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Lecture

21. Protein Structure function and separation strategies.pdf

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Department
Biology (Sci)
Course Code
BIOL 200
Professor
Richard Roy

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Naveen Sooknanan McGill Fall 2011 Protein Structure, Function and Separation Strategies: As we have seen over throughout this course, proteins have a wide variety of functions which depend on their structure. Some specific domains of proteins can carry out independent functions o An example is the DNA binding domain found in transcriptional activators such as homeodomain proteins Other proteins have structures which confer very unusual and peculiar functions o An example if the green fluorescent protein (GFP) whose barrel-like structure allows fluorescence when shone with specific frequencies of light o GFP can be fused into a desires protein by placing the GFP gene directly downstream of the proteins gene This causes the mRNA to have the GFP mRNA causing the translation of a fusion protein by the ribosome During transcription, RNA polymerase II (or whichever is used) skips the stop site of the desired proteins gene and performs read- through right to the end of the GFP gene X-ray diffraction and crystallography are two techniques widely used to accurately determine the structure of proteins High concentrations of a purifies proteins, perhaps made from bacterial overexpression, tend to form crystal lattice structures o This structure corresponds to the proteins lowest energy state These crystals are bombarded with high energy beams, such as X-rays which reflect and scatter once they hit the protein o These beams are supplied by a piece of machinery called an electron collider o These radioactive scatter patterns are unique in every proteins and are directly relates to the proteins structure Complex programs are able to take this scatter data and determine the function of the protein through prediction of electron density This process is analogous to determining the shape of a rock from examining its ripple pattern in water The product of this technique is an electron density map which can be analyzed in order to determine structural aspects of the protein Through these maps, skeletons and models can be built by placing corresponding amino acids into place which correspond to various areas on the map First, the amino acid chain is produces and then any secondary, tertiary and quaternary structure is determined through hydrophobic, hydrophilic and other types of interactions 1 Naveen Sooknanan McGill Fall 2011 o This has been useful in molecular biology because we can manipulate proteins by slightly altering their shape and seeing what this does to the function Looking at the amino acid sequence, however, can only give scientists a crude approximation of what the actual protein will look like Thus, looking at the amino acid sequence alone can be deceptive and lead to false assumptions with respect to the function By determining the function of the entire protein through a method such as X-ray crystallography, it is possible to get a better understanding of the function of the protein as a whole o For example, a characteristic helix structure in membrane proteins (such as ion channels) give these proteins the function of embedding themselves within the plasma membrane As we have seen time and time again, protein can have catalytic activities in the body. These are known as enzymes The reaction between two (or more) substrates for the formation of a product (or products) always requires an input of energy to enter a transition state o This is known as the activation energy of this reaction Once the activation energy barrier is reached, the reaction proceeds downhill (releasing energy) until it reaches product formation o The product is either a higher or lower energy state than the product, classifying the reaction as endothermic (using energy) or exothermic (releasing energy) respectively The purpose of an enzyme is to lower the activation energy of a specific reaction, allowing it to happen spontaneously within the binding sites of enzymes o This usually happens by placing the substrates in very close proximity to one another o An enzyme is specific to one set of substrates and perform one specific reaction o These substrates dont just have to be other proteins, they can be any molecule in the body The enzyme gets is activity from characteristic tertiary structure which allow binding of substrates and helps drive the reaction forwards As we have also discussed, RNA molecules can also have catalytic activity with their specific secondary structure o These are called ribozymes, such as the 23S rRNA which catalyzes the peptidyl- transferase reaction during translation The efficiency of enzymes can be quantized (measured) and reflects specific inherent properties of the protein The rate of product formation the in presence of an enzyme is dependent on the concentration of substrate o At first, the reaction speeds up very quickly, but eventually plateaus when the solution becomes saturated in substrate 2Naveen Sooknanan McGill Fall 2011 The maximal speed of conversion from substrate to product is also dependent on the concentration of enzyme o Needless to say, more enzyme means faster product formation o The maximal speed, however, is not very important because it does not give any characteristic information about the protein There is, however, a specific substrate concentration called m , or the Michaelis Constant, which corresponds to an enzyme reaching half of its maximal speed o This value is significant because it is INDEPENDENT of substrate concentration o K thmrefore, is an intrinsic property of a protein and is unique to the enzyme being tested Protein can also bind to other molecules in a non-catalytic manner. These specific interactions happen between a protein and a binding entity known as a ligand. Some common ligands include growth hormones, steroid hormones and cytokines, which all bind to specific protein receptors Another very important protein-protein interaction is between an antibody and an antigen; the basis of the immune system and many protein isolation techniques as we will see soon GTP-binding proteins are also considered ligand (GTP) binding Ligand binding can act as allosteric switches because they can considerably change the
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