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Volume ~ 1 µm x 1 µm x 1 µm a single molecule of any kind inside an E. coli cell is at a concentration of 1.6 nM! most nucleic acid-binding proteins possess.

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Presentation on theme: "Volume ~ 1 µm x 1 µm x 1 µm a single molecule of any kind inside an E. coli cell is at a concentration of 1.6 nM! most nucleic acid-binding proteins possess."— Presentation transcript:

1 volume ~ 1 µm x 1 µm x 1 µm a single molecule of any kind inside an E. coli cell is at a concentration of 1.6 nM! most nucleic acid-binding proteins possess dissociation constants K d ~ M M. Thus, as an example, a single DNA binding protein molecule of any kind in E coli, with dissociation constant of M or smaller, would be 94% or more of the time bound to its DNA binding site. So, it is not surprising that many proteins in a bacterial cell are present only in single or in very few copies. When some proteins are present in some tens or hundreds of copies, it is often because they are being used quite effectively by the cell to favor certain processes over others, or because the cell must simultaneously control may processes at once, as happens in transcription regulation of certain critical genes during the exponential growth of the cell. A nanochemical approach to biology: the cell is not a macroscopic system E. Coli cell (http://history.nih.gov/exhibits/nirenberg/HS6_univ.htm) ( from C. Bustamante “Physical Biology at the crossroads” Welch Conference 2008)

2 e.g. at pH = 7 (i.e. at H + concentrations of ~ 1 x 10 –7 M), there are only ~ 60 free protons in the whole cell. The pH concepts inside the cell, although strictly correct, can neither properly describe the acid-base balance in the cell interior, nor the local fluctuations of proton concentration away from the average that are necessary for the myriads of cell functions and that must involve the unique donor-acceptor properties of H 2 0. Cell processes do not always display the smooth varying nature of the observables that we have come to associate with the reactions of large numbers of molecules where fluctuations are all but averaged out. Rather, inside the cell, fluctuations dominate and any real attempt to get an insight into its organization must take into account the discrete nature of the chemical transformations taking place among few reacting species. Macroscopic definitions and concepts from macroscopic chemistry are insufficient to describe the interior of the cell ( from C. Bustamante “Physical Biology at the crossroads” Welch Conference 2008) Single mol

3 Single molecule methodologies Instead of to investigate the fluctuating nature of individual molecules and their reactions 2 ex

4 Single molecule methods to overcome the limitation of the traditional ensemble- averaged methodologies conformational averaging The structural complexity and the conformational equilibria of biological molecules temporal averaging Biological molecular motors Conf aver

5 Single molecule methods overcome the conformational averaging of the traditional methodologies Large observation volume An “averaged” picture is obtained: they look all the same but they are not ! + ….. IUP ?

6 Single molecule methods to overcome the limitation of the traditional ensemble- averaged methodologies conformational averaging The structural complexity and the conformational equilibria of the intrinsically unstructured proteins temporal averaging Biological molecular motors Tem. aver

7 The stochastic nature of chemical transactions implies that it is not possible to synchronize molecules undergoing a chemical process except for very short initial times. As a result, bulk experiments provide only averages over all the phases of the process represented by the molecules in the population. This is what in chemistry has been traditionally called “kinetics,” i.e., the description of the dynamics of the mean of the population. In direct contrast, single molecule methods are ideal for investigating the dynamics of complex reactions, for, unlike their bulk counterparts, they make it possible to follow the trajectories of the individual molecules as they undergo their reactions in real-time, thus avoiding the ensemble average over the unsynchronized signals of the molecules in a population. ( from C. Bustamante “Physical Biology at the crossroad” Welch Conference 2008) to overcome the temporal averaging limitation Mol motors

8 Most essential cellular functions such as chromosomal segregation, transport of organelles from one part of the cell to another, or the maintenance of a voltage across the membrane, all involve directional movement of chemical species. Essential processes such as replication, transcription, and translation require the information encoded in the sequence of linear polymers to be read and copied in a directional manner, and cells must often move and orient in response to external chemical gradients and other signals. None of these processes can be carried out by simple diffusion and random collision of the reacting species. The cell needs to insure that these processes are carried out directionally. To overcome the randomizing effect of Brownian motion and perform these directional processes, cells possess molecular entities that have evolved to behave as tiny machine-like devices. These devices operate as molecular motors, converting chemical energy into mechanical work. Molecular motors ( from C. Bustamante “Physical Biology at the crossroad” Welch Conference 2008)

9 The study of molecular motors, has become possible only recently through the use of methods of single molecule manipulation. Using these techniques, quantities that are most natural to these molecular entities and their reactions, i.e., forces, displacements, torques and twist angles can now be directly monitored at the single molecule level and in real-time. These methods are still in an early phase of development, but they are becoming an increasingly powerful tool for investigating the fluctuation dynamics of individual molecules. The study of molecular motors by methods of single molecule manipulations OT

10 Optical tweezers OT

11 (see Davenport et al. 2000). Measure directly the forces in processes in which stresses and strains are developed along the reaction coordinate Optical tweezers RNAP

12 RNA polymerase is a molecular motor DNA sequence is copied by RNA polymerase to produce a complementary nucleotide RNA strand, called messenger RNA (mRNA), because it carries a genetic message from the DNA to the protein-synthesizing machinery of the cell RNA Polymerase translocates along the DNA, exerting a linear force of pN. This forward motion is dependent on the presence of the next incoming ribonucleotide triphosphate (NTP), which is encoded by the DNA template strand sequence.

