1/40. Cellular mechanics I nd term

Transcription

1 1/40 Cellular mechanics I nd term

2 Various Science 2/40 Biology Physics Chemistry Biology is the science of life.... Biologists study the structure, function, growth, origin, evolution and distribution of living organisms. Biology is the natural science that studies life and living organisms, including their physical structure, chemical processes, molecular interactions, physiological mechanisms, development and evolution The dictionary definition of physics is the study of matter, energy, and the interaction between them, but what that really means is that physics is about asking fundamental questions and trying to answer them by observing and experimenting. Physics is the natural science that studies matter and its motion and behavior through space and time and that studies the related entities of energy and force. Chemistry is the scientific discipline involved with compounds composed of atoms, i.e. elements, and molecules, i.e. combinations of atoms: their composition, structure, properties, behavior and the changes they undergo during a reaction with other compounds. Chemistry is the study of matter, its properties, how and why substances combine or separate to form other substances, and how substances interact with energy.

3 Physics is about great laws Newton s 3 Laws (Mechanics) (Stat. Mechanics of Entropy) S= k log W 3/40 (Electricity & Magnetism) Maxwell s 4 Equations (Quantum Mechanics) Schrodinger s Equation Ludwig Boltzmann Isaac Newton James Clerk Maxwell (Thermodynamics) Gibb s Free Energy Erwin Schrödinger J. Willard Gibbs

4 Does biology have any great theories or law? 4/40 Evolution -- Life evolved from simpler forms --One of the best tested scientific theories around Evolution is a series of tricks/random events Charles Darwin, Age 51, 1860, On the Origin of Species Build complex beings from simpler parts Often many ways of doing this Our life form is just one. Gregor Johann Mendel ( )

5 Biophysics 5/40 Biophysics is an interdisciplinary science that applies the approaches and methods of physics to study biological systems. Biophysics applies the principle of physics and chemistry and the methods of methodical analysis and computer modeling to biological system, with the ultimate goal of understanding at a fundamental level the structure, dynamics, interactions, and ultimately the function of biological system. Biophysics covers all scales of biological organization, from molecular to organismic and population.

6 Biophysics_New fields 6/40 A new filed of sciences 1892 Karl Pearson termed biophysics Modern, interdisciplinary field of science for understanding biological phenomenon. Explanations based on physical and mathematical concepts Investigations and experiments with physical instruments Bragg at the Cavendish Lab Cambridge Mechanics Kinetics Electricity Magnetism Bio Acoustics Thermodynamics Hydrodynamics Mathematics Optics

7 Bio-physics-engineering 7/40

8 What is the goal of biophysics? 8/40 Create simplified models of biological systems - If all life follows the same basic rule, what is it? Make quantitative predictions and experimental test Develop new technology Advance understanding about bio-system

9 What are biophysicist working on? 9/40 Research in biochemistry and biophysics is focused on numerous process central to understanding life. Biophysicists study life at every level, from atoms and molecules to cells, organisms, and environments Several groups use biochemical and structural approaches to address the basic principle governing protein folding, function and biological recognition. Using in vitro approaches, the central steps in biological information transfer are being analyzed, from maintenance of the genome to protein synthesis, sorting and processing. - How do protein machines work? - How do systems of never cells communicate? - How do proteins pack DNA into viruses? - How do viruses invade cells? - How do cells move? - What are the mechanical properties of cells? How does this relate to disease?

10 Some aspects of biophysics 10/40 1. Biophysics at the molecular level: Determination and prediction of protein structures Single-molecule spectroscopy Molecular motors The protein folding problem DNA-protein interactions.. 2. Biophysics at the cellular level: Transport within and across cell membranes Structure and properties of cell membranes Propagation of neural signals Cytoskeleton and cell movements Cytokinesis. 3. Biophysics at multicellular and higher levels: Tissue and biomedical engineering Physical and mathematical physiology Biomechanics and bio-rheology Population dynamics and theory of evolution Mathematical epidemiology.

11 Luigi Galvani 11/40 Electro-Physiology Discovered Bio-electricity 1787, Frog muscle experiment Physicist, biologist, and philosopher Luigi Galvani touched the nerves of a frog's spinal cord with metal electro des which caused contractions of the leg muscles. Electricity is the language of the nervous system.

