Review Article
Determination of thermodynamics and kinetics of RNA reactions by force
- Ignacio Tinoco, Pan T. X. Li, Carlos Bustamante
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- Published online by Cambridge University Press:
- 16 October 2006, pp. 325-360
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1. Introduction 326
2. Instrumentation 328
2.1 Instruments to study mechanical properties of RNA 328
2.1.1 AFM 328
2.1.2 Magnetic tweezers 328
2.1.3 Optical tweezers 330
2.2 Optical trap instrumentation 330
2.3 Calibrations 332
2.3.1 Calibration of trap stiffness 332
2.3.2 Calibration of force 333
2.3.3 Calibration of distance 334
2.4 Types of experiments 334
2.4.1 Force-ramp 334
2.4.2 Force-clamp or constant-force experiments 335
2.4.3 Extension-clamp or constant extension experiments 335
2.4.4 Force-jump, Force-drop 336
2.4.5 Passive mode 336
3. Thermodynamics 336
3.1 Reversibility 336
3.2 Gibbs free energy 337
3.2.1 Stretching free energy 338
3.2.1.1 Rigid molecules 338
3.2.1.2 Compliant or flexible molecules 339
3.2.2 Free energy of a reversible unfolding transition 339
3.2.3 Free energy of unfolding at zero force 340
3.2.4 Free energy of an irreversible unfolding transition 340
3.2.4.1 Jarzynski's method 341
3.2.4.2 Crooks fluctuation theorem 343
4. Kinetics 345
4.1 Measuring rate constants 345
4.1.1 Hopping 345
4.1.2 Force-jump, Force-drop 347
4.1.3 Force-ramp 348
4.1.4 Instrumental effects 350
4.2 Kinetic mechanisms 351
4.2.1 Free-energy landscapes 351
4.2.2 Kinetics of unfolding 353
5. Relating force-measured data to other measurements 354
5.1 Thermodynamics 354
5.2 Kinetics 357
6. Acknowledgements 357
7. References 358
Single-molecule methods have made it possible to apply force to an individual RNA molecule. Two beads are attached to the RNA; one is on a micropipette, the other is in a laser trap. The force on the RNA and the distance between the beads are measured. Force can change the equilibrium and the rate of any reaction in which the product has a different extension from the reactant. This review describes use of laser tweezers to measure thermodynamics and kinetics of unfolding/refolding RNA. For a reversible reaction the work directly provides the free energy; for irreversible reactions the free energy is obtained from the distribution of work values. The rate constants for the folding and unfolding reactions can be measured by several methods. The effect of pulling rate on the distribution of force-unfolding values leads to rate constants for unfolding. Hopping of the RNA between folded and unfolded states at constant force provides both unfolding and folding rates. Force-jumps and force-drops, similar to the temperature jump method, provide direct measurement of reaction rates over a wide range of forces. The advantages of applying force and using single-molecule methods are discussed. These methods, for example, allow reactions to be studied in non-denaturing solvents at physiological temperatures; they also simplify analysis of kinetic mechanisms because only one intermediate at a time is present. Unfolding of RNA in biological cells by helicases, or ribosomes, has similarities to unfolding by force.
The structure of aquaporins
- Tamir Gonen, Thomas Walz
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- Published online by Cambridge University Press:
- 11 December 2006, pp. 361-396
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1. Introduction 362
1.1 The elusive water pores 362
1.2 CHIP28 362
2. Studies on AQP-1 363
2.1 Expression of AQP1 cDNA in Xenopus oocytes 363
2.2 Reconstitution of purified AQP1 into artificial lipid bilayers 364
2.3 Structural information deduced from the primary sequence 365
2.4 Evolution and mammalian AQPs 365
3. Chronological overview over AQP structures 368
3.1 AQP1 – the red blood cell water pore 368
3.2 GlpF – the E. coli glycerol facilitator 371
3.3 AQPZ – the E. coli water pore 372
3.4 AQP0 – the lens-specific aquaporin 373
3.5 AQP4 – the main aquaporin in brain 377
3.6 SoPiP2;1 – a plant aquaporin 379
3.7 AQPM – an archaeabacterial aquaporin 379
4. Proton exclusion 380
5. Substrate selectivity 382
6. Pore regulation 385
6.1 Hormonal regulation of AQP trafficking 385
6.2 Influence of pH on AQP water conduction 386
6.3 Regulation of AQP pore conductance by protein binding 387
6.4 Pore closure by conformational changes in the AQP0 pore 388
7. Unresolved questions 390
8. Acknowledgments 390
9. References 391
The ubiquitous members of the aquaporin (AQP) family form transmembrane pores that are either exclusive for water (aquaporins) or are also permeable for other small neutral solutes such as glycerol (aquaglyceroporins). The purpose of this review is to provide an overview of our current knowledge of AQP structures and to describe the structural features that define the function of these membrane pores. The review will discuss the mechanisms governing water conduction, proton exclusion and substrate specificity, and how the pore permeability is regulated in different members of the AQP family.