Macromolecular Structure BCH 701
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This 54 page Class Notes was uploaded by Kian Berge on Thursday October 15, 2015. The Class Notes belongs to BCH 701 at North Carolina State University taught by Staff in Fall. Since its upload, it has received 18 views. For similar materials see /class/223863/bch-701-north-carolina-state-university in Biochemistry at North Carolina State University.
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Date Created: 10/15/15
Gene Isolation This is beyond the scope of this class so assume it is easy From this pomt its pretty straightforward to express it and make large quantities of the protein for which it codes The segment of DNA corresponding to the coding region need only be inserted is the correct reading frame into suitable DNA expression vector Such vectors are usually small circular DNA molecules that include all the nucleic acid sequences needed to ensure replication of the vector and efficient transcription and translation of the gene in an appropriate host cell There are a whole bunch ofsuch expression system which differ primarily in their efficiency of translation and is the way that they process the protein synthesized Amplify DNA using isolate and cut vector PCR techniques with restriction enzymes Cut DNA with restriction enzymes Ligate insert and vector Transform into suitable bacterial host SeieCt tranSfOI39mantS gt Subclone into expression vector Isolate recombinant DNA and confirm by DNA sequencing For large scale production of proteins bacteria such as Ecoi are favored because of the wealth of knowledge about the genetics of this system and the availability of very efficient expression vectors Synthesis of a desired protein by an expressing vector should require only that the protein have an initiating methionine residue because all other known signals for transcription and translation lie outside the proteincoding region of a gene and are part of the expression vector Anyway leads us onto site directed mutagenesis We can make a change at the gene level to code for a different residue at a particular place in the sequence can have serious effects on structure 1 structure assembled a long run ofamino acids linked by a polypeptide bond Arg Thr Val Ala Phe Pro Cys Val Glu In reality these are not just linear The large size of polypeptide chains enables them to fold back on themselves not completely randomly so that many simultaneous interactions take place among different parts of he molecule A complex 3D structure results putting functional groups in the appropriate places The activities of proteins also relies on interactions with their environment water salts membranes other proteins nucleic acids etc All of these interactions arise from a limited set of fundamental noncovalent forces but with many variations on the theme Noncovalent interactions within a protein A lot of these are mediated by water 1 Short Range repulsions Molecules get close they repel Electron orbitals overlap and can t be in the same space Pauli exclusion principle2 things can t occupy same space The repulsion is strong varies exponentially with the inverse of the distance and limits how close atoms can come Because this repulsive energy is so strong and rises so steeply we can consider atoms and molecules as having definite dimensions and occupying volumes that are impenetrable to other atoms at ordinary temperatures Individual atoms are modeled as spheres and their volumes are defined by Van der Waal s radii VDW VDW radii smallest distance I 1 between noncovalent atoms 39I ll l I I ll A range of values is usually given for VDW radii because the observed radius depends on the way in which the atom is covalently bonded ex H bonded to an aromatic carbon VDWR1 A BUT when bonded to a negative ion VDWR 154A That s a big variation VDWR is the minimal estimates of the size of an atom or molecule They define the VDW surface area and volume More practical approach to surface area is the concept of accessible surface area area defined by the solvent molecules in contact Important for areas of proteins exposed for attacktargeting by other molecules 2 Electrostatic Forces These dominant everything All intermolecular forces are thought to be essentially ELECTROSTATIC in origin Point charges Coulomb s aw the force of attraction or repulsion acting along a straight line between two electric charges is directly proportional to the product of the charges and inversely to the square of the distance between them Energy of interaction AEZAZBE2 rAB Z of charges Doesn t matter is E electron charge 1r rAB distance This attractiverepulsive force is excellent over long distance and is strong Coulomb s law is only good for 2 point charges in a vacuum To accommodate the electronic environment we use the dielectric constant D the electrostatic constant that takes into