12 DNA structure 12/ X-rays discovered by Roentgen 1914 First diffraction pattern of a crystal made by Knipping and von Laue 1915 Theory to determine crystal structure from diffraction pattern developed by Bragg 1953 DNA structure solved by Watson and Crick Max Theodor Felix von Laue German physicist who won the Nobel Prize in Physics in 1914 for his discovery of the diffraction of X-rays by crystals Sir William Lawrence Bragg, 31 March July 1971 Australian-born British physicist and X- ray crystallographer, discoverer (1912) of Bragg's law of X-ray diffraction, which is basic for the determination of crystal structure

13 Double slit interference 13/40 Coherence of light waves mean they have the same phase and frequency Diffraction: Spreading of light passing through a small aperture or around a sharp edge

14 14/40 Constructive interference ΔL = nλ n = 0, 1, 2, Destructive interference ΔL = (2n+1)λ/2 n = 0, 1, 2,

15 X-ray interacts with matter 15/40

16 Coherent scattering by an atom 16/40 Incoming X-rays are electromagnetic waves that exert a force on atomic electrons. The electrons will begin to oscillate at the same frequency and emit radiation in all directions. Coherent scattering by an atom is the sum of this scattering by all of the electrons. Electrons are at different positions in space, so coherent scattering from each generally has different phase relationships. At higher scattering angles, the sum of the coherent scattering is less. e - 2q

17 Diffraction patterns 17/40 Two successive CCD detector images with a crystal rotation of one degree per image:

18 Diffraction of X-rays by crystals 18/40 The science of X-ray crystallography originated in 1912 with the discovery by Max von Laue that crystals diffract X-rays. Von Laue was a German physicist who won the Nobel Prize in Physics in 1914 for his discovery of the diffraction of X-rays by crystals. X-ray diffraction pattern from a single-crystal sample Max Theodor Felix von Laue ( )

19 Diffraction of X-rays by crystals 19/40 After Von Laue's pioneering research, the field developed rapidly, most notably by physicists William Lawrence Bragg and his father William Henry Bragg. William Henry Bragg In , the younger Bragg developed Bragg's law, which connects the observed scattering with reflections from evenly-spaced planes within the crystal. William Lawrence Bragg

20 Diffractogram 51 20/40 The best X-ray scattering on DNA (Franklin and Gosling, 1952). Only diffractograms with enough order can be analyzed quantitatively by applying the theory if X-ray scattering on helical molecules. Rosalind Elsie Franklin ( ) Her X-ray diffraction produced the most beautiful pattern that could be analyzed quantitatively.

21 DNA is a double helix! Saturday February 28, /40 A model is built in the Cavendish laboratory in Cambridge consistent with all the discernible features of the diffractogram 51...

22 22/40 The distinctive X in this X-ray photo is the telltale pattern of a helix Because the X-ray pattern is so regular, the dimension of the helix must be constant. For example, the diameter of the helix stays the same.

23 23/40 The helix s pitch, or its degree of rise, can be calculated from the angle the X makes with the horizontal axis. If we distort the helix, you can get an idea how the helical pitch is related to the X-ray pattern.

24 In an X-ray diffraction pattern, the closer the spots, the larger the actual distance. So the horizontal bars actually correspond to helical turns. The vertical distance between the bars is a measure of the height of the one helical turn. 24/40

25 Since, we know the height of one helical repeat (34 Å ) and we know the distance between the stacked base pairs (3.4 Å ), there must be 10 nucleotides per helical repeat. 25/40

26 Central dogma of molecular biology 26/40 DNA molecules serve as templates for either complementary DNA strands during the process of replication or complementary RNA during the process of transcription. RNA molecules serve as a template for ordering amino acids by ribosomes during protein synthesis. DNA mrna protein DNA TRANSCRIBES to mrna Process is called transcription mrna TRANSLATES to proteins Process is called translation Actually makes amino acids, which come together to make proteins

27 Molecular biophysics 27/40 For biological systems to function, interactions occur between many different types of molecules: DNA, RNA, Protein, Lipids, etc. To ensure that biological systems function appropriately, such interactions must be carefully regulated Wide range of biophysical chemistry approaches are useful for studying these interactions Interactions between molecules are central to how cells detect and respond to signals and affect: Gene expression (transcription & translation) DNA replication, repair and recombination Signalling And many other processes... Interactions are mainly mediated by many weak chemical bonds (van der Waals forces, hydrogen bonds, hydrophobic interactions) Accumulation of many bonds influences affinity and specificity of interactions

28 Examples of molecular biophysics 28/40 Structure and Conformation of Biological Molecules Structure Function Relationships Conformational Transitions Ligand Binding and Intermolecular Binding Diffusion and Molecular Transport Membrane Biophysics DNA and Nucleic Acid Biophysics Protein Biophysics Energy Flow and Bioenergetics Thermodynamics Statistical Mechanics Kinetics Molecular Machines Allosterics

29 Protein structure 29/40 Primary structure Assembly Secondary structure Folding Tertiary structure Packing Quaternary structure Interaction

