BCHM 3P02 2024 Fall Lecture 1 Introduction to Protein PDF

Instructor : Dr. Shasha Hu [email protected] Protein Structure and Function BCHM 3P02 2024 Fall Grade Evaluation Project 1 15% Midterm 20% Assignment 10% Project 2 20% Final Exam 35% Total 100% ...

Instructor : Dr. Shasha Hu [email protected] Protein Structure and Function BCHM 3P02 2024 Fall Grade Evaluation Project 1 15% Midterm 20% Assignment 10% Project 2 20% Final Exam 35% Total 100%  Nov. 05 is the date for withdrawal from the course without academic penalty. Oct. 28 is the date you will be notified of 15% of your course grade Important Oct. 14 to 18 is the scheduled reading Dates week. Oct. 29 is for the midterm exam. Dec. 07 to 19 are set aside for formal examination periods. Couse Schedule  Lecture PowerPoint files will be posted on Brightspace. Structure and function is a cornerstone topic in this course – it will be expected that students know and appreciate the structure/function of key molecules of interest Key areas of course: Composition, chemistry, structure & function of proteins Application (in lab and computationally) of proteins in research How is structure/function determined, analyzed? How does structure relate to function? How is proteomic information stored/accessed/visualized? How do proteins function, evolve, operate? Kinetics, chemistry, structure orthologs, paralogs, homologs, convergent/divergent evolution of domains Important Notes: You can print out the lecture slides and take notes as you follow along through the lecture. You can also bring your own laptop or tablet to take notes. Technical issues with your computer and/or internet connection or loss of data do not constitute a valid excuse for inability to perform a given task. It is expected that you log in to check for updates, forum posts, etc. at a minimum of every two days. There are no supplemental assignment to perform should you miss an assignment/project report or obtain a low mark. Top tips for excelling in this course Read and understand all course policies and procedures Keep up to date on when things are due. Keep up to date on lecture content. Utilize the instructor and TA to seek clarification. Start the assignment or report as early as you can. Lecture1: Introduction to Protein  Functions of Protein Structure Proteins: Keratins, actin Enzyme: Catalyst Antibody: recognition Signaling/regulation: receptors Transporter: ion channels Transducer: eg: chemical to mechanical energy, muscle contraction, kinesins, ATP synthase  Structural Proteins Fibrous proteins Also known as structural proteins Appear in body structures Examples include collagen and keratin Stable Actin Protein a highly conserved protein polymerizes to produce filaments that form cross-linked networks in the cytoplasm of cells cell motility and contraction Functional Proteins Globular proteins Also known as functional proteins Function as antibodies or enzymes Can be denatured Enzymes Act as biological catalysts Increase the rate of chemical reactions Bind to substrates at an active site Proteins as Catalysts Nearly every biological function (w.r.t. metabolism) can occur “naturally” without the use of proteins (enzymes) The rates of these reactions are many orders of magnitude (105 to 1020) too slow to satisfy biological demands Enzymes represent a class of protein which serve as a catalyst to greatly increase the rate of very specific reactions within (or sometimes outside of) a biological system Without enzymes most, if not all biological life would not be possible How enzymes work Structure of Amino Acid - COO CHR H3 C + NH3 H Gly Ala General structure of an amino acid -often called proteinogenic amino acids  Zwitterions Amino acids have both a basic amino group and an acidic carboxyl group Carboxyl group can donate a proton to solution or amino group, depending on pH Amino group can accept a proton depending on the pH Zwitterions have no net charge, but do have polar regions http://chemistry.tutorvista.com/biochemistry/essential-amino-acids.html The side chain of histidine has a pKa of 6.04 →what does this mean? Ka: acid dissociation constant (aka acidity constant) You have likely used this