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Lecture 5

BIOC12 Lecture 5.docx

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Department
Biological Sciences
Course Code
BIOC12H3
Professor
Shelley A.Brunt

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BIOC12 Lecture 5 +NMR Spectroscopy - Useful in protein analysis -> can be used in fluids, not crystals - Within a magnetic field, proteins absorb electromagnetic radiation differently - Absorbance primarily influenced by neighbor atoms -> allows study of atoms close together - MUST be combined with amino acid sequence proteins. - Useful only for small proteins -> X-rays are a lot faster Bovine ribonuclease A NMR structure Figure 1: NMR structure of Bovine Ribonuclease. Yellow shows disulfide bridges - Shows the protein backbone! NMR allows the viewing of disulfide bridges, which are absent in crystal structure - RNAse A o Very small and stable protein o First used to understand naturation and denaturation o Often times, thought to be too stable! -> Requires both heat+chemicals to denature Figure2: Comparison of bovine and human ribonulceases. Note the similarity! - When comparing the structure of Ribonuclease between bovine and humans o The structures are very similar! Thus, have very similar active sites! o Very slight differences (note the location of the beta sheets o If analyzed, will show high sequence similarity Structure of Peptide Bonds - Peptide bonds are between carbonyl oxygen + amide hydrogen, on adjacent alpha carbons - Rigid planar structure gives resonance! o 40% double bond character! Resonance - Peptide bonds can be shown as both single or double bonds - Double bond nature gives rigidity! Cannot rotate about the C-N bond. - Double bond nature shown below Figure3. Double bond nature of the C-N caused by resonance! - As a result, atoms of a peptide bond always lie on the same plane! - Rigidity of protein from the R-groups, not peptide bonds! Figure4. Note theat the peptide bonds ALWAYS lie on the same plane! Rigidity by double bond nature - Due to double bond nature, side chain (R group) of alpha carbons point away from each other! - Almost ALWAYS in trans confirmation unless Proline ->due to the ring structure - Cis-trans conformations arise during protein synthesis, and can NOT be changed to the other conformation o The exception here is Proline. Due to the cis structure of proline, it can change! - Cis is typically less favourable due to steric interactions between alpha carbons o Almost all are going to be trans! Except proline Figure5: Showing steric hinderance of non-proline amino acids on the left. On the right, it shows that steric interactions will exist equally in both cis and trans conformations of Proline - Specific enzyme required for proline: - Peptidylprolyl – cis-trans isomerase o Very important in protein structure and function! o Groups together with chaperones to alter proteins! (covered in Lecture 6) o Transient destabilization of resonance hybrid structure: allows free rotation of the atom  Basically, makes the double bond nature at C-N unstable, favoring C=O, allowing for more rotation  Due to equal steric interference, most Cis bonds are X-pro linkages. Double bond conveys a rigid planar structure - The rigid nature of the peptide determins bond angles! - There’s limited rotation between the C-N due to double bond nature o You don’t need to know double bond lengths and angles, but DO need to relative sizes o C-Cα (Psi) > Cα-N (Phi)> C-N (aka omega/ amide bond) (C = Carbon, Cα = Alpha Carbon, N = Nitrogen) Figure 6. Shows the different bond distances and angle between phi, psi, and omega bonds - There’s some free rotation about the Ca-N (phi) and the Ca-C (psi) bonds. - This is where the flexibility of amino acid chains come from! - If bonds are at 180 degrees, then it becomes a very stiff protein! o Extended polypeptides form very rigid polypeptide groups. - However, rotation between Psi and Phi bonds can be easily restricted by side chain interactions, as well as carbonyl oxygen interference – shown in Figure B below. - Note: C-N (amide) bonds are referred to as omega bonds. - Figure 7: In (a), the peptide bond is in extended conformation, which allows some free rotation between the atoms. In (b), there is steric interference caused by the two adjacent carbonyl oxgyens. Sample question: What is the configuration of most alpha carbon atoms of amino acids linked in a peptide bond? Why is this so? Ans: Trans to reduce steric hinderance. Ramachandran Diagrams - They are simply predictions, given what we know about the steric interactions. - We can look at what is sterically allowed, and calculate all possible (distance) values of the tripeptide for all values of phi and psi bonds. Figure 8: Sample ramachandran diagram. - You do not know how to understand! This is generated algorithmically! - Can determine most likely bond angles! - There are variations of helical structures possible between two bond angles o This shows the most likely structure as well as the range of the bond angle o This shows each possible tripeptide combo – potential for bond bongles o The outer shows the maximum. The darker shows the most likely range - Note: It is rare to find bond angles for the left-handed helix! Alpha Helix - Mostly right handed, but can sometimes be left handed (rare) - Stabilized by intrachain hydrogen bonds - C=O forms a hydrogen bond with the fourth C-N residue towards the C-terminus of the polypeoptide chain! The i+4 principle, which means there are a total of 5 amino acids involved. Example, AA2-AA6, AA5-AA9, etc. - Figure 9. Within an alpha helix, C=O of atom i will bond with the N-H of atom i+4. - The hydrogen bonding are parallel to the long axis of the helix. Many Hydrogen bonds come together – forms additional stability o Allows for us to predict the structure given information about the hydrogen bonding o Especially stable when H bonds are in the hydrophobic interior! - All carbonyl groups point towards the C-terminus o Because all point in the same direction, entire alpha helix is a dipole! N is positive, C is negative! (amphipathic property) - KNOW! 3.6 amino acid residues per turn - For a right handed helix, backbone turns in a clockwise direction o Phi and psi angles of each residue are the same! Provides for similar repeating structure -> conveys stability o Number of residues per turn CAN change, but it changes the conformation of the helical structure.  