Biochem2_Proteins

CHAPTER 2PROTEINSCentral molecules of biochemistry include:Nucleic Acid (DNA/RNA)SugarsLipidsProteinsPROTEINSA.What is a protein and what does it do?1.The term protein is derived from the Greek term Proteios meaning ofthe first rank [Jöns Berzelius]2.All functions of proteins are important for life; some more than others3.Without proteins, life as we know it, would not exist. Withoutproteins the chemical changes which are necessary to sustain lifewould occur much too slowly4.Proteins come in basically two “flavors” Fibrous and Globulara)Fibrous – water insoluble structural proteins (polymeric)b)Globular – mostly water soluble (variety of functions)B. Protein functions:1.Enzymatic catalysis – lowers activation energy and thus increases therate of chemical reactions without increasing temperature. Increasedrate of at least million fold 106a) Important for sight, thought, warmth, movement – in generallife cannot be sustained without enzymes2. Transport and Storage – Transport of oxygen and other nutrients isfacilitated by proteinsa) Ions such as Ca+2, K+, Na+ are transported across membranesand are important in nerve signal transmission, musclecontraction and inter and intra-cellular communication.b) Transport of sugar, fats and other proteins is essential forenergy production and storagec) Storage of Fe, starch, glycogen, fats, amino acids and otherions and molecules is maintained and regulated by proteins3. Motion – Proteins such as myosin and actin form muscle fibers.a) Proteins are also essential for the movement of sperm cells,bacteria and other single celled organismsb) Proteins facilitate the movement of DNA during cellularmitosis and meiosis.4. Structural and Mechanical Support – Fibrous proteins are essentialcomponents of all organisms, especially among the more complexeukaryotes such as plants and animalsa) Cell walls, Hair, fingernails, muscles, wings, tendons and otherconnective tissuesb) Cytoskeleton – inner framework of animal cells which definesshapec) Proteins provide the machinery to make other proteins5. Protection – proteins play an integral role in protecting organismsfrom invasion by other organisms and in repairing extraneous damagea) Antibodies and complement are proteins which are involved ineliminating foreign substances.b) MHC complexes are proteins which provide for selfrecognitionc) Fibrinogen and thrombin are proteins which are involved inblood clotting6. Regulatory Functions – Some proteins function as hormones and areinvolved in systemic communicationa) Insulin signals the fed state and initiates many intracellularevents, one of which culminates in the uptake of glucose.b) Human growth hormone, oxytocin and vasopressin are allexamples of protein hormonesc) Cell cycle, growth and differentiation are all controlled at somelevel by proteins. Proteins play a key role in replicating,packaging, winding and unwinding DNA7. Nervous System – proteins are actively involved in the generation,transmission and resetting of nerve impulses.Note:Many inherited diseases occur due to faulty or missing proteins which are required for a specific taskC.We have still not answered the question -- What is a protein?1. A protein is a very large molecule (macromolecule) which is made-upof mostly C, H, N and O ----- some S2. A protein is a polymer made-up of repeating subunits (similar tonylon or polystyrene)a)each subunit (often called a residue) is analogous to a singlecar on a train; where the train represents the entire proteinb)Subunits of proteins (residues) are called amino acids due tothe functional groups present in the molecules.i)amine functional group R-NH2ii)carboxylic acid group R-COOHiii) R - is the distinguishing group for each amino acidR = variable groupC = called the "-carbonc)There are basically 20 different R-groups -- corresponding to20 different amino acidsi)some species modify the basic amino acids for differentreasonsii)Humans ----> bacteria use the same repertoire of 20 a.a.for the construction of proteinsiii) Humans and other animals are not capable ofsynthesizing all 20 amino acids and must thereforeconsume certain amino acids which are termed essential(there are 9 