as a measure of the strength of an acid in solution. Written as an equilibrium constant for a chemical reaction where a proton dissociates from its conjugate base, releasing a proton to solution (altering the pH of the solution) The larger the Ka value, the more readily the proton in question dissociates and thus the stronger the acid http://n-pharmacology.blogspot.ca/2013/06/absorption-of-drugs.html http://www.calpoly.edu/~cbailey/125LabExperiments/Titration/Titration.html Note that HA, A− and H+ are in equilibrium – both the forward and reverse reactions take place. So far we have covered Ka, what about pKa? http://www.syncytiabet a.org/~syncyt5/sy ncy tiabeta/images/b/b6/Henderson-Hassel balch_Equati on.png http://www.shimadzu.com/an/hplc/support/qn50420000002e5p-img/qn50420000006nbn.gif pKa is the pH at which there is an equal amount of protonated and depronated molecules (for our acid/base of interest) 1 14 The molecule of interest will lose more protons (shift to mostly depronated) in the solution with increases in pH such that pH > pKa In other words, the higher the pH value is above pKa, the more likely a molecule will lose a proton The side chain of histidine has a pKa of 6.04 →what does this mean? At pH 6.04 histidine side chain exists in equal states of protonation and deprotnation 50% of His a.a has a +1 charge, 50% are neutral Importantly at physiologically relevant pHs, small shifts in pH will change the proportion of charged vs uncharged His a.a If pH < 6.04 , the R group of most His a.a. will be protonated (+1) The R group ring will have two N-H bonds and a net positive charge If pH > 6.04 , the R group of most His a.a. will be deprotonated The R group ring will have one NH bond only and no net charge. In this case His has donated a proton (possibly to a molecule). http://physiologyonline.physiology.org/content/22/1/30/F2 pI (Isoelectric Point ) pI of a protein is determined by the pKa of every constituent amino acid. The pH at which a given amino acid has a neutral charge. At its pI, the positive and negative charges on the amino acid balance. It moves neither to the anode nor cathode in the electric field. + - - COOH -H COO + COO -H + pK1 ' + H3N C H H3N C H pK2 ' H2N C H + + R +H R +H R  Amino acids with neutral charged side chain: pI = (pK’1 + pK’2 )/2 Amino acids with negative charged side chain: pI = (pK’1 + pK’R-COO- )/2 Amino acids with positive charged side chain: pI = (pK’2 + pK’R-NH2 )/2 + - - COOH -H COO + COO -H + pK1 ' + H3N C H H3N C H pK2 ' H2N C H + + R +H R +H R  Amino Acid Isomerization Amino acids can have different arrangements of atoms about the alpha carbon Convention is to name different orientations either D or L isomers  L & D amino acids http://bioweb.wku.edu/courses/Biol220CAR/4AA/ http://bioweb.wku.edu/courses/Biol220CAR/4AA/  22 amino acids are used across the entire animal kingdom 20 are used “universally” in all biological systems Exceptions: 1)pyrolysine: used for methane generation in some organisms 2)Selenocysteine: used as a cysteine alternative in some organisms Diversity seen in: Hydrophobicity Hydrophilicity Charge flexibility Diversity in size Long/short Planar Kink vs straight chain Ring Diversity in “targeting” Phosphorylation: Ser, Tyr, Thr Glycolyslation: Asn, Ser, Thr Hydroxylation: Pro, Lys, Asp Myristoylation: Gly Classification of Amino acids http://lipidlibrary.aocs.org/Lipids/protlip/Figure1.png http://what-when-how.com/wp-content/uploads/2011/05/tmp1C12_t humb.jpg Optical Absorption of Protein Commonly, the optical absorption of proteins is measured at 280 nm. At this wavelength, the absorption of proteins is mainly due to the amino acids tryptophan, tyrosine Overview of amino acid metabolism What are amino acids used for? Proteins Nucleotides Coenzymes Essential amino acids-from diet: PHILL of MTV (Phe, His, Ile, Lys, Leu, Met, Trp, Thr, Val) Nonessential amino acids: synthesize them Amino acids converted to common intermediates Ala → Pyruvate Asp → Oxaloacetate Glu → α-ketoglutarate Amino acids are precursors of glucose, fatty acids, ketone bodies Therefore Amino acids are metabolic fuels in addition to building blocks for protein Other Amino