Comparison between slow, anisotropic LE4PD fluctuations and the Principal Component Analysis modes of Ubiquitin

Proteins' biological function and folding mechanisms are often guided by large-scale, slow motions, which involve crossing high energy barriers. In a simulation trajectory, these slow fluctuations are commonly identified using a principal component analysis (PCA). Despite the popularity of this method, a complete analysis of its predictions based on the physics of protein motion has been so far limited. This study formally connects the PCA to a Langevin model of protein dynamics and analyzes the contributions of energy barriers and hydrodynamic interactions to the slow PCA modes of motion. To do so, we introduce an anisotropic extension of the Langevin Equation for Protein Dynamics, called the LE4PD-XYZ, which formally connects to the PCA 'essential dynamics'. The LE4PD-XYZ is an accurate coarse-grained diffusive method to model protein motion, which describes anisotropic fluctuations in the protein's alpha-carbons. The LE4PD accounts for hydrodynamic effects and mode-dependent free-energy barriers. This study compares large-scale anisotropic fluctuations identified by the LE4PD-XYZ to the mode-dependent PCA's predictions, starting from a microsecond-long alpha-carbon molecular dynamics atomistic trajectory of the protein ubiquitin. We observe that the inclusion of free-energy barriers and hydrodynamic interactions has important effects on the identification and timescales of ubiquitin's slow modes.

The T4 TerL Prohead Packaging Motor Does Not Drive DNA Translocation by a Proposed Dehydration Mechanism

A “DNA crunching” linear motor mechanism that employs a grip-and-release transient spring like compression of B- to A-form DNA has been found in our previous studies. Our FRET measurements in vitro show a decrease in distance from TerL to portal during packaging; furthermore, there is a decrease in distance between closely positioned dye pairs in the Y-stem of translocating Y-DNA that conforms to B- and A- structure. In normal translocation into the prohead the TerL motor expels all B-form tightly binding YOYO-1 dye that cannot bind A-form. The TerL motor cannot package A-form dsRNA. Our work reported here shows that addition of helper B form DNA:DNA (D:D) 20mers allows increased packaging of heteroduplex A-form DNA:RNA 20mers (D:R), evidence for a B- to A-form spring motor pushing duplex nucleic acid. A-form DNA:RNA 25mers, 30mers, and 35mers alone are efficiently packaged into proheads by the TerL motor showing that a proposed hypothetical dehydration motor mechanism operating on duplex substrates does not provide the packaging motor force. Taken together with our previous studies showing TerL motor protein motion toward the portal during DNA packaging, our present studies of short D:D and D:R duplex nucleic acid substrates strongly supports our previous evidence that the protein motor pushes rather than pulls or dehydrates duplex substrates to provide the translocation into prohead packaging force.