essential a.a. required by humans)d)Two absolute configurations possible R or S The "-carbon ischiral [exception glycine]3.Fischer Notation ---- biochemists replace R and S notationsa)Simplifies stereochemistry (R and S dependent on first atom onR-group)b)L and D notations are independent of the R-group!EnantiomersD isomer L isomerc)Book states that only L amino acids are constituents of proteins Not always true —It appears that all amino acids placed into a newly synthesized protein are all L, however some species change certain residues from L to D for specific reasons.One and three letter codes:most of the three letter codes are simply abbreviations of the name4.Acidity / Basicity of amino acids a)Amine group is a Bronstead/Lowery BASENH 2 accepts proton --------> NH 3+b)Carboxyl group is a B/L ACIDCOOH donates proton -------> COO -c)R-groups --- some acidic ------ some basicNote:The ionization state of any of these groups is dependent on the pH of the solution pH 1.0 ------------->totally protonated amino acidsCHARGED +pH 7.0 -------------->Partially deprotonated amino acidCHARGE is Neutral pH 9.0 -------------->Totally deprotonated amino acidCHARGED -REVIEW ACID/BASE CALCULATIONSA.Water constant 2 H2O <======> H3O+ + OH-sometimes writtenas simple dissociation H2O <======> H+ + OH-[H2O] is 55.5M -- ionization is comparably insignificant[H2O] is considered constantpure water -----[H+] = 1 X 10-7 and [OH-] = 1 X 10-7 Therefore-----Kw = 1 X 10-14(Kw is equilibrium constant)B.If we add exogenous [H+] or [OH-] to water we will disrupt the equilibriumand establish a new equilibrium based on the constant value of water(Example)Say we add HCl so that the final concentration of [H3O+] isnow much greater than 10-7 ---------> Let's say 10-3MThe [OH-] is automatically adjusted to 10-11 M so that theproduct [H3O+] [OH-] will equal the Kw of 10-14C.Weak acids ---- DO NOT donate ALL H+ to waterACID + H2O <=======> [H3O+] + [A-]Rearrange the above equation and solve for [H+]Take the - Log of both sides of the equation- Log [H+] is defined as the pHBy definition the - Log Ka is called the pKaIf we invert the A- / Acid ratio then the sign is changed to +Henderson/ Hasslebalch equationQuestion:When does the pH equal the pKa?Answer:When [A-] = [Acid] concentrationLog [1] = 0When 1/2 of the H+ has been donated the pH of the solution is = pKa. InO+] when 1/2 of the acid is ionized other words the pKa describes the [H3The pKa value gives us a quick look at the strength of the weak acidD.If we consider the ionization state [A-] to [Acid] ratio at a fixed pH we canuse the above equation also.1.What is the ionization state of the acid at 1.5 pH units above the pKa?a)mostly deprotonated (almost completely ionized)[A-] predominates2.What is the ionization state of the acid at 1.5 pH units below the pKa?a)Mostly protonated (mostly unionized)[Acid] predominatesE.Biological systems are buffered to fix charges on amino acids and adsorbany H+ produced during chemical reactions1.Buffers are usually weak acids which maintain a fixed pH bydonating or scavanging H+ as needed ---- they are most effective atpH values close to the pKa of the acida)WHY ?ACID/BASE RELATIONSHIPS OF AMINO ACIDSA.Each amino acid has at least two pKa values:1 -- for the amine group1 -- for the carboxyl group1.Some amino acids have a third pKa value contributed by the R-group2.pKa COOH Low(acidic)pH = 1.8 to 2.3+High (basic)pH = 9.0 to 11.03.pKa NH3(pKa values are dependent on other factors)B.If we exclude the R-group for a moment and look at protonation of anamino acid at pH 7.01.More than 1.5 pH units above pKa of COOH group (deprotonated)More than 1.5 pH units below pKa of NH3+ group(protonated)At pH 7.0 amino acids aredipolar ions with oppositecharges on the same moleculeCalled a ZWITTER ION(Hybrid)C.Consider titration curve for amino acid with 2-pKa valuesKa1Ka2Gly+Gly o Gly-We must consider 2 equations1.2.Titration curve(example problem)100 ml of a 0.250 M solution of glycine-HCl (Gly+) is titrated with 10 N NaOH to pH 3.20 . What are the relative concentrations of Gly+ and Gly o at this pH? Do not consider Gly- concentration (pH is well below the second pKa)1.First