Homodimeric KIF17 and heterotrimeric KIF3AB are processive, kinesin-2 family motors that act jointly to carry out anterograde intraflagellar transport (IFT), ferrying cargo along microtubules (MTs) toward the tips of cilia. How IFT trains attain speeds that exceed the unloaded rate of the slower, KIF3AB motor remains unknown. By characterizing the motility properties of kinesin-2 motors as a function of load we find that the increase in KIF3AB velocity, elicited by forward loads from KIF17 motors, cannot alone account for the speed of IFT trains in vivo. Instead, higher IFT velocities arise from an increased likelihood that KIF3AB motors dissociate from the MT, resulting in transport by KIF17 motors alone, unencumbered by opposition from KIF3AB. The rate of transport is therefore set by an equilibrium between a faster state, where only KIF17 motors move the train, and a slower state, where at least one KIF3AB motor on the train remains active in transport. The more frequently the faster state is accessed, the higher the overall velocity of the IFT train. We conclude that IFT velocity is governed by () the absolute numbers of each motor type on a given train, () how prone KIF3AB is to dissociation from MTs relative to KIF17, and () how prone both motors are to dissociation relative to binding MTs.
While protein conformational heterogeneity plays an important role in many aspects of biological function, including ligand binding, its impact has been difficult to quantify. Macromolecular X-ray diffraction is commonly interpreted with a static structure, but it can provide information on both the anharmonic and harmonic contributions to conformational heterogeneity. Here, through multiconformer modeling of time- and space-averaged electron density, we measure conformational heterogeneity of 743 stringently matched pairs of crystallographic datasets that reflect unbound/apo and ligand-bound/holo states. When comparing the conformational heterogeneity of side chains, we observe that when binding site residues become more rigid upon ligand binding, distant residues tend to become more flexible, especially in non-solvent exposed regions. Among ligand properties, we observe increased protein flexibility as the number of hydrogen bonds decrease and relative hydrophobicity increases. Across a series of 13 inhibitor bound structures of CDK2, we find that conformational heterogeneity is correlated with inhibitor features and identify how conformational changes propagate differences in conformational heterogeneity away from the binding site. Collectively, our findings agree with models emerging from NMR studies suggesting that residual side chain entropy can modulate affinity and point to the need to integrate both static conformational changes and conformational heterogeneity in models of ligand binding.
New X-ray crystallography and cryo-electron microscopy (cryo-EM) approaches yield vast amounts of structural data from dynamic proteins and their complexes. Modeling the full conformational ensemble can provide important biological insights, but identifying and modeling an internally consistent set of alternate conformations remains a formidable challenge. qFit efficiently automates this process by generating a parsimonious multiconformer model. We refactored qFit from a distributed application into software that runs efficiently on a small server, desktop, or laptop. We describe the new qFit 3 software and provide some examples.
The thiamine pyrophosphate (TPP) riboswitch is a cis-regulatory element in mRNA that modifies gene expression in response to TPP concentration. Its specificity is dependent upon conformational changes that take place within its aptamer domain. Here, the role of tertiary interactions in ligand binding was studied at the single-molecule level by combined force spectroscopy and Förster resonance energy transfer (smFRET), using an optical trap equipped for simultaneous smFRET. The ‘Force-FRET’ approach directly probes secondary and tertiary structural changes during folding, including events associated with binding. Concurrent transitions observed in smFRET signals and RNA extension revealed differences in helix-arm orientation between two previously-identified ligand-binding states that had been undetectable by spectroscopy alone. Our results show that the weaker binding state is able to bind to TPP, but is unable to form a tertiary docking interaction that completes the binding process. Long-range tertiary interactions stabilize global riboswitch structure and confer increased ligand specificity.DOI: http://dx.doi.org/10.7554/eLife.12362.001
The line shape problem in the near-infrared spectrum of self-colliding CO 2 molecules: Experimental investigation and test of semiclassical models J. Chem. Phys. 130, 184306 (2009) Frequency-stabilized cavity ring-down spectroscopy was used to study CO 2 lineshapes in the (20013) ← (00001) band centered near 2.06 μm. Two rovibrational transitions were chosen for this study to measure non-Voigt collisional effects for air-broadened lines over the pressure range of 7 kPa-28 kPa. Lineshape analysis for both lines revealed evidence of simultaneous Dicke (collisional) narrowing and speed-dependent effects that would introduce biases exceeding 2% in the retrieved air-broadening parameters if not incorporated in the modeling of CO 2 lineshapes. Additionally, correlations between velocity-and phase/state changing collisions greatly reduced the observed Dicke narrowing effect. As a result, it was concluded that the most appropriate line profile for modeling CO 2 lineshapes near 2.06 μm was the correlated speed-dependent NelkinGhatak profile, which includes all of the physical effects mentioned above and leads to a consistent set of line shape parameters that are linear with gas pressure. © 2014 AIP Publishing LLC.
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