30 Protein folding 30/40 Transcription Translation Folding DNA RNA Polypeptide Functional protein Linear nucleic acid Linear nucleic acid Linear amino acid sequence Three dimensional structure Proteins fold spontaneously under physiological conditions - In the equilibrium between the denatured state (unfolded or partially unfolded) and the native state (folded, biologically functional), under physiological conditions the vast majority of molecules are in the native state Renaturation Denaturation Primary structure Unfolded structure Inactive collection of many random structures Tertiary structure Folded structure Active 3-dimensional structure of protein

31 Protein structure analysis 31/40 Experimental determination and analysis Repositories Protein Data Bank Molecular Modeling DataBase Resolution X-Ray Crystallography NMR Spectroscopy Mass Spectroscopy (next week) Fluorescence Resonance Energy Transfer Computational determination and analysis Databases CATH (Class, Architecture, Topology, Homologous superfamily) SCOP (Structural Classification Of Proteins) FSSP (Fold classification based on Structure- Structure alignment of Proteins) Prediction Ab-initio, theoretical modeling, and conformation space search Homology modeling and threading Energy minimization, simulation and Monte Carlo

32 Protein Data Bank 32/40 The Protein Data Bank was established at Brookhaven National Labs in 1971 as an archive of biological macromolecular crystal structures. Since October 1998, the PDB database has been managed by the Research Collaboratory for Structural Bioinformatics (RCSB), which is a consortium consisting of Rutgers, the State University of New Jersey; The San Diego Supercomputer Centre at the University of California, San Diego; and the National Institute of Standards and Technology.

33 33/40 The PDB archive contains macromolecular structure data on proteins, nucleic acids, protein-nucleic acid complexes, and viruses. Files in its holdings are deposited by the international user community and maintained by the RCSB PDB staff. Approximately new structures are deposited each week. They are annotated by RCSB and released upon the depositor's specifications. PDB data is freely available worldwide. As of 14 March 2017, structures have been deposited in the PDB

34 X-Ray crystallography 34/40 Crystallize and immobilize single, perfect protein Bombard with X-rays, record scattering diffraction patterns Determine electron density map from scattering and phase via Fourier transform: Use electron density and biochemical knowledge of the protein to refine and determine a model

35 NMR Spectroscopy 35/40 Nuclear Magnetic Resonance Protein in aqueous solution, motile and tumbles/vibrates with thermal motion NMR detects chemical shifts of atomic nuclei with non-zero spin, shifts due to electronic environment nearby Determine distances between specific pairs of atoms based on shifts, constraints Use constraints and biochemical knowledge of the protein to determine an ensemble of models determining constraints

36 Fluorescence resonance energy transfer 36/40 FRET described as a molecular ruler segments of a protein are tagged with fluorophores energy transfer occurs when donor and acceptor interact, falls off as 1/d 6 where d is separation between donor and acceptor donor and acceptor must be within 50 Å, acceptor emission sensitive to distance change can determine pairs of side chains that are separated when unfolded and close when folded

37 Optical trap 37/40 Conservation of linear momentum

38 Nucleic-acid polymerases 38/40 Optical tweezers configurations used to study RNA polymerase. (a) A single trap configuration is shown. One end of the DNA is attached to a glass coverslip via a digoxigenin antidigoxigenin linkage, while RNA polymerase is attached to an optically trapped bead via an avidin biotin bond. The bead position is maintained by stage motion to provide constant tension (typically from right to left). (b) A schematic of a dual-trap configuration is shown. An RNA polymerase DNA complex is trapped by two optical traps simultaneously. The left DNA end is manipulated via a digoxigenin antidigoxigenin linkage, while RNA polymerase is attached via an avidin biotin interaction. In this figure, the upstream end of DNA is linked to the left bead so that RNAP transcribes from right to left. The tension of the DNA RNAP complex was kept constant during transcription by moving left stronger trap. Single molecule studies of DNA binding proteins using optical tweezers, Analyst, 2006

39 39/40 Single monomers of HIV-1-PR were manipulated as depicted in Fig. Themolecules were stretched and relaxed by moving the pipette relative to the optical trap, while the applied force and molecular extension were measured. As the protein unfolds and refolds under tension its extension suddenly changes, generating discontinuities (rips) in the stretching and relaxation traces that can be analyzed to gain insight into the kinetic and mechanical properties of the molecule.

40 Magnetic tweezer 40/40 The energy of a superparamagnetic particle in a magnetic field B is given by: U = m B The force experienced by the particle is given by the negative gradient of the energy. F = U= m B For small external fields, the magnetic moment is linear in the external field. In this case, the force is proportional to the gradient of the square of the magnetic field F = χv μ 0 B 2 F For large fields, the magnetic moment of the beads reaches the saturation value m sat and the force is proportional to the gradient of the magnetic field F = m sat B