calculate the total moles of Gly+ found in 100 mL of solution2.After titration some of the Gly+ will be converted to Gly o, but none of it willbe lost. Therefore:O + Na+3.Gly+ + NaOH --------------> Gly o + H20.0250 - X X X4.0.0250 moles Gly+ - X will remain after titration5.X = Gly o X Gly o will be formed during titration6.Plug values for weak acid Gly+ and its conjugate base Gly o into theHenderson/ Hasslebalch equation and solve for X7.PROTEINS --- Polymers of amino acidsA.20 different amino acids (monomers or subunits). They each vary in shape,size, charge (given pH), H-bonding capacity and chemical reactivity1.different physical and chemical propertiesa)some ---- hydrophobicb)some ----hydrophilicc)some ----aromatice)some ----contain Sf)some ----acid R groupg)some ----basic R groupB.Amino acids link together to form long chains (polymers). The linkagebetween the amino acids is called an amide bond.1.The polymer is called a polypeptide or protein2.Linkage occurs as follows:3.Amino terminus is considered the beginning of the polymer or+)protein chain (NH34.Carboxyl terminus is considered the end of the polymer or proteinchain (COO-)5.Main Chain (regular repeating portion) of the protein is called thebackbone6.Variable portion of the protein ---- R groupsC.The overall charge of a protein is independent of the ends. At physiologicalpH the ends are nearly always charged and the charges cancel each other1.The charge of a protein is dependent on R-groups and pH of themedium.a)Charge determines chemical reactivityb)Charges may participate in protein structure (Example problem)1.What is the net charge on the following protein at a given pH 7.5?G H W S F M L E E A R small peptide (11 a.a.)First assume that deprotonation of the R-group is predominates at 1- pH unit above the pKa and protonation of the R-group predominates at 1- pH unitbelow the pKa.+G H W S F M L E E A R-o - - +(SEE pKa Table)His pKa 6.0 -- 6.5Glu pKa 4.4Arg pKa12.0pH of 7.5 is well below the pKa of Arg --- Arg must be protonated (+ charge)pH of 7.5 is 1-full pH unit above the pKa of His --- His deprotonated (no charge) pH of 7.5 is well above the pKa of Glu --- Glu deprotonated (- charge)NET CHARGE AT pH 7.5 = -12.What is the net charge at pH 5.5?+G H W S F M L E E A R-+ - - +The only change is His ---- protonated below pKa ---- His is + chargedNET CHARGE AT pH 5.5 = 0(close to isoelectric point)PROTEIN MASSA.The mass of a protein is described in Daltons1. 1 - Dalton is simply 1-amu12C mass = 12.00000 Daltons2.Typical proteins contain between 50 to 2000 amino acids linkedtogether. The majority falls somewhere between 200 - 1000 a.a.3.The average atomic mass of all amino acids is . 110 Daltons50 a.a. X 110 Daltons = 5,500 Daltons or 5.5 kdamino acidAgain most proteins fall between ----- 10 to 100 kdB.Peptides ----- Small chains of amino acids1.No absolute defined line between a protein and a peptidea)generally 2 a.a -------> 50 a.a chains are called peptides(Have seen 8.0 kd and below called peptides) PRIMARY PROTEIN SEQUENCEA.In 1953 Frederick Sanger determined the first sequence of amino acidswithin a small protein called bovine insulin******SHOW OVER HEAD*****1.Bovine insulin consists of two peptide chains called " and $a)Sanger showed that proteins have a defined order ofamino acids within a peptide or proteinb)Sanger showed that proteins are made-up of only L a.a.and not D2.It is now known that the " and $ chains are initially synthesized asonly one single chain called Proinsulin and then processed into twoseparate chainsB. Sequencing peptides: Almost exclusively automated º Simply applysample to Automatic Sequencer and the sequence of a small peptide can be determined!(10-15 amino acids with very good accuracy) See Edman Degredation.1. N-Terminusa) FDNB (5-fluoro-1,3-dinitrobenzene)(Sanger’s Technique) Forms bright yellow derivative with N-Terminal residue.b) Dabsyl Chloride - commonly used now - more sensitive - candetect smaller quantities.c) Dansyl Chloride - very sensitive- forms fluorescentsulphonamides.(Note) After reaction - perform amino acid composition. Look foramino acid linked to label.* Only good for N-Terminal Residue. Must be repeated for entire protein Auung!! 