Using Single-Molecule Approaches to Understand the Molecular Mechanisms of Heat-Shock Protein Chaperone Function

The heat-shock proteins (Hsp) are a family of molecular chaperones, which collectively form a network that is critical for the maintenance of protein homeostasis. Traditional ensemble-based measurements have provided a wealth of knowledge on the function of individual Hsps and the Hsp network; however, such techniques are limited in their ability to resolve the heterogeneous, dynamic and transient interactions that molecular chaperones make with their client proteins. Single-molecule techniques have emerged as a powerful tool to study dynamic biological systems, as they enable rare and transient populations to be identified that would usually be masked in ensemble measurements. Thus, single-molecule techniques are particularly amenable for the study of Hsps and have begun to be used to reveal novel mechanistic details of their function. In this review, we discuss the current understanding of the chaperone action of Hsps and how gaps in the field can be addressed using single-molecule methods. Specifically, this review focuses on the ATP-independent small Hsps and the broader Hsp network and describes how these dynamic systems are amenable to single-molecule techniques.     

A comparison of soil core sampling and minirhizotrons to quantify root development of field-grown potatoes

Root growth of potato (Solanum tuberosum L.) is sensitive to soil conditions. A reduced root system size can result in reduced uptake of water and/or nutrients, leading to impaired crop growth. To understand the mechanisms by which soil conditions affect crop growth, study of temporal and spatial development of roots is required.In field experiments, effects of soil temperature, soil compaction and potato cyst nematodes (Globodera pallida) on root growth of potato cultivars were studied using two methods: core sampling and vertically oriented minirhizotrons.Minirhizotrons showed relatively more roots in deeper soil layers than core sampling, probably because of preferential root growth along the tube. Spatial distribution of roots should therefore be analysed by core sampling.To eliminate differences in spatial distribution, total root systems as measured by both methods were compared. Nematodes, cultivars and time did not affect the relationship between both methods. Soil compaction, however, affected it because of a strong response of root length in bulk soil and small differences in root number against the minirhizotron, suggesting that soil coring has to be used to study effects of different bulk densities.With both methods, sequential measurements of roots give the net effect of root growth and decay. Data on root turnover can only be obtained with minirhizotrons by comparing video recordings of different dates. Other information obtained with minirhizotrons is the average orientation of roots. Moreover, the minirhizotron method has the advantage of demanding less labour.     

Single-Molecule Imaging at High Fluorophore Concentrations by Local Activation of Dye

Single-molecule fluorescence microscopy is a powerful tool for observing biomolecular interactions with high spatial and temporal resolution. Detecting fluorescent signals from individual labeled proteins above high levels of background fluorescence remains challenging, however. For this reason, the concentrations of labeled proteins in in vitro assays are often kept low compared to their in vivo concentrations. Here, we present a new fluorescence imaging technique by which single fluorescent molecules can be observed in real time at high, physiologically relevant concentrations. The technique requires a protein and its macromolecular substrate to be labeled each with a different fluorophore. Making use of short-distance energy-transfer mechanisms, only the fluorescence from those proteins that bind to their substrate is activated. This approach is demonstrated by labeling a DNA substrate with an intercalating stain, exciting the stain, and using energy transfer from the stain to activate the fluorescence of only those labeled DNA-binding proteins bound to the DNA. Such an experimental design allowed us to observe the sequence-independent interaction of Cy5-labeled interferon-inducible protein 16 with DNA and the sliding via one-dimensional diffusion of Cy5-labeled adenovirus protease on DNA in the presence of a background of hundreds of nanomolar Cy5 fluorophore.     

Real-time single-molecule observation of rolling-circle DNA replication

We present a simple technique for visualizing replication of individual DNA molecules in real time. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, we are able to monitor the progression of single-DNA synthesis events and accurately measure rates and processivities of single T7 and Escherichia coli replisomes as they replicate DNA. This method allows for rapid and precise characterization of the kinetics of DNA synthesis and the effects of replication inhibitors.     

