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1. EVERYTHING IS CONNECTED IN CELL
METABOLISM? |
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Definition
Two opposing streams of chemical reactions occur in cells: (1) the catabolic pathways break down foodstuffs into smaller molecules, thereby generating both a useful form of energy for the cell and some of the small molecules that the cell needs as building blocks, and (2) the anabolic, or biosynthetic, pathways use the energy harnessed by catabolism to drive the synthesis of the many other molecules that form the cell. Together these two sets of reactions constitute the metabolism of the cell (Figure 2-36).
In fact, the metabolic balance of a cell is amazingly stable. Whenever the balance is perturbed, the cell reacts so as to restore the initial state. The cell can adapt and continue to function during starvation or disease. Mutations of many kinds can damage or even eliminate particular reaction pathways, and yet—provided that certain minimum requirements are met—the cell survives. It does so because an elaborate network of control mechanisms regulates and coordinates the rates of all of its reactions. These controls rest, ultimately, on the remarkable abilities of proteins to change their shape and their chemistry in response to changes in their immediate environment. The principles that underlie how large molecules such as proteins are built and the chemistry behind their regulation will be our next concern.
EXAMPLE: METHOTREXATE INHIBITS DHFR: is a good example of how what’s done in one place in the cell can lead to numerous changes and trigger unusual events within the cell. |
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2.METHOTREXATE (METH-TREX=drug) changes how
DHFR (an enzyme)
acts and what it makes????? (fxn&production)
HOW? (HINT: gene duplicatation and stabilization) |
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Definition
| the gene for the enzyme dihydrofolate reductase (DHFR) often becomes amplified in response to cancer chemotherapy with the folic-acid antagonist methotrexate.To make matters worse, cells that are exposed to one anticancer drug often develop a resistance not only to that drug, but also to other drugs to which they have never been exposed. This phenomenon of multidrug resistance is frequently correlated with amplification of a part of the genome that contains a gene called Mdr1 |
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2.METHOTREXATE BLANKS DHFR CAUSING
SO...HOW DOES THE CELL DEAL WITH THE PROBLEM
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Definition
INHIBITS; Inhibiting this enzyme cause rapidly proliferating cells to preferentially die off
•DHFR is a key enzyme in many cellular processes the cell attempts to deal with the situation in a number of ways.
•Novell pathways and processes not normally used by the cell are activated to deal with the problem.
SO TO DEAL WITH PROB:
•One of the first things that happens is a stabilization of the mRNA for DHFR. This allows more copies of the protein to be made from a single mRNA molecule and has the effect of increasing the amount of enzyme in the cell.
•This stabilization occurs in an interest manner – the enzyme DHFR actually binds to a portion of the non-coding region of it’s own message and stabilizes it – we’ll see more examples of this kind of feedback regulation later in the course.
•Second and more importantly ( and more interesting) is that complex series of events are triggered that lead to a process called gene amplification. Here the cell begins to make multiple copies of the DHFR gene- often several hundred to a thousand tandem (end to end repeats) of the gene. This leads to a vast increase in the amount of DHFR enzyme in the cell and almost total resistance to the drug.
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3.p450 ENZYMES are imPortant??? WHY, HOW?
EXAMPLE????
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Definition
| The amount and type of p450 isoform one has is vitally important in how one react to drugs, medications, environmental toxins, vitamins, alcohol and even coffee. |
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4.MAGNIFICATION RANGE?
MY EYE
VS.
LIGHT MICROSCOPE |
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Definition
LOOK AT SLIDE PICTURE
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the eye near point is usually about 25 cm; as people age, Because humans can't focus on anything beyond the near point, the maximum magnification of the human eye---in terms of the size of the image that forms on the retina as compared to the size of the object itself---.068 cm.
Light Microscopes which are limited by the physics of light to 500x or 1000x magnification
Read more: What Is the Maximum Magnification of the Human Eye? | eHow.com http://www.ehow.com/about_6622019_maximum-magnification-human-eye_.html#ixzz1YuqHk2yg
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| 5.What limits what we can see thru as scope? What is optical diffraction? |
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Definition
LIMITS:
1) The wavelength of the light source
2) The numerical aperture of the lens system
3) The quality of the optics themselves
Since optics aren’t perfect
Light actually travels a variety
From the light source to your
Eye. This phenomena is called
Optical diffraction. Optical
Diffraction can actually be
used to improve resolution.
