Background:The catalytic mechanism of Trichoderma reesei cellobiohydrolase I (TrCel7A) is still unclear. Results: TrCel7A exhibited similar reaction kinetics during crystalline cellulose I ␣ and III I hydrolysis. Conclusion: Not differences in kinetic parameters but surface properties of the crystalline cellulose influence the susceptibilities of cellulose I ␣ and III I to hydrolysis by TrCel7A. Significance: Single-molecule measurements further our understanding of TrCel7A mechanism.

High-speed atomic force microscopy (HS-AFM) and total internal reflection fluorescence microscopy (TIRFM) have mutually complementary capabilities. Here, we report techniques to combine these microscopy systems so that both microscopy capabilities can be simultaneously used in the full extent. To combine the two systems, we have developed a tip-scan type HS-AFM instrument equipped with a device by which the laser beam from the optical lever detector can track the cantilever motion in the X- and Y-directions. This stand-alone HS-AFM system is mounted on an inverted optical microscope stage with a wide-area scanner. The capability of this combined system is demonstrated by simultaneous HS-AFM∕TIRFM imaging of chitinase A moving on a chitin crystalline fiber and myosin V walking on an actin filament.

High-speed atomic force microscopy (HS-AFM) has been established and used, which can visualize biomolecules in dynamic action at high spatiotemporal resolution without disturbing their function. Various studies conducted in the past few years have demonstrated that the dynamic structure and action of biomolecules revealed with HS-AFM can provide greater insights than ever before into how the molecules function. However, this microscopy has still limitations in some regards. Recently, efforts have been carried out to overcome some of the limitations. As a result, it has now become possible to visualize dynamic processes occurring even on live cells and perform simultaneous observations of topographic and fluorescent images at a high rate. In this review, we focus on technical developments for expanding the range of objects and phenomena observable by HS-AFM as well as for granting multiple functionalities to HS-AFM.

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High-speed atomic force microscopy (HS-AFM) has enabled observing protein molecules during their functional activity at rates of 1–12.5 frames per second (fps), depending on the imaging conditions, sample height, and fragility. To meet the increasing demand for the great expansion of observable dynamic molecular processes, faster HS-AFM with less disturbance is imperatively needed. However, even a 50% improvement in the speed performance imposes tremendous challenges, as the optimization of major rate-limiting components for their fast response is nearly matured. This paper proposes an alternative method that can lower the feedback control error and thereby enhance the imaging rate. This method can be implemented in any HS-AFM system by minor modifications of the software and hardware. The resulting faster and less-disturbing imaging capabilities are demonstrated by the imaging of relatively fragile actin filaments and microtubules near the video rate, and of actin polymerization that occurs through weak intermolecular interactions, at ∼8 fps.

Highlights d SRP RNA exhibits robust co-transcriptional folding invariant to transcription rates d SRP RNA adopts a non-native obligatory intermediate prior to its functional fold d The obligatory intermediate enables SRP RNA maturation during early transcription d Altering the non-native-to-native transition in SRP RNA impacts cell viability

SummarySignal recognition particle (SRP) in Escherichia coli comprises protein Ffh and SRP RNA. Its essential functionality-co-translational protein-targeting/delivery to cellular membraneshinges on the RNA attaining a native long-hairpin fold that facilitates protein conformational rearrangements within the SRP complex. Since RNA folds co-transcriptionally on RNA polymerase, we use high-resolution optical tweezers to first characterize the mechanical unfolding/refolding of incrementally lengthened RNAs from stalled transcription complexes until reaching the full-length transcript. This analysis allows identification of folding intermediates adopted during the real-time co-transcriptional folding of SRP RNA. The cotranscriptional folding trajectories are surprisingly invariant to transcription rates, and involve formation of an obligatory non-native hairpin intermediate that eventually resolves into the native fold. SRP RNA variants designed to stabilize this non-native intermediate-likely sequestering the SRP ribonucleoprotein complex in an inactive form-greatly reduce cell viability, indicating that the same co-transcriptional folding mechanism operates in vivo and possible alternative antibiotic strategies.

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