400-500 residues. (If you could do 1 per day, 1-2 years!)2. Edman Degredation - Method applied by automated sequenators!Chemical used Phenylisothiocyanate.Label at N–Terminus º Brief acidic cleavage leaving newN-Terminus for another reaction. (Organic Chemistry)Requires very little sample 10 picomoles (10-12 moles protein!!)C. Larger Proteins: Divide and Conquer1. Cleavage of Proteins: Not into amino acids but into smallersections . peptides.a) CNBrº Very dangerous - work with in hood only.i) Seems to be a specific chemical cleavage of a protein.(unusual)ii) Splits the peptide bond on the carboxyl side of metresiduesiii) Separate and Sequence pieces!b) Trypsin - Enzyme (protein) produced by pancreas whichdegrades proteins.i) Specifically cleaves on the COOH side of Lys or Arg.c) Endo Proteinase Lys C - Enzymei) Specifically cleaves on the COOH side of Lys (only)Very Populard) Chymotrypsin - Enzymei) Non specific cleavage on the COOH side of aromatic andsome other non polar residues.Note * digestion with two or more reagents is used to determine therelatedness of proteins - called peptide mapping.Show example: Show how to put a peptide back together using piecesD. Multiple Chains:1. Separate chains first using reducing agent i.e., $-mercaptoethanol orDTT (dithiothreitol) and denaturants, i.e., Urea or Guanidine HCla) isolate each polypeptide, digest into pieces, isolate each of thepieces and then sequenceE. Importance of Primary sequence:1. Protein Folding – All the information required for proper proteinfolding and function must be found in the primary sequence of aminoacids2. Inherited Disease– Many hereditary diseases are caused byalterations in the primary structure of proteins. Comparing theprimary amino acid sequence of a functional protein with that of anonfunctional or abnormal protein helps establish the underlyingcause of a given diseaseExamples:Cystic Fibrosis, Sickle cell anemia3. Nonfatal Alterations– Primary amino acid differences andsimilarities in common proteins among related and non relatedspecies are used to establish relationships among species:Evolutionary Lineages4. Protein Families–Proteins with similar or related primary structuresoften have similar functions. We group these related proteins into“families.” The primary structure of these related proteins helpsestablish structure/function relationshipsa) Cancer causing viruses often contain DNA segments whichencode for proteins that are similar to growth regulationproteins found in normal cells5. 3D Structure– The primary protein sequence is very helpful, if notessential, in the determination of the complete 3-dimensionalstructure by X-ray diffractiona) The X-ray diffraction gives electron density data only. The datalooks a lot like a 3D topographical map6. Repetitive Sequences– Short stretches of amino acids common todifferent proteins, often provide a similar function to these differentproteins. The stretches are called amino acid motifsExample:The binding of Ca+2 or Zn+2 is carried out in differentproteins by similar stretches of amino acids.Glycosylation sites are similar among different proteinsAsn – X – Ser Asn – X – Thr7. Cloning Genes– The primary amino acid structure can be used tomake DNA probes. These DNA probes are used to identify genesthat encode for the protein of interest8. Generate Antibodies – In some cases short protein sequences(peptides) can be used to elicit an immune response. The antibodiesgenerated can be used to study the production of the original proteinin different species or tissues.F.Changes in the amino acid sequence may cause serious phenotypicdisorders!1.Depends on the protein, location and type of amino acid changea)substitutions of similar a.a. in noncritical locations may notaffect the function of a protein at allb)Insulin ---- several changes -----> same functionc)Hemoglobin ---- one a.a. change -----> sickle cellG.Protein sequencing of an entire protein is rarely done anymore (exceptionsmall peptides 10 to 50 a.a). The work is very tedious and expensive1.Most researchers sequence small portions of a protein and then usethat information to find the DNA sequence (gene). The DNAsequence