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6.What is one of the main uses for phase contrast microscopy?
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Definition
•Much of the work done with a microscope is done on samples that have been fixed and stained to either provide contrast or to make specialized features stand out. This is OK for many uses but sometimes you need to work with living tissue.
•Phase contrast or differential interference microscopy can be used to look at living tissues.
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Term
7.Describe fluorescent microscopy. How does it work and what is it useful for?
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Definition
•A chemical is fluorescent if it absorbs light at one wavelength, and emits light at longer specific wavelength. In Fluorescence Microscopy, fluorescent dyes are used to treat purified antibodies. The fluorescent dye-antibody complex is then added to a cell or tissue section, whereupon it binds to the corresponding antigens.
•The cell or tissue section is then placed within the microscope and illuminated by a specific wavelength of light. The light causes the dyed antibodies to fluoresce. The light is captured photographically, producing an image. Since the chosen dye-antibody complex only binds to specific proteins in the cell, the fluorescence acts as a structural marker for regions of interest. By treating a specimen with multiple differently-colored dyed antibodies, multiple proteins can be localized within the same cell. When imaged with multiple corresponding wavelengths of light, multiple images produced, one per dye.
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8.What is indirect immuno fluorescence and how does it work?
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Definition
| from textbook picture explanation: This detection method is very sensitive because the primary antibody is itself recognized by many molecules of the secondary antibody. The secondary antibody is covalently coupled to a marker molecule that makes it readily detectable. Commonly used marker molecules include fluorescent dyes (for fluorescence microscopy), the enzyme horseradish peroxidase (for either conventional light microscopy or electron microscopy), colloidal gold spheres (for electron microscopy), and the enzymes alkaline phosphatase or peroxidase (for biochemical detection). |
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9.Describe Nomarski Interference Microscopy. Why would you use it?
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Definition
•Instead of fluorescence, Nomarski optics use light refraction to highlight cell structure. In this technique, a prism splits an incoming beam of plane-polarized light into two beams. Both beams are projected through the cell but at slightly different angles. After passing through the cell, the beams are combined and exhibit bright and dark interference patterns that highlight areas in the cell that have differing thickness and refractive indexes.
•Nomarskioptics also enable multiple images to be captured, each with a different plane of focus. The resulting images each contain a cross-section of the cell
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10.Explain confocal microscopy and what is it used for?
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Definition
Confocal microscopy is simpler fast and easier- a laser is used to provide a coherent light source on a very small portion of the sample being observed- by this method out of focus light is removed leading to a very sharp image.
Multiple confocal images can be used to produce three dimensional reconstructions |
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11.Explain TEM and SEM. How do they differ?
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Definition
•TEM or transmission electron microscopy can be used to look at details below the level of resolution of light microscopes- down to about 2nM or about 100 times better than the best light microscopes.
•The major drawbacks are that your section needs to be fixed, and extremely thin- this makes it more difficult.
•SEM can be used to obtain three dimensional images of objects.
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12.Describe Indirect immunoflorescence – what would you use it for? |
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Definition
| REPEATED QUESTION/same as question8 |
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13.Describe, briefly, describe the various mechanism we talked about to get materials into living cells. |
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Definition
•Injection – by using an extremely fine needle you can inject directly into single cells. This is how nuclei are transferred in most cloning procedures.
•Electroporation – using short intense bursts of current you can get proteins, RNA and DNA into a cell.
•Lipid vesicles – by encapsulating substance in artificial lipid membranes you can get them into a cell.
•Gene guns – link your DNA of interest to a heavy inert molecule- usually gold- and simply blast it into the cell. This is a very common technique in agricultural molecular biology.
