Gwangrog Lee

Nanospring behaviour of ankyrin repeats

Ankyrin repeats are an amino-acid motif believed to function in protein recognition; they are present in tandem copies in diverse proteins in nearly all phyla. Ankyrin repeats contain antiparallel α-helices that can stack to form a superhelical spiral. Visual inspection of the extrapolated structure of 24 ankyrin-R repeats indicates the possibility of spring-like behaviour of the putative superhelix. Moreover, stacks of 17–29 ankyrin repeats in the cytoplasmic domains of transient receptor potential (TRP) channels have been identified as candidates for a spring that gates mechanoreceptors in hair cells as well as in Drosophila bristles. Here we report that tandem ankyrin repeats exhibit tertiary-structure-based elasticity and behave as a linear and fully reversible spring in single-molecule measurements by atomic force microscopy. We also observe an unexpected ability of unfolded repeats to generate force during refolding, and report the first direct measurement of the refolding force of a protein domain. Thus, we show that one of the most common amino-acid motifs has spring properties that could be important in mechanotransduction and in the design of nanodevices.

Elastic Coupling Between RNA Degradation and Unwinding by an Exoribonuclease

Loading a Spring To regulate cellular RNA levels, transcription must be balanced by RNA degradation. An important player is the exosome, which can unwind and degrade structured RNA. Lee et al. (p. 1726) used single-molecule fluorescence analysis to investigate how degradation and unwinding are coupled in the catalytic subunit of the yeast exosome complex, Rrp44. Rrp44 apparently digests several base pairs without unwinding, accumulates the energy, which it then uses to unwind four to five base pairs in a burst. Similar spring-like behavior has been proposed for conventional helicases, except that the stored energy comes from hydrolysis of adenosine triphosphate rather than the RNA polymer. Rrp44 stores the energy from snipping off four bases and then uses it to unwind duplex RNA spasmodically. Rrp44 (Dis3) is a key catalytic subunit of the yeast exosome complex and can processively digest structured RNA one nucleotide at a time in the 3′ to 5′ direction. Its motor function is powered by the energy released from the hydrolytic nuclease reaction instead of adenosine triphosphate hydrolysis as in conventional helicases. Single-molecule fluorescence analysis revealed that instead of unwinding RNA in single base pair steps, Rrp44 accumulates the energy released by multiple single nucleotide step hydrolysis reactions until about four base pairs are unwound in a burst. Kinetic analyses showed that RNA unwinding, not cleavage or strand release, determines the overall RNA degradation rate and that the unwinding step size is determined by the nonlinear elasticity of the Rrp44/RNA complex, but not by duplex stability.

Fast and Forceful Refolding of Stretched α-Helical Solenoid Proteins

Anfinsens thermodynamic hypothesis implies that proteins can encode for stretching through reversible loss of structure. However, large in vitro extensions of proteins that occur through a progressive unfolding of their domains typically dissipate a significant amount of energy, and therefore are not thermodynamically reversible. Some coiled-coil proteins have been found to stretch nearly reversibly, although their extension is typically limited to 2.5 times their folded length. Here, we report investigations on the mechanical properties of individual molecules of ankyrin-R, beta-catenin, and clathrin, which are representative examples of over 800 predicted human proteins composed of tightly packed alpha-helical repeats (termed ANK, ARM, or HEAT repeats, respectively) that form spiral-shaped protein domains. Using atomic force spectroscopy, we find that these polypeptides possess unprecedented stretch ratios on the order of 10-15, exceeding that of other proteins studied so far, and their extension and relaxation occurs with minimal energy dissipation. Their sequence-encoded elasticity is governed by stepwise unfolding of small repeats, which upon relaxation of the stretching force rapidly and forcefully refold, minimizing the hysteresis between the stretching and relaxing parts of the cycle. Thus, we identify a new class of proteins that behave as highly reversible nanosprings that have the potential to function as mechanosensors in cells and as building blocks in springy nanostructures. Our physical view of the protein component of cells as being comprised of predominantly inextensible structural elements under tension may need revision to incorporate springs.

Single-molecule analysis reveals three phases of DNA degradation by an exonuclease.

λ exonuclease degrades one strand of duplex DNA in the 5’-3’ direction to generate a 3’ overhang required for recombination. Its ability to hydrolyze thousands of nucleotides processively is attributed to its ring structure and most studies have focused on the processive phase. Here, we use single molecule FRET to reveal three phases of λ exonuclease reactions: initiation, distributive and processive phases. The distributive phase occurs at early reactions where the 3’ overhang is too short for a stable engagement with the enzyme. A mismatched base is digested five times slower than a Watson-Crick paired base and concatenating multiple mismatches has a cooperatively negative effect, highlighting the crucial role of basepairing in aligning the 5’ end toward the active site. The rate-limiting step during processive degradation appears to be the post-cleavage melting of the terminal base pair. We also found that an escape from a known pausing sequence requires enzyme backtracking.