account any modulation in the environment due to electrostatic interactions AEZAZBE2 D rAB Dielectric constant decreases the interactions and is usually between 2110 ex water80 41 V V Hydrogen bonds involved in the interaction betWEen ion he tw 281 A teft and 289 A right Adapted from D D Bray39 e 24414 418 1984 Dipoles Some charge can be localized if the atoms have different electronegativties Big electronegative atoms have negative charges others have positive charges Electronegativity having a tendency to attract electrons The electronegativities of the atoms in proteins are 0345 N298 C255 S 253 H213 Most Least OH NH 6 6 6 6 Also the TE electrons in the aromatic rings of Phe Tyr Trp are localized above and below the ring g N Negative clouds Each face has a net negative charge of O15E Whereas the hydrogens have a net positive charge of about the same value This creates an electrostatic sandwich These partial charges dominate ring interactions E Ring interactions can occur at right angles or just slightly offset on top and below The separation ofcharge in a molecule determine its dipole moment D It has magnitude and direction ex peptide bond Dipoles interact with point charges other dipoles any charged basically Dipole are weaker than those between ions Why 3 Hydrogen Bonds Occur when 2 electronegative atoms compete for the same hydrogen atom 3D HA Donor Acceptor Favorable interaction 6 6 6 D HA Partial positive charge due to electronegativit effects We can expect a lot of Hbond N H OC Approx 103A Approx 1920 A Hbond N 3 A The lengths and strengths of hydrogen bonds depends on the electronegativities of the acceptor and donor of course Water Solubilities To be soluble in water a molecule must occupy a certain volume thereby disrupting the water structure at least within that volume The solubility of a molecule in water depends on how mucn of the unfavorable aspects of creating a cavity in water are compensated by favorable interactions with the surrounding water molecules A measure of favorable interactions of a molecule is its hydrophilicity Arg gt Asp Glu His gt Trp Tyr gt Thr Ser gt Met Cys Phe gt Val Ala Gly Asn Lys Gln Leu e Polar hydrogen bond donors and acceptors like to Hbond to water Like to be on surface N0 POlaFS SUCk39 HATE water of protein Hydrophobic Interaction Nonpolar groups hate water hydrophobic and prefer to hang out in nonpolar environments sticky places The preference of nonpolar moieties for non polar places has become known as the nyaropnomc Interacuons and is a major factor in the stabilities of proteins nucleic acids and membranes Avoiding much of the thermodynamics the hydrophobic interaction does not result from repulsions between water and nonpolar entities as implied by the name The hdro hobic interaction really is a tendency of nonpolar Vrou s to interact with each other rather than with water So which residues are most hydrophobic ILV gt GAF gt CM gt ST gt WYPH Most gt Least The amino acid side chains with polar groups are amphiphilic have both polar and nonpolar segments They tend to interact in aqueous solution in such a way that their nonpolar segments interact with other nonpolar groups and their polar groups contact water This is the basic principle of the formation of membranes lipid bilayers and micelles A similar phenomenon can be seen to produce the folded conformations of proteins Sticky patch on surface of protein want to cover with another sticky patch of same type wants to search out another hydrophobic target usualy Binding sites Detection of amino acids and proteins 1 Ultraviolet absorbance 280nm Recognizes Phe Tyr Trp and disulfide bonds To quantitate need molar absorbance coefficient of the protein Luckily a fully unfolded protein has same properties of absorbance close to its constituents in the aromatic range So if you know the number of aromatics and disulfides you can calculate this coefficient 2 Staining Commonly use Coomassie Blue a dye which doesn t react with protein but rather sticks to it forming a noncovalent complex It makes the noncovalent complex using a mix of nonpolar and ionic interactions however this is not true for all proteins No one really knows how it works R250 RH G250 RCH3 oc H 166 2 5 Two types 6250 used to quantify the amount of protein in solution because complex formation changes their absorbance properties In acidic conditions the absorbance max moves from 465 to 595 nm Very simple and widely used The Rgroup on the picture before is a methyl group R250 used to stain proteins in gels This procedure relies on the proteins being made insoluble and binding the dye tightly Most of the excess dye is removed washing the gel methanol acetic acid water destain Approximately 01mg of protein can be detected on a polyacrylamide gel The Rgroup on the picture before is a Hydrogen For more sensitive detection use silver staining 10399 g but in general for any