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14.What is green fluorescent protein and how is it used. |
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Definition
•The green fluorescent protein (GFP) is a protein composed of 238 amino acids (26.9kDa), which exhibits bright green fluorescence when exposed to blue light
•The GFP gene can be introduced into organisms and maintained in their genome through breeding, injection with a viral vector, or cell transformation. To date, the GFP gene has been introduced and expressed in many bacteria, yeast and other fungi, fish (such as zebrafish), plant, fly, and mammalian cells, including human.
•Martin Chalfie, Osamu Shimomura, and Roger Y. Tsien were awarded the 2008 Nobel Prize in chemistry on 10 October 2008 for their discovery and development of the green fluorescent protein.
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Term
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Definition
•Fluorescence Resonance Energy Transfer (FRET)
•Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. Thus, FRET is an important technique for investigating a variety of biological phenomena that produce changes in molecular proximity. When FRET is used as a contrast mechanism, colocalization of proteins and other molecules can be imaged with spatial resolution beyond the limits of conventional optical microscopy.
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16.How does fura-2 work? Why would it be useful in nerve cells? |
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Definition
Molecules like fura-2 can be used to determine the relative concentrations of substance within the cell
ANSWER from book (below):
Ca2+-sensitive fluorescent indicators, such as aequorin or fura-2 (discussed in Chapter 9), are often used to monitor cytosolic Ca2+ in individual cells after the inositol phospholipid signaling pathway has been activated. When viewed in this way, the initial Ca2+ signal is often seen to be small and localized to one or more discrete regions of the cell. These signals have been called Ca2+ blips, quarks, puffs, or sparks, and they are thought to reflect the local opening of individual (or small groups of) Ca2+-release channels in the ER and to represent elementary Ca2+ signaling units. If the extracellular signal is sufficiently strong and persistent, this localized signal can propagate as a regenerative Ca2+ wave through the cytosol, much like an action potential in an axon (see Figure 15-37). Such a Ca2+ “spike” is often followed by a series of further spikes, each usually lasting seconds (Figure 15-39). These Ca2+ oscillations can persist for as long as receptors are activated at the cell surface. Both the waves and the oscillations are thought to depend, in part at least, on a combination of positive and negative feedback by Ca2+ on both the IP3-gated Ca2+-release channels and the ryanodine receptors: the released Ca2+ initially stimulates more Ca2+ release, a process known as Ca2+-induced Ca2+ release. But then, as its concentration gets high enough, the Ca2+ inhibits further release.
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17.What is SDS? Where could you find it in your house? |
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Definition
Gel electrophoresis can be used to separate molecules by both size and charge. Proteins are first separated by iso-electric focusing and then by size using standard SDS-PAGE.
Sodium dodecyl sulfate (SDS or NaDS), sodium laurilsulfate or sodium lauryl sulfate (SLS) is an organic compound with the formula CH3(CH2)11OSO3Na). It is an anionic surfactant used in many cleaning and hygiene products. The salt is of an organosulfate consisting of a 12-carbon tail attached to a sulfate group, giving the material the amphiphilic properties required of a detergent. Being derived from inexpensive coconut and palm oils, it is a common component of many domestic cleaning products.
NOTE: SDS is a substance but SDS PAGE is a method of separating proteins.
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18.Describe in detail three ways to get something into a eukaryotic cell. |
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Definition
This pretty much the same question as 13:
•Injection – by using an extremely fine needle you can inject directly into single cells. This is how nuclei are transferred in most cloning procedures.
•Electroporation – using short intense bursts of current you can get proteins, RNA and DNA into a cell.
•Lipid vesicles – by encapsulating substance in artificial lipid membranes you can get them into a cell.
•Gene guns – link your DNA of interest to a heavy inert molecule- usually gold- and simply blast it into the cell. This is a very common technique in agricultural molecular biology.
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19.
Describe the three types of column chromatography and their uses.
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Definition
Column chromatography can be used to separate molecules based on size, charge affinity or a combination of factors.