Atomic cranks and levers control sugar ring conformations

In this paper we review the conformational analysis of sugar rings placed under tension during mechanical manipulations of single polysaccharide molecules with the atomic force microscope and during steered molecular dynamics simulations. We examine the role of various chemical bonds and linkages between sugar rings in inhibiting or promoting their conformational transitions by means of external forces. Small differences in the orientation of one chemical bond on the sugar ring can produce significantly different mechanical properties at the polymer level as exemplified by two polysaccharides: cellulose, composed of β--linked D-glucose, and amylose, composed of α--linked D-glucose. In contrast to β-glucose rings, which are mechanically stable and produce simple entropic elasticity of the chain, α-glucose rings flip under tension from their chair to a boat-like structure and these transitions produce deviations of amylose elasticity from the freely jointed chain model. We also examine the deformation of two mechanically complementary -linked polysaccharides: pustulan, a β--linked glucan, and dextran, a α--linked glucan. Forced rotations about the C5–C6 bonds govern the elasticity of pustulan, and complex conformational transitions that involve simultaneous C5–C6 rotations and chair–boat transitions govern the elasticity of dextran. Finally, we discuss the likelihood of various conformational transitions in sugar rings in biological settings and speculate on their significance.

Reversible and Controllable Nanolocomotion of an RNA-Processing Machinery

Molecular motors have inspired many avenues of research for nanotechnology but most molecular motors studied so far allow only unidirectional movement. The archaeal RNA-exosome is a reversible motor that can either polymerize or degrade an RNA strand, depending on the chemical environments. We developed a single molecule fluorescence assay to analyze the real time locomotion of this nanomachine on RNA. Despite the multimeric structure, the enzyme followed the Michaelis-Menten kinetics with the maximum speed of ∼3 nucleotides/s, showing that the three catalytic cylinders do not fire cooperatively. We also demonstrate rapid directional switching on demand by fluidic control. When the two reaction speeds are balanced on average, the enzyme shows a memory of the previous reaction it catalyzed and stochastically switches between primarily polymerizing and primarily degrading behaviors. The processive, reversible, and controllable locomotion propelled by this nanomachine has a promising potential in environmental sensing, diagnostic, and cargo delivery applications.

A stochastic, cantilever approach to the evaluation of solution phase thermodynamic quantities

A cantilever device based on competitive binding of an immobilized receptor to immobilized and soluble ligand and capable of measuring solution-phase thermodynamic quantities is described. Through multiple binary queries, the device stochastically measures the probability of the formation of a bound complex between immobilized protein and immobilized ligand as a function of soluble ligand concentration. The resulting binding isotherm is described by a binding polynomial consisting of the activities of soluble and immobilized ligand and binding constants for the association of immobilized protein with free and immobilized ligand. Evaluation of the polynomial reveals an association constant for the formation of a complex between immobilized ligand and immobilized protein close to that for the formation of complex between soluble protein and soluble ligand. The methodology lays the foundation for construction of practical portable sensing devices.

DNA looping-dependent autorepression of LEE1 P1 promoters by Ler in enteropathogenic Escherichia coli (EPEC).

Significance Ler [locus of enterocyte effacement (LEE)-encoded regulator], encoded by the first gene of the LEE1 operon in enteropathogenic Escherichia coli (EPEC), represses its own transcription driven by two promoters separated by 10 bp. We found that Ler does this repression through a DNA loop of 16 helical turns, in which RNA polymerase is trapped as open promoter complex, although this complex should be most readily transformed into productive initiation complex. Ler, a homolog of H-NS in enteropathogenic Escherichia coli (EPEC), plays a critical role in the expression of virulence genes encoded by the pathogenic island, locus of enterocyte effacement (LEE). Although Ler acts as an antisilencer of multiple LEE operons by alleviating H-NS–mediated silencing, it represses its own expression from two LEE1 P1 promoters, P1A and P1B, that are separated by 10 bp. Various in vitro biochemical methods were used in this study to elucidate the mechanism underlying transcription repression by Ler. Ler acts through two AATT motifs, centered at position −111.5 on the coding strand and at +65.5 on the noncoding strand, by simultaneously repressing P1A and P1B through DNA-looping. DNA-looping was visualized using atomic force microscopy. It is intriguing that an antisilencing protein represses transcription, not by steric exclusion of RNA polymerase, but by DNA-looping. We propose that the DNA-looping prevents further processing of open promoter complex (RPO) at these promoters during transcription initiation.

Allosteric ring assembly and chemo-mechanical melting by the interaction between 5′-phosphate and λ exonuclease

Phosphates along the DNA function as chemical energy frequently used by nucleases to drive their enzymatic reactions. Exonuclease functions as a machine that converts chemical energy of the phosphodiester-chain into mechanical work. However, the roles of phosphates during exonuclease activities are unknown. We employed λ exonuclease as a model system and investigated the roles of phosphates during degradation via single-molecule fluorescence resonance energy transfer (FRET). We found that 5′ phosphates, generated at each cleavage step of the reaction, chemo-mechanically facilitate the subsequent post-cleavage melting of the terminal base pairs. Degradation of DNA with a nick requires backtracking and thermal fraying at the cleavage site for re-initiation via the formation of a catalytically active complex. Unexpectedly, we discovered that a phosphate of a 5′ recessed DNA acts as a hotspot for an allosteric trimeric-ring assembly without passing through the central channel. Our study provides new insight into the versatile roles of phosphates during the processive enzymatic reaction.

A graphene oxide-based tool-kit capable of characterizing and classifying exonuclease activities

Enzyme kinetics and classification are key topics in modern biochemistry. Here, we have developed a simple, rapid, and quantitative assay via a terminally capped DNA probe, reaction-specific-labeling and the fluorescence-quenching capability of graphene-oxide. Our technique provides not only characterization of kinetic information but also the immediate classification of an exonuclease without needing to design a set of DNA substrates.