type of accurate structure determination we need more than this xray and NMR Determination of protein sizes A peptide becomes a protein when it has more than 50 residues 50 what s the molecular weight of the protein If you know the amino acid sequence then you simply add it up 1 Sedimentation analysis Analytical Ultracentrifugation Runs N 250000rpm Under a given set of conditions the rate of sedimentation of a protein depends on its MW density shape and interactions with the solvent A protein that is large is more likely to migrate rapidly but it might be aggregating A small protein may move slower or it may have an unusual shape Measure the time it takes for large molecules to move to bottom to determine the size 2 Gel Filtration Size of a protein determines its rate of passage through a molecular sieve Molecular Sieve small particles with a network of pores into which molecules of less than some maximum size can penetrate The smaller the proteinthe greater the probability it will enter the internal volume of the particles Pour proteins into a molecular sieve Then wash with a buffer The first proteins to elute TOO LARGE TO ENTER PORES The volume of buffer required to elute them Void Volume Vo volume of column outside the particles Other proteins are eluted in DECREASING order of the molecular size A gel filtration column is calibrated using proteins of known size Note Funny shapes can mess you up 3 SDS Polyacrylamide Gel Electrophoresis SDSPAGE Extremely popular method Check protein movement in polyacrylamide gel electrophoresis in the presence of the detergent sodium dodecyl sulfateSDS CH3OSOS Na This method is related to gel filtrations in that the size of a protein is estimated by its migration through the small pores of a gel matrix In this case the gel matrix is continuous rather than particulate so smaller proteins move through faster Use a set of standard molecular weight markers to compare your protein migration to those The eletrophoretic mobility how fast something moves in relation to charge of a protein is determined by its weight charge and shape Charge and shape are standardized for all proteins by SDS electrophoresis SDS binds to proteins somehow disrupts their structure and shape dissociates them into polypeptide chains and imposes comparable shapes and net charge densities on them Don t really know how It just works ProteinSDS complexes have electrophoretic mobilities thru polyacrylamide gels that are inversely proportional to the logarithm of the length of the polypeptide chain Compare to standards Get your weight Original mixture of large and small protein molecules G l e Migration through filtration continuous gel Migration Gel filtration Polyacryl resin amide gel Pattern in gel Elllmon from after given period co um of migration c O v0 v3 ve m g z 3 J 1 l l g 39 o o E C D O U U E F 2 E 9 9 EL 0 Elution volume a Distance migrated a Determining the primary amino acid sequence The 1 sequence identifies a protein unambiguously determines all its cnemlcal and biological properties and specifies its structure 1 Edman degradation sequencing from the Nterminus It removes and identifies one residue at a time Requires that the terminal qamino group be free so it can be reacted with phenylisothiocyantate Must be in alkaline conditions R1 I II QNCS NHz CH C NH peptide lpH 9 l R NH C NH CH C NH peptide itri uoroacelic acid NH S NHZ peptide NH CO l EH next cycle R1 ll M HC rats C NH 0 CH IL PTHamino acid 2 Mass Spectrometry Can be used for MW determination and with Edman degradation to help solve the primary sequence 3 Enzyme digest Use chymotrypsin to cleave after every Tyr Phe Trp and Leu Use tryspin to cleave after ever Lys Arg Once the sequence is cleaved using one of various enzymes or procedures use Mass Spec on the fragments A database is used to tell you what each fragment is However most sequencing done from gene sequences Table 15 Methods for Cleaving Polypeptide Chains Sequence cleaveda AlaYaa ArgYaa AsnGly AspYaa AspPro XaaAsp XaaCys GluYaa GlyYaa LeuYaa XaaLeu LysYaa MetYaa PheYaa XaaPhe ProYaa TrpYaa TyrYaa Procedure or enzyme Elastase bromelain Trypsin endoproteinase ArgC clostripain Hydroxylamine V8 protease Mild acid AspN protease Cyanylation V8 protease Elastase Pepsin Thermolysin Trypsin endoproteinase LysC bromclain CNBr Chymotrypsin pepsin Thermolysin Prolylendopeptidase Iodosobenzoic acid N chlorosuccinimide chymotrypsin Chymotrypsin bromelain The speci c residues are indicated by bold type Xaa and Yaa can be almost any amino acid except for Pro in many instances Cleavage is Cterminal to residue Xaa Nterminal to Yaa Assembly of 1 structure Coded information for the structures of proteins is contained in the genetic material ofthe chromosome usually DNA in the form of linear sequences of nucleotide bases 3 sequential nucleotides contain the code for a single amino acid residue The genetic info