Acids More than 500 amino acids are currently known (as of ~1983) More than 240 occur in nature Generally called nonstandard, noncoding or nonproteinogenic Purpose of other amino acids -metabolic intermediates -plant defense against herbivores -other have widespread function: eg: taurine aids in membrane stabilization, calcium signaling, cardiovascular function, second messenger signaling, development of retina Protein Synthesis Central Dogma of biology? DNA makes RNA makes amino acid sequence makes protein structure makes protein function  Protein synthesis mRNA: template, codons tRNA and aminoacyl-‐tRNA synthetases - pairing between A.A. and tRNA Ribosomes: machinery for protein synthesis rRNA as enzymes https://www.science.org/content/article/there-are-millions-protein-factories-every-cell-surprise-they-re-not-all-same  (a) Primary structure. A protein’s primary structure is the unique sequence of amino acids in the Amino acids polypeptide chain. Hydrogen bonds Amino acids (b) Secondary structure. Two types of secondary structure are named alpha-helix and beta- pleated sheet. Secondary structure is reinforced by hydrogen bonds. Dashed lines represent the hydrogen bonds in this figure. -pleated sheet Alpha- helix Figure 2.18a-b Figure 2.18c-d  tRNA does not take on the “standard” 2D structure in 3D space Loops and folds similar to a protein http://img.ffffound.com/static- data/assets/6/b99ff4391a549b86ee2ef7693b0a4754a65a3201_m.jpg The crystal structure of the bacterial 70S ribosome https://www-science-org.proxy.library.brocku.ca/doi/10.1126/science.1131127  Energetic cost of 1 ATP per tRNA charged How is the right a.a. added to the proper tRNA? →through unique conformational shape of the folded tRNA  Previous figures show an “uncharged” tRNA which lacks an amino acid Each tRNA has a unique acceptor arm to which an amino acid binds through an acyl linkage High energy bond which helps drive the reaction forward when linking multiple amino acids together Process of charging or linking amino acids to tRNAs is performed by aminoacyl-tRNA synthetases Separate enzyme for each amino acid  Each aminoacyl-tRNA synthetase attaches a specific A.A. to 1 or more tRNAs with the corresponding anticodons by recognizing the unique structural features of A.A. and the related tRNAs (99.9% accuracy) One to one relationship between the 20 AAs and aminoacyl-tRNA synthetases. Thought to require ~80% of the cells energy stores, resources, etc. 64 codons (4*4*4)→ 47 tRNAs (human) →20 aminoacyl-tRNA synthetases for 20 A.A.s (1 to 6 tRNAs for each A.A.)  Aminoacyl-tRNA Synthetases Fall into Two Classes Class I tRNA synthetase contacts tRNA at Class II aminoacyl-tRNA synthetase the minor groove of the acceptor stem and contacts tRNA at the major groove of at the anticodon. the acceptor helix and at the anticodon loop. The Ribosome https://www.visiblebody.com/blog/dna-and-rna-basics-replication-transcription-and-translation  N-formyl-methionyl-tRNA (tRNAfMet ) – The aminoacyl-tRNA that initiates bacterial polypeptide translation. N-formyl-methionyl-tRNA (fMet-tRNAf) is generated by formylation of methionyl-tRNA  An initiation site on bacterial mRNA consists of the AUG initiation codon, preceded with a gap of ~10 bases by the Shine–Dalgarno polypurine sequence.  The stop codons UAA , UAG, and UGA terminate translation. In bacteria they are used most often with relative frequencies UAA>UGA>UAG. Termination codons are recognized by protein release factors Eukaryotic protein synthesis follows same general theme but with many more interactions involved. 5’ methylated cap on mRNA in eukaryotes recruits a large number of proteins which assemble as one large complex The recruitment of the ribosome in eukaryotes is very complex with many factors involved  Prokaryotic vs eukaryotic mRNA has some key differences Prokaryotic mRNA can be polycistronic; coding for many functional proteins Most (if not all) eukaryotic mRNA is monocistronic; coding for a single protein Regulation in bacteria: Physical interference blocking ribosomal subunit assembly due to secondary mRNA structure Regulation in eukaryotes?  