will reveal the entire protein sequencea)Also very complicated and time consuming -- advantage,you have the gene to work with when you are done AMINO ACID MODIFICATIONS IN PROTEINSA.Conversion of Proline into hydroxyProline1.H-pro is an important constituent of collagen (Vit C is required forthe conversion)------------------->a)Vit C deficiency ---- Bad collagen ---- ScurvyB.Carboxy Glutamate (dicarboxylic acid)1.Prothrombin (blood clotting protein) converted to thrombin (active)(Vit K is important for this conversion)--------------->a)Vit K deficiency --- inefficient clotting, bleeding disordersC.Phosphorylation(molecular switch)1.Regulation of enzyme and other protein functions can be controlledby phosphorylationa)signals activation or deactivation via phosphoester bond to oneof the hydroxyl-containing R-groups --- Ser, Thr or Tyr------------->b)Phosphorylation is reversible --- switch on or offD.Glycosylation(Sugar added to protein)1.Modified sugars are attached to either Asn, Ser or Thra)Asn --- N-linkedb)Ser, Thr --- O-linked2.Important for solubility of some hydrophobic proteins3.Important for localization --- usually targeted to outer membraneE.Signal Sequences1.The first 10 - 30 amino acids of some proteins are removed from anacent protein chaina)Targets protein to cellular organellesb)Targets proteins for insertion into membranesF.Proteolysis1.Specific cleavage of one protein by another protein (molecularswitch)a)Proinsulin -----> insulin (active)b)Prothrombin -----> thrombin (active)SECONDARY PROTEIN STRUCTUREA.The peptide unit is defined as the amide bond (fairly rigid structure)1.Resonance stabilized structure. The hybrid structure demonstrates thatboth the O and N bonds have double bond characteristica)Limits rotation about the C-N bondN RThe O from the carbonyl and the H are nearly always in the trans positions(exception is Pro)2.Two Planar rigid structures called the N and R planes can freely rotateabout the central "-carbona)rotation and rigidity allows proteins to assume well defined 3-D conformations (Not like a string)i)Proteins are not a random mess of amino acids3.Linus Pauling and Corey built precise models of proteins based onexperimental bond angles. (daunting task)a)Linus Pauling decided to see where H-bonds might form byrolling the paper (Nobel Prize)4.After much work two possible structures were proposeda)"-helix and $-SheetNote:Both structures were proven correct when the first X-rayreconstruction of myoglobin and other proteins were solved THE"-HELIXA.Proteins can be nearly 100% "-helices or nearly devoid of them1.Right handed helix ---- look up through the bottom of the spiral ----turns right or clockwise away from you(Example)Phone cord2. 3.6 residues (a.a) per complete 360o turn of the helix3. Pitch: rise (1.5 Å) X 3.6 residues = 5.4 Å rise/ turn4.Local proximity of amino acids — interactions among amino acids islocalized to 5 amino acidsa) H-bonding pattern is a very important force which holds thestructure in placeb) Amine H shared with the carbonyl O every 4th residue away3.Coiled/coils---- compound coils (example twisted telephone cord)a)Strong structures found in keratin ---- porcupin quilsb)Found in cytoskeleton proteinsTHE$-SHEETA.The $-sheet(often called a $ pleated sheet due to the folds in the structure)1.$-sheets require a 180o turna) $ -turn, reverse turn or hairpin turn are all names for the samestructureb)$ -turn is a small structure usually 4 a.a. in lengthc)Gly is nearly always found in the structure (small R-group)2.Several strands can be involved in a $-sheet structure******SHOW OVER HEAD*****3.H-bonding pattern is also a very important force which holds thestructure in placea)Amine H interacts with the carbonyl Ob)Distant interactions --- several a.a may intervene between theH-bonding pair (No set distance)4.There are basically two types of $-sheets --- Parallel and antiparallela) Parallel – all strands are facing the same directionb) antiparallel – all strands alternate in directionD.Super secondary Structures – combinations of " and $ secondary structures******SHOW OVER HEADS*****1. "$ Saddle2. $$ Sandwich3. "$ Barrel4. Four helix bundle5. " turn "LEVELS OF