ofthe DNA is constantly checked and edited by the cell to ensure that it is altered as little as possibleonly the correct amino acid is placed in the correct place mm worm mum is seam c A W m mm a e Gene Structure brief background All the info for synthesizing the 1 structure of a protein is encoded in the genetic material of the chromosomes which is double stranded DNA Into is encoded in sequences of4 nucleotides on one strand AAdenine CCytosine G Guanine TThymine For RNA T 9 U Uracil The sequence of the other strand or DNA is chemically complementary to the first A pairs with T AGCCT G pairs with C TCGGA Form genes Code for proteins DNA Promoter i lntron Intron 539runtranslated 339Lmtranslated 39 region region lTranscription mRNA precursor 53 lsoiiclng mRNA JTranslation Protein precursor H7N cojH ll rocessmg modificatlon Final protein product HEN WT COEH FIGURE 21 Steps in the expression of genetic information in biosynthe sis ofa protein At the top is a simpli ed typical cukai39yotic gene with the promoter and other regulatory regions of the gene on the left upstream The segments to be transcribed into mature messenger RNA mRNA are shown as boxes those segments coding for protein are shaded whereas those unshaded correspond to the 5 and 3 untranslated re gions of the mRNA After transcription of the gene into the precursor mRNA second line the introns are removed by splicing Prokaryotic genes differ in not having introns and not undergoing splicing The mature mRNA line three is translated into polypeptide chain line four which may be the same as the nal form olthe protein or a precursor form that is modi ed Parts ofthe polypeptide chain can he re moved proteolytically from a precursor form and a variety of other covalent modi cations of the chain may also occur the branched structures here indicate glycosyl groups added to speci c Asn residues In an additional process not iridi cated here the polypeptide chain must fold into the appro priate threedimensional conformation Chapter 7 to yield the final biologically active gene product Expression of genetic info in biosynthesis of protein 1Chromosoma DNA undergoes transcription to a complementary messenger RNA mRNA molecule A bunch of regulatory proteins are present which bind to the upstream part of the gene promoter or all over enhancer regions Binding of the regulatory proteins to the DNA can have positive or negative effects on transcription they are there to ensure that the gene is expressed only under appropriate conditions When all is fine and dandy for the expression of a gene it is transcribed by RNA polymerase enzymes which copies one strand of DNA into its complementary RNA strand a39 uptranslned ragi 2 Translation Mature mRNA molecules are translated into polypeptide chains in the cytoplasm by a complex apparatus of ribosomes tRNA and various factors Translation of mRNA is initiated by the formation of complexes of mRNA the two subunits or ribosomes and 3 initiation factor proteins 1 FIGURE 31 I 1 a v Steps in th expression of39geneiic infori nu m biqsyn 39 39ois proigin At the 16pi a simpli ed typical e ukaiyo 39 guncnwilh ihe promoter nnd dthcr regulatory region39s f thequot I 39genc39 onthgl39 quot 39 V L e imo manire pstream Th segments a transcnb d I I qc gcr39RNA mRNA m sh v vn p quotf hos segments coding foipr ot ih shad d thosg unshn39ded c rpspondjo the 5 and 3 untririsl u A b 5r glans infthe wk 7 jiccu39rsor mRN 39 1 splicing Prolg i39 hm ndergszinzs translated into pp p 73thquot s m as th39e39l39 alf aumi39uf ic39pfofeinvo H 39 39 batik modi quotParts ofthc39 39olypqptidgc can to Ribosomes are big consisting of at least 3 RNA molecules and 55 different proteins Prokaryotic cells have one RNA polymerase that transcribes all genes but its made specific for different genes by various sigmafactor proteIns tukaryotlc cells have 3 enzymes RNA polymerase I II and III which transcribes different genes land Ill genes that code for stable RNA 9 proteins Goal is to get mRNA 1 Eukaryotic gene splicing of useless portionsintrons 2 Prokaryotic gene no splicing MATURE mRNA Amino acids couple to appropriate tRNA molecules which adapt them and supply them in the proper sequence to the ribosome mRNA complex where they are attached to the polypeptide being synthesized SO 5 3 nucleotides 3 dictate what amino acid is added Second a position U a g a E 8 LL CL Phe Ser Tyr Cys U U Phe Ser Tyr Cys C Leu Ser Terminate Terminate A Leu Ser Terminate Trp G Leu Pro His Arg U C Leu Pro His Arg C Leu Pro Gln Arg A Leu Pro Gln Arg G Ile Thr Asn Ser U A He Thr Asn Ser C 116 Thr Lys Arg A Met Thr Lys Arg G Val Ala Asp Gly U G Val Ala Asp Gly C Val Ala Glu Gly A Val Ala Glu Gly G This is very important feature because we can change one amino acid in a primary sequence to determine which residues are the most important Car analogy Macromolecular Structure Proteins They are a relatively