A Generic Equation for Nitrogen-limited Leaf Area Index and its Application in Crop Growth Models for Predicting Leaf Senescence

Appropriate quantification of leaf area index (LAI) is importantfor accurate prediction of photosynthetic productivity by cropgrowth models. Estimation of LAI requires accurate modellingof leaf senescence. Many models use empirical turnover coefficients,the relative leaf-death rate determined from frequent fieldsamplings, to describe senescence during growth. In this paper,we first derive a generic equation for nitrogen-determined photosyntheticallyactive LAI (LAI_N), and then describe a method of using thisequation in crop growth models to predict leaf senescence. Basedon the theory that leaf-nitrogen at different horizons of acanopy declines exponentially, LAI_N, which is counted from thetop of the canopy to the depth at which leaf-nitrogen equalsthe minimum value for leaf photosynthesis, is calculated analyticallyas a function of canopy leaf-nitrogen content. At each time-stepof crop growth modelling, LAI_Nis compared to an independentcalculation of the non-nitrogen-limited LAI assuming no leafdeath during that time-step (LAI_NLD). In early stages, LAI_Nishigher than LAI_NLD; but with the advancement of crop growth,LAI_Nwill become smaller than LAI_NLD. The difference betweenLAI_NLDand LAI_N, whenever LAI_Nis smaller than LAI_NLD, gives theestimate of leaf area senesced at the time-step; the senescedleaf area divided by specific leaf area (SLA) gives the estimateof senesced leaf mass. The method was incorporated into twocrop models and the models adequately accounted for the LAIobserved in field experiments for rice and barley. The novelfeatures of the approach are that: (1) it suggests a coherent,biologically reasonable picture of leaf senescence based onthe link with photosynthesis and leaf nitrogen content; (2)it avoids the use of empirical leaf-turnover coefficients; (3)it avoids over-sensitivity of LAI prediction to SLA; and (4)it is presumably of sufficient generality as to be applicableto plant types other than crops. The method can be applied tomodels where leaf-nitrogen is used as an input variable or issimulated explicitly. Copyright 2000 Annals of Botany Company Leaf area index, leaf senescence, canopy nitrogen, modelling     

Single-molecule approaches to characterizing kinetics of biomolecular interactions

Single-molecule fluorescence techniques have emerged as powerful tools to study biological processes at the molecular level. This review describes the application of these methods to the characterization of the kinetics of interaction between biomolecules. A large number of single-molecule assays have been developed that visualize association and dissociation kinetics in vitro by fluorescently labeling binding partners and observing their interactions over time. Even though recent progress has been significant, there are certain limitations to this approach. To allow the observation of individual, fluorescently labeled molecules requires low, nanomolar concentrations. I will discuss how such concentration requirements in single-molecule experiments limit their applicability to investigate intermolecular interactions and how recent technical advances deal with this issue.     

Pyrovanadolysis, a pyrophosphorolysis-like reaction mediated by pyrovanadate, Mn2+, and DNA polymerase of bacteriophage T7

DNA polymerases catalyze the 3'-5'-pyrophosphorolysis of a DNA primer annealed to a DNA template in the presence of pyrophosphate (PP(i)). In this reversal of the polymerization reaction, deoxynucleotides in DNA are converted to deoxynucleoside 5'-triphosphates. Based on the charge, size, and geometry of the oxygen connecting the two phosphorus atoms of PP(i), a variety of compounds was examined for their ability to carry out a reaction similar to pyrophosphorolysis. We describe a manganese-mediated pyrophosphorolysis-like activity using pyrovanadate (VV) catalyzed by the DNA polymerase of bacteriophage T7. We designate this reaction pyrovanadolysis. X-ray absorption spectroscopy reveals a shorter Mn-V distance of the polymerase-VV complex than the Mn-P distance of the polymerase-PP(i) complex. This structural arrangement at the active site accounts for the enzymatic activation by Mn-VV. We propose that the Mn(2+), larger than Mg(2+), fits the polymerase active site to mediate binding of VV into the active site of the polymerase. Our results may be the first documentation that vanadium can substitute for phosphorus in biological processes.     

Spotting the mistakes, one molecule at a time

In this issue of Structure, Cho and colleagues provide intriguing insight into the first steps of the DNA mismatch repair process. By using single-molecule techniques, they show that the protein MutS undergoes two different types of diffusion on error-containing DNA in an ATP-dependent way.     

Uncoupling of sister replisomes during eukaryotic DNA replication

The duplication of eukaryotic genomes involves the replication of DNA from multiple origins of replication. In S phase, two sister replisomes assemble at each active origin, and they replicate DNA in opposite directions. Little is known about the functional relationship between sister replisomes. Some data imply that they travel away from one another and thus function independently. Alternatively, sister replisomes may form a stationary, functional unit that draws parental DNA toward itself. If this "double replisome" model is correct, a constrained DNA molecule should not undergo replication. To test this prediction, lambda DNA was stretched and immobilized at both ends within a microfluidic flow cell. Upon exposure to Xenopus egg extracts, this DNA underwent extensive replication by a single pair of diverging replisomes. The data show that there is no obligatory coupling between sister replisomes and, together with other studies, imply that genome duplication involves autonomously functioning replisomes.