mRNAs store the code for specifying the polypeptide chain in a continuous, non- overlapping string of 3-letter codons called an open reading frame (ORF) An ORF starts from a start codon and ends at a stop codon Perspective on speed…. DNA polymerase speed: 200-1000dNTP/sec RNA polymerase speed: 50-100 rNTP/sec Ribosome speed: 1-20 a.a./sec Why such a difference in speed? In part due to many potential combinations (ie: ¼ vs ¼ vs 1/20 units can be inserted into ribosome) Speed difference partially compensated for by polyribosome or polysome  In eukaryotes initiation is much more complex, so “restarting” a ribosome is a slower event Cell circularizes mRNA to facilitate the rapid restarting of ribosomes which just completed translation Some initiation factors required for ribosome recruitment also interact with poly a tail binding proteins, causing a circular formation of the mRNA  Error rate of ribosome thought to be 1:1,000-10,000 Lower rates are problematic for protein folding/function One main error proofing property of ribosomes: 16S component has two A residues which aid in anticodon hydrogen bonding incorrect codon/anticodon binding will have weaker affinity and likely be removed/displaced  AA interacts in minor grove of anticodon/codon double strand http://www.biogem.org/codon.jpg Related Codons Represent Related Amino Acids Sixty-one of the 64 possible triplets encode 20 amino acids. Three codons (stop codons) do not represent amino acids and cause termination of translation.  The genetic code was frozen at an early stage of evolution and is nearly universal. Most amino acids are represented by more than one codon. Some correlation of the frequency of amino acid use in proteins with the number of codons specifying the amino acid is observed. Codon–Anticodon Recognition Involves Wobbling The wobble in pairing between the first base of the anticodon and the third base of the codon results from looser monitoring of the pairing by rRNA nucleotides in the ribosomal A site. Wobble in base pairing allows G-U pairs to form between the third base of the codon and the first base of the anticodon. Summary: as long as the first 2 nucleotides in an mRNA strand bind with the anticodon, the 3 rd nucleotide selection is less stringent  Many mutations in the 3rd codon position are “silent” in that they still code for the same a.a. Typically other single mutations within the genetic code are conservative →tend to maintain key function/theme in protein sequence Eg: make a nonsilent mutation from Valine Make a nonsilent mutation from Valine G – U – X →make G and U permutations (for simplicity lets keep X as a U) G permutations: U – U – U →phe C – U – U →Leu A – U – U →iso U permutations: G – C – U → ala G – G – U → gly G – A – U → asp If we continued we would get….. Mutation to single base for ANY codon for valine will produce: Phenylamine leucine isoleucine methionine alanine Asparagine Glutamate glycine Mutation to single base for ANY codon for valine will produce: Phenylamine leucine isoleucine methionine alanine Asparagine Glutamate glycine Of these 8 possible mutations 5 a.a. are similar chemically, and would not cause substantial changes to the protein unless in a constrained area (Eg active site) *arguably 6/8 would be conservative Remaining 2 or 3 a.a. mutations would be nonconsertative Key: many mutations are either silent or conservative, making the otherwise “redundant” genetic code very resilient/flexible to mutation/change More on Mutations & Genetic Code Codons with pyrimidines (U,C) at the 2nd position mostly specify hydrophobic a.a.s (Ala, Val, Ile, Pro, Phe, Leu, etc.) Codons with with purines (A,G) at the 2nd position correspond mostly to polar a.a.s (Thr, Tyr, Asn, etc.) Types of changes seen: purine to purine →(a/g) transition Pyrimidine to pyrimidne (c/u)→transition Purine to pyrimidine or vise versa → transversion Typically a single transition (most likely to occur) tends to result in conservative mutations In general: when both the 1st and 2nd positions are G or C, a change at the 3rd position, does not change the a.a. (see: Pro, Ala, etc) When both the 1st and 2nd positions are A or U, then 3rd position changes do change the a.a. (see: Met, Phe, Tyr, etc)