13 The RNA polymerase polymerase is bound nonspecifically to the glass slide. Transcription can be followed in real time: as it transcribes the DNA, the molecule must thread the DNA through itself, thus pulling the DNA and the bead away from the optical trap. Transcription by RNA polymerase Understanding regulatory mechanism of transcription elongation by which transcription factors modify the mechanical performance of RNAP, allowing it to operate against higher loads and possible roadblocks, such as nucleosomes. (Bustamante, C., Macosko, J.C., and G.J.L. Wuite, “Grabbing the Cat by the Tail: Manipulating Molecules One by One” Nature Reviews Molecular and Cell Biology, 1, 130 – 136 (2000). DNAP

14 DNA polymerase is a molecular motor is an enzyme that assists in DNA replication, is responsible for the synthesis of a new double strande (ds) DNA strand on a single-stranded (ss) template.

15 A ssDNA molecule bound with a primer connects a bead fixed at the end of a micropipette and a bead in the optical trap. A feedback circuit is used to keep the DNA molecule at a fixed tension, F. Replication by DNA polymerase Since ssDNA and dsDNA have different extension at that force, conversion of ssDNA into dsDNA during replication require the pipette to adjust its position relative to the optical trap by an amount proportional to the movement of the enzyme over the template. (Bustamante, C., Macosko, J.C., and G.J.L. Wuite, “Grabbing the Cat by the Tail: Manipulating Molecules One by One” Nature Reviews Molecular and Cell Biology, 1, 130 – 136 (2000). virus

16 The life cycles of many viruses include a self-assembly stage in which a powerful molecular motor packs the DNA genome into the virus's preformed shell (the capsid). DNA translocation motor

17 The DNA passes into the shell through a channel formed by a structure called the connector Biochemical and biophysical studies have identified essential components of the packaging machinery and measured various characteristics of the packaging process, while crystallography and electron microscopy have provided snapshots of viral structure before and after packaging. Structurally motivated models over the past 30 years have coupled DNA movement to rotation of the connector relative to the capsid. cryo-em reconstruction of the full motor complex ATPase in red, capsid in gray with a top view cartoon of the ATPase ring alone (below, gray). The packaging motor

18 To probe the dynamics of the packaging motor of  29, single prohead-DNA complexes are tethered between two polystyrene beads held in two optical traps. DNA translocation by the motor is determined from the decrease in the contour length of the DNA tether and is followed with base-pair-scale resolution Representative packaging traces collected under low external load, ~8 pN, and different [ATP]: 250 µM, 100 µM, 50 µM, 25 µM, 10 µM, and 5 µM in purple, brown, green, blue, red, and black, respectively.

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20 The DNA inside some viruses is packed so tightly that the internal pressure reaches ten times that in a champagne bottle The motor can pack DNA to a pressure of about 60 atmospheres. A bottle of champagne typically is under pressure of five to six atmospheres It is stronger than the motors that move our muscles or the nanoscale molecular motors that duplicate DNA or transcribe it into RNA this high pressure helps the virus spurt its DNA into a cell once it has latched onto the surface

21 The 10-bp Bursts are Composed of Four 2.5-bp Steps

22 Schematic diagram of the two-phase mechanochemical cycle of  29 overlaid on a sample packaging trace. (b) Detailed kinetics of ATP binding. ATP binding occurs in two steps, ATP docking (green, T) followed by tight-binding (red, T * ).

23 Schematic diagram of the communication between subunits during ATP binding. Upon tight-binding of an ATP, the binding pocket of the next subunit, formerly inactive (gray), is activated for docking (green). (d) Schematic depiction of the full mechanochemical cycle of j29. During the burst phase, ADP may remain on the ring (blue) to be released in the dwell phase. One subunit must be distinct from the others (purple) in order to break the symmetry of the motor and generate only 4 steps per cycle. The identity of this subunit may change each cycle. Un motore a cinque cilindri

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25 Understanding the code that controls the processes of biological self- assembly and what the physical determinants (stereo-specificity, strength, directionality, reversibility, cooperativity of assembly, etc…) will not only reveal one of the essential features of the living state, but also will make it possible for scientists to apply the lessons learned from biology to non-biological building blocks and endow them with some of the same properties to obtain ever more complex supra- molecular architectures. ( from C. Bustamante “Physical Biology at the crossroads” Welch Conference 2008) Understanding the code that controls the processes of biological self-assembly DNA

26 We chemists want to understand molecules and their intrinsic essence, and to be able to predict what happens when molecules meet and do they attach weakly to each other or do they react passionately to form new molecules? Not least, we want to understand the complicated chemistry called life. Through a revolution in knowledge, molecules today take center stage in all fields, from biology and medicine through environmental sciences, and technology The world of molecules


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