1. ion-exchange chromatography
2.gel-filtration chromatography
3.affinity chromatography
LOOK at pics on slides, the rest of the info is from the book:
In ion-exchange chromatography (A) the insoluble matrix carries ionic charges that retard the movement of molecules of opposite charge. Matrices used for separating proteins include diethylaminoethylcellulose (DEAE-cellulose), which is positively charged, and carboxymethylcellulose (CM-cellulose) and phosphocellulose, which are negatively charged. Analogous matrices based on agarose or other polymers are also frequently used. The strength of the association between the dissolved molecules and the ion-exchange matrix depends on both the ionic strength and the pH of the solution that is passing down the column, which may therefore be varied systematically (as in Figure 8-12) to achieve an effective separation. In gel-filtration chromatography (B) the matrix is inert but porous. Molecules that are small enough to penetrate into the matrix are thereby delayed and travel more slowly through the column. Beads of cross-linked polysaccharide (dextran, agarose, or acrylamide) are available commercially in a wide range of pore sizes, making them suitable for the fractionation of molecules of various molecular weights, from less than 500 to more than 5 × 106. Affinity chromatography (C) uses an insoluble matrix that is covalently linked to a specific ligand, such as an antibody molecule or an enzyme substrate, that will bind a specific protein. Enzyme molecules that bind to immobilized substrates on such columns can be eluted with a concentrated solution of the free form of the substrate molecule, while molecules that bind to immobilized antibodies can be eluted by dissociating the antibody-antigen complex with concentrated salt solutions or solutions of high or low pH. High degrees of purification are often achieved in a single pass through an affinity column.
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20.
Describe how centrifugation can be used to fractionate cells.
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Definition
Centrifugation is a useful tool in analyzing and purifying molecules of all sorts.
Centrifugation is the first step in most fractionations, but it separates only components that differ greatly in size. A finer degree of separation can be achieved by layering the homogenate in a thin band on top of a dilute salt solution that fills a centrifuge tube. When centrifuged, the various components in the mixture move as a series of distinct bands through the salt solution, each at a different rate, in a process called velocity sedimentation (Figure 8-9A). For the procedure to work effectively, the bands must be protected from convective mixing, which would normally occur whenever a denser solution (for example, one containing organelles) finds itself on top of a lighter one (the salt solution). This is achieved by filling the centrifuge tube with a shallow gradient of sucrose prepared by a special mixing device. The resulting density gradient—with the dense end at the bottom of the tube—keeps each region of the salt solution denser than any solution above it, and it thereby prevents convective mixing from distorting the separation.
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21. How does SDS PAGE work? |
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Definition
from book: (LOOK AT PICTURE)
Figure 8-14SDS polyacrylamide-gel electrophoresis (SDS-PAGE)
(A) An electrophoresis apparatus. (B) Individual polypeptide chains form a complex with negatively charged molecules of sodium dodecyl sulfate (SDS) and therefore migrate as a negatively charged SDS-protein complex through a porous gel of polyacrylamide. Because the speed of migration under these conditions is greater the smaller the polypeptide, this technique can be used to determine the approximate molecular weight of a polypeptide chain as well as the subunit composition of a protein. If the protein contains a large amount of carbohydrate, however, it will move anomalously on the gel and its apparent molecular weight estimated by SDS-PAGE will be misleading.
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22.
What’s proteonomics? Why is it useful?
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Definition
answer from online source; nothing in book or powerpt
Proteomics is the large-scale study of proteins, particularly their structures and functions.[1][2] Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term "proteomics" was first coined in 1997[3] to make an analogy with genomics, the study of the genes. The word "proteome" is a blend of "protein" and "genome", and was coined by Marc Wilkins in 1994 while working on the concept as a PhD student.[4][5] The proteome is the entire complement of proteins,[4] including the modifications made to a particular set of proteins, produced by an organism or system. This will vary with time and distinct requirements, or stresses, that a cell or organism undergoes.
After genomics, proteomics is considered the next step in the study of biological systems. It is much more complicated than genomics mostly because while an organism's genome is more or less constant, the proteome differs from cell to cell and from time to time. This is because distinct genes are expressed in distinct cell types. This means that even the basic set of proteins which are produced in a cell needs to be determined.
