group-free, chemically denatured polypeptides deviate significantly from a power-law relationship with polymer length. The R(G) of the remaining 26 polypeptides, which range from 16 to 549 residues, are well fitted (r(2) = 0.988) by a power-law relationship with a best-fit exponent, 0.598 +/- 0.028, coinciding closely with the 0.588 predicted for an excluded volume random coil. Therefore, it appears that the mean dimensions of the large majority of chemically denatured proteins are effectively indistinguishable from the mean dimensions of a random-coil ensemble.