Exam 1 Study guide PCB3134 Fall 2013 Lacey Nilles 1 Four tenets of the cell theory 1 The cell is the basic unit of life 2 All organisms are made of one or more cells 3 New cells arise only from division of pre existing cells not necessarily true anymore synthetic biology has come a long way three branches of an evolutionary tree 4 Cells evolved from more primitive common progenitor cells along 2 Principles of modern microscopy methods a The optical path of a conventional light microscope Conventional light refers to bright field microscopy The specimen is mounted on a transparent glass slide and positioned on the movable specimen stage Light goes through the specimen and is focused by the objective lens It is projected through the eyepiece b How to determine the resolution of an objective lens Know that resolution is different from magnification Resolution is the ability to distinguish between two very closely positioned objects The resolution of a microscope is represented by D the minimum distance between two distinguishable objects The smaller the value of D the better the resolution Here is the equation to determine resolution D 0 61 NA NA stands for Numerical Aperture or N sin OR D 0 61 Nsin o is the wavelength of the incident light o N is the refractive index of the medium between the o is the angular aperture or half angle of the cone of specimen and the objective lens light entering the objective lens from the specimen For a higher resolution or smaller D shorten wavelength or increase N or both gather more light The higher the Numerical Aperture the higher the lens resolution For dry lenses the NA can t be larger than 1 0 If using oil the NA is 1 4 The highest NA could be is 1 7 c Fluorescence and principles of fluorescence microscopy Fluorescence when a chemical absorbs light at one wavelength and emits it at a specific and longer wavelength of less energy Therefore it absorbs wavelength and gives off heat energy In modern fluorescence microscopes only fluorescent light emitted by the sample is used to form an image Light from the UV source of the exciting wavelength induces the fluorescence but is then not allowed to pass the filters placed between the objective lens and the eye or camera This filter is a dichroic mirror that refracts UV light and passes fluorescent light The objective lens serves as a condenser and transmits light to the detector When proteins are tagged with naturally occurring green fluorescent protein GFP or its variants are expressed in live cells they can be visualized in a fluorescence microscope Fluorescent microscopy has a greater resolution than bright light microscopy because fluorescent light has a shorter wavelength 400nm vs 600nm respectively Immunofluorescence labeling of antibodies antigens with fluorescent probes The FC domain is labeled with fluorescent dye fluorophore d Applications of fluorescence microscopy FRAP FRET Fluorescence Resonance Energy Transfer FRET is used to detect protein protein interactions by identifying changes in wavelength FRAP Fluorescence Recovery After Photobleaching Scientists label membrane proteins with a fluorescent reagent fluorophore Then they bleach a section of membrane proteins This bleaching ruins the fluorescent activity By tracking the bleached proteins or phospholipids without fluorescent activity over time scientists can see lateral movement in the membrane This experiment FRAP supports the Fluid Mosaic Model of biomembranes components of membranes such as proteins and lipids are laterally mobile only 2 D ie lipids and proteins do not move from one leaflet to the other spontaneously The results showed that about 50 of the labeled lipids and proteins moved laterally This means that some molecules are mobile and some are constrained Below is a picture of FRAP e Transmission electron microscopy resolution and sample preparation In TEM electrons are emitted from a filament and accelerated in an electric field A condenser lens focuses the electron beam onto the sample objective and projector lenses focus the electrons that pass through the specimen and project them onto a viewing screen or other detector Electrons used in TEM have a much shorter wavelength than light used in light microscopy Therefore TEM provides a much higher resolution of cell structure Preparation of TEM is similar to LM preparation In TEM samples are commonly stained with heavy metals such as lead and uranium The stained areas appear dark because the metals scatter most of the incident electrons and scattered electrons are not focused by the electromagnetic lenses and do not contribute to the image Areas that take up less stain appear lighter Because electrons are easily scattered by air molecules the entire column is maintained at a very high vacuum LM TEM 3 Protein structure and function a 20 AAs and properties of their R groups elongation of a polypeptide by peptide bond The basic structure of an amino acid is an amine group a carboxyl group and an R group side chain R groups that are hydrophilic aspartate and glutamate are acidic positive charge lysine arginine histidine are basic negative charge serine threonine asparagine and glutamine are polar no charge R groups that are hydrophobic alanine valine isoleucine leucine methionine phenylalanine tyrosine and tryptophan R groups that are special cysteine glycine and proline Elongation peptide bond forms between carboxyl group of aan and amine group of aan 1 Primary amino acid structure is linked by covalent peptide bonds that form through dehydration reactions Peptidyl transferase catalyzes this reaction GTP is used not ATP This amino acid synthesis occurs on ribosomes b Primary structure sequence of AAs secondary structure local folding helix and sheet tertiary structure overall conformation of a polypeptide three dimensional structure quaternary structure number and relative positions of subunits in a multimeric protein complex Types of bonds interactions required for stabilizing these structures From structure to function case studies sickle cell anemia Primary Structure Linear structure consisting of single amino acids linked by covalent peptide bonds Secondary Structure local interaction of amino acids helices the protein backbone is stabilized H bonds between C O of aan and N H of aan 4 There are 3 6 residues per turn of the helix R groups stick out from the side of the helix The chemical nature of R groups determines the nature of helix surface or region of surface