In the past this was done by mRNA analysis, but this was found not to correlate with protein content.[6][7] It is now known that mRNA is not always translated into protein,[8] and the amount of protein produced for a given amount of mRNA depends on the gene it is transcribed from and on the current physiological state of the cell. Proteomics confirms the presence of the protein and provides a direct measure of the quantity present. |
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23.Describe the GST system |
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Definition
| GST fusion proteins, generated by standard recombinant DNA techniques, can be captured on an affinity column containing beads coated with glutathione. To look for proteins that bind to protein X, cell extracts can be passed through this column. The hybrid protein and its binding partners can then be eluted with glutathione. The identities of these interacting proteins can be determined by mass spectrometry (see Figure 8-20). In an alternative approach, a cell extract can be made from a cell producing the GST fusion protein and passed directly through the glutathione affinity column. The GST fusion protein, along with proteins that have associated with it in the cell, are thereby retained. Affinity columns can also be made to contain antibodies against GST or another convenient small protein or epitope tag (see Figure 8-48). |
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24.How does gentamyacin interact with defective CF chloride channels to get them to function? |
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Definition
•Currently much of the emphasis in drug discovery and clinical treatment is shifting away from sequence analysis to the field of protein interactions. By being able to modify the shape of a protein you can alter it’s activity- gentamyacin will interact with some defective CF channel proteins and restore channel activity.
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25.
Describe the formation of a peptide bond- does it make or require energy?
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Definition
The shape of a protein is initially determined by it’s primary or amino acid sequence. Amino acids are hooked together by peptide bonds- a type of condensation reaction (a removal of water so .
Peptide bonds lead to a repeating sequence of atoms along the core of the protein called the polypeptide backbone from which specific amino acids R or side groups protrude. These side chains are what differentiate the individual amino acids and give them their specific characteristics.
from internet:
A peptide bond (sometimes mistakenly called amino bond) is a covalent bond that is formed between two molecules when the carboxyl group of one molecule reacts with the amino group of the another molecule, releasing a molecule of water. This is a a condensation reaction and usually occurs between amino acids. The resulting CO-NH bond is called a peptide bond, and the resulting molecule is an amide.
- Peptide bond formation is an endothermic reaction. This means that the reaction requires the absorption of energy: It does not take place spontaneously.
- Peptide bond formation is a condensation reaction. It hence involves the net removal of a water molecule. So not only can this reaction not happen spontaneously in an aqueous medium, but, in fact, the presence of water inhibits the reaction.
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26.
Describe the backbone of a primary peptide structure
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Definition
| A polypeptide chain consists of a repeating backbone of N-C-C-N-C-C |
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Definition
R groups or side chains protrude from this backbone. Some are polar ( water loving) others nonpolar ( water hating) and others neither. The relative polarity and charge of the R groups does much to help determine a proteins three dimensional shape.
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28.What are the types of R groups and why are they important|?
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Definition
The type of side chain present is an important factor in protein folding- nonpolar side chains tend to cluster in the interior of a protein in aqueous solution and polar side chains tend to be exposed to the aqueous environment.
So two types: polar and nonpolar
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29.
How are these R(residue/side group) groups used in active sites?
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Definition
Alpha helix’s can also wrap around each other to produce coiled strands. This happens when the non-polar R groups of two separate helixes are predominantly interacting with each other and the polar R-groups face the aqueous environment. Keratins in the skin and hair are prime examples of this type of coiled structure. This interaction of alpha helix’s is also used in some types of receptor interactions.
Turns are the third type of "classical" secondary structures. Approximately one-third of all residues in globular proteins are contained in turns that serve to reverse the direction of the polypeptide chain.
Three types of turns are designated I, II, and III are known. Type III is simply a single turn of 3.10 helix . Each type contains a hydrogen bond between the carbonyl oxygen of residue i and the amide nitrogen of i+3.Since a turns have several unsatisfied backbone hydrogen bond donors and acceptors, they are polar, and are usually found near the surface of the protein. Proline is very common in turns, as it always has the correct phi angle. Glycineis also very common in turns because its R group presents little steric hindrance
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