PROTEIN STRUCTUREA.1o structure ---linear sequence of a.a. in a protein chain: The types ofa.a. present and their unique order in the chainB.2o structure ---Substructures of a protein; "- helix, $-sheet or somecombination of the two (super secondary structure)C.3o structure ---The total unique 3-D structure of a single protein chain(polypeptide) ----> interactions (disulfide bonds,dipole/dipole, H-bonds) involve residues which are farapart in the primary sequenceD.4o structure ---Interactions between two or more completely foldedpolypeptide chains. Many proteins contain subunitpolypeptides of differing types. A tetramer is a proteinformed by four polypeptide chains. Each polypeptidemay be identical in primary structure or they may be verydifferent. Often the protein contains two sets of identicalpolypeptides called "2$ 2Summary of 2o structures and the forces which are involved in both the 2o and 3o structure of a proteinNote:disulfide bond is a covalent linkage of S--S atoms between Cys residues. Reversible oxidation/reduction reactionPROTEIN FOLDINGA.It is believed, with some strong experimental evidence, that the 3o structureand ultimate function of a protein is completely coded by the primary a.a.sequence1.Proteins cannot possibly fold into a 3o structure merely by a randomsearch of all possible conformations !2.Consider a small protein of 100 a.a. ---- Cyrus Levinthal calculatedthat if each residue could only assume 3 different conformations(positions in space), then the total number of possible structureswould be 3100 ---- 5 X 1047 different possible structures(whoaaaaaaa!!!!!)3.If it took only 10-13 sec (incredibly fast) to try each possible structureallowing for no repeated structures --- it would take 1.6 X 1027years for a protein to fold !!!a)Actual time is less than 0.1 secB.Richard Dawkins asked --- how long would it take a monkey to randomlytype a short line from one of the sonets of Shakespear's plays (Hamlet)?1.He calculated that at random keystrokes it would take 1040 keystrokes2.Now, retain correct keystrokes and allow the monkey to retype onlythe wrong ones ---- calculated 2 - 3,000 keystrokesa)difference of 1037 keystrokes by retaining correct lettersStryer --- "The essence of protein folding is the retention of partiallycorrect intermediates"C.Protein folding --- area of intense research ---- very complex subject!!1.Impetus for such research --- thousands of primary structures are nowknown --- very few 3o structures are known.a) Scientists would like to predict 3o from 1o information FOLDING AND FUNCTION RELATIONSHIPA.Christian Anfinsen: worked to understand the folding/function relationshipof an enzyme called ribonuclease (degrades RNA)1.Chemicals used:a)Urea (8M): chemical which disrupts the H-bonding within the2o structure of a protein ---- denaturant (unfolds proteins)b)Guanidine HCl: strong denaturant --- probably disrupts H-bonding as wellc)$-mercaptoethanol ($-ETSH): mild reducing agent ---reduces S--S bonds to SH SH (unlinks the disulfide bonds)2.Treated RNase with Urea or g-HCl (+ $-ETSH) ----- completelyunfolded "denatured" protein chaina)Dialysis: removes all denaturants (size exclusion)i)Complete retoration of enzyme activty (24 hr)3.Conclusion: Protein folded into native conformation on its own (noother proteins were needed to intercede)a)S--S bonds probably reformed by oxidation with airB.Alternate procedure1.Remove $-ETSH first, allow S--S bonds to form, then remove Ureaa)retain denatured state but allow S--S bonds to reform2.Removal of the Urea --- 1% of the total activity remained ?3.Conclusion: The RNase was scrambled by the random formation ofS--S bondsa)8 - SH (Cys) groups taken in pairs = 105 combinationsb) 1 out of 105 RNase molecules formed correct S--S bondsbefore they were allowed to fold --- 1% activity4.Added trace of $-ETSH to 1% active sample ---- total activity slowlyreturned --- ($-ETSH is volatile)C.Native (active) form of RNase must be the most thermodynamically stableconformation (conformation of lowest energy) and water must be driving force in foldingNote:The folding of some proteins is assisted by enzymes which do catalyze the lowest energy state conformation。