If you stretch it out and add more amino acids per turn -> it looks like a ribbon! - Figure 10: C terminus is towards the left, N terminus towards the right. Yellow shows the parallel pature of the hydrogen bonds. It will be easier to see if you draw an axis similar on figure to the right. Pitch = how much space it takes up per turn. Rise = how much space it takes up per amino acid. From a Ramachandran diagram - We can see that almost all alpha helicies are right handed! Left handed helicies are very rare! - This is due to the side groups which interact with other amino acids and molecules - This often results with alpha helicies being amphipathic – Hydrophobic one side, charged on the other. There are different ways of thinking of alpha helicies, as shown below. All forms are correct given certain circumstances. Figure 11: Unlabeled figure shows interactions. A is ball-stick method. B shows alpha helicies as a ribbon. C shows alpha helicies as being cylindrical. All are correct! What we want to see depends on context! Right handed helicies - Their sizes vary greatly – on average, it’s 12 residues long, but it varies from 4-40. - It requires 20 amino acids to cross a membrane, from hydropathy plot. - Stability of the alpha helix greatly depends on the side chains! o Bulky side chains typically do not show up in alpha helicies o Small uncharged amino acids are often found!  Glycine is an exception: Due to destabilization properties, it’s not typically normally within the alpha helicies! Found either at beginning or the end, due to its free rotation  Proline is also typically not found-> disrupts right handed helix/secondary structure - Typically, in alpha helixes, one side is hydrophobic, other is hydrophilic! o Can be seen in the helical wheel. Figure 12: You can see the 3.6 amino acids per turn. You can also see glycine near the beginning and end of the helix. You can also see a clear separation of charges! Bottom left = uncharged. Top right = charged! Secondary structure prediction Figure 13: Secondary structure prediction given an amino acid sequence. Arrows (denoted by , E) are Beta sheets, Cylindrical structures (denoted by ==, H) are Alpha Helicies. Unlabled are random coils (Denoted by C) - Allows us to quickly find whether it’s predominantly alpha helixes of beta sheets. - However, this doesn’t provide information regarding folding, or the direction of the B-sheets - Require a lot more information to find this out! Liver Alcohol Dehydrogenase Figure 14: Liver dehydrogenase. Active site is an alpha helix! We can identify isoleucine, phenylalanine, and leucine buried in the active site! - We can see many alpha helicies and beta-sheets. - Primarily Beta-sheets. We can count 15-16 beta sheets - However, active site is on an alpha helix: where outside is primarily hydrophilic, and the inside is hydrophobic (blue) A single protein sequence prediction won’t provide accurate information! Figure 15: First, protein isolated from different organisms to create a consensus sequence, which is then used to create secondary sequence information via different programs. Remember!  are beta sheets. === are alpha helicies. --- are random coils! - We must use different sequences from the protein of different organisms! o We can use this to create a concensus sequence o We then run this consensus sequence through different prediction programs  There will always be differences! However, typically, they usually show the same structure, albeit at different lengths! Thus, we use this information to create an “average” protein Leucine zipper - It’s a protein->Dna interactions! o Two alpha helicies (amphipathic) interact with each other and create a structure o Inner area interacts w/DNA -> Positively charged b/c DNA is negatively charged. o Hydrophobic leucine interact with each other, and hydrophilic faces are exposed! 3 helix 10 - Secondary structure that’s rarely found in protein o Like the alpha helix, it is also a right handed o Each AA is a full 120 degree rotation, uses n+3 as opposed to n+4  As a result, it has a tighter hydrogen-bonded ring structure  10 atoms rather than 13,fewer residues per turn, and longer pitch o Howeever, less stable than the alpha helix because of steric hinderance and awkward geometry o Typically, only occurs at the C-terminal end of an a-helix. o Due to geometry, phi and psi angles will be found in different regions of Ramachandran plot Figure 16. Comparison of 10helix (left) and an alpha-helix. Note that the hydrogen bond (pink) forms between amide and carbonyl three positions away. Alpha helixes form this same bond 4 positions away Beta-structure - B-sheet/strands take up more space per residues than alpha helixes - B-sheets and B-strands o Strands are polypeptides that are fully extended -> 0.32nm/
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