homogeneous class of molecules All are the same type of linear polymer built of various combinations of the same 20 amino acids differing only in the sequence Their functional diversity lies in the threedimensional structures that these linear polymers can make by simply being linked in different sequences What do proteins do Pretty much everything Store and transport a variety of molecules Guide flow of electrons in processes such as I hotos nthesis Transmit information between specific cells within an organ Control passage of molecules across membranes of compartmentalized cells and organelles Function in immune system to defend against intruders antibodies Control gene expression by binding to specific sequences of nucleic acids to turn them onoff Structural stability within cells including hair nails tendons and bones of animals 19 Amino Acids have this structure Exception PmHne mu cH ooo mo cu W On ythe RVrou changes A ways the LIsomer 5mquot Nuckeuphlllc OH H L HQNXCOOH HZN coon H2111 COOH Glycmz my a Alanine Na A Senna Sen 5 MW 5705 Mw71os MW 97011 pKa 15 Hydrophobic Hgnjfcoon HEN600H H211 COOH Vanna m1 I Lemme Leu L 1551911211191119 1 MW 9914 MW 11316 MW 11315 Arumalm H CH N 1 HEN COOH HZN COGH HEN coon Phenyla anrne1PheF Tymsme Ty V Trymophanmp W Mw 147111 Mw15311a MW 111621 Amide Ham 0 o NHZ HN NHf NH2 H2N COOH HZN cooH HEN CDOH Aspaya91neAsn N Glmzmme em a H1shdme H15 H Mw11411 MW 121114 Mw 13711111 504 I HZN c OOH Thleomne Th1 T MW 1u111 pKa 15 HEN coo Methmmne Mei M 13119 MW Acidic 0 0H HZN COOH Aspamc Amd 1A5p D MW 11509 pKa 9 HZN Lysine L ys K Mw 1231711va NHJ cooH 1079 SH 1 HEN COUH Cysteine Cys 0 MW39 103161 1125 CL N H COOH Pmlme Pm 12 MW 9712 HEN CODH G mzm1cAmdGluE Mw 129121 407 HEN YNHQquot NH HZN cooH Avgmme A19 R Mw1551gplta12411 In proteins the amino acid is linked together by PEPTIDE BONDS Peptide Dunc Each amino acid in a polypeptide chain is referred to as a residue Usually between 50 to 3000 linked together to form a polypeptide chain gt 50 protein lt50 polypeptide This polymeric linear linkage Primary sequence structure The sequence of amino acids in a proteinpolypeptide chain generally identifies a protein unambiguously Peptide bond appears to have 40 double bond character which makes it strong Rotation about this bond is restricted and pretty planar 4 Asymmetric center Trans Conformer Shorter bonds are stronger and have less movement Cis Conformer 1 4 This conformation is not very well liked except with proline 2 3 Cis conformations tend to create steric hindrance and repulsions with the side H H chains of the amino acids R groups All about energy Trans is favored energetically fewer repulsions However if the residue that follows a peptide bond is a proline its cyclic side chain diminishes the repulsive effects and Cis trans isomers are approximately the same energetically maybe around a factor of4 difference rather than 1000 This could cause problems from a structural analysis viewpoint example D9K Resonance of the peptide bond tend to redistribute its electrons and the polypeptide backbone is correspondingly polar The H and N atoms appear respectively to have positive and negative charges of 020 electrons whereas C and 0 respectively have positive and negative charges of 042 electron The positive and negative charges help to give the protein its functionality This gives the peptide bond a permanent dipole moment of 35 Debye units The peptide backbone of each residue contains one potent hydrogen bond donor NH and a hydrogen bond acceptor CO this is critical for the 3D architecture of proteins The peptide backbone is not very reactive chemically The only groups usually ionized are the terminal 0 amino and carboxyl groups which normally have pKa values of about 74 to 39 respectively depending on the nature of the terminal amino acid residue A proton is added or lost to internal peptide bonds only at extremes of pH The apparent pK value of the amide NH for deprotonation is between 15 and 18 and is in the region of 8 to 12 for protonation The oxygen atom of the carbonyl group is protonated more readily with an apparent pK of about 1 These properties facilitate the exchange of hydrogen Isotopes between the backbone and aqueous solvents which is important to the study of protein fluctuations in solution The amide proton has the ability to exchange with the solution to create a constant exchange process Glycine DH It has no OlC asymmetry Glycine is a very flexible residue because there is no steric hindrance This allows glycine to be dynamic It is very common in loops Aliphatics 0W rm Hydrophobic They hate water but love each other They also like other nonpolar atoms They are referred to as STICKY They help stabilize the folded conformations of proteins Cyclic imino residue Rigid aboutthe N C bond slighin puckered with gamma Carbon raised a little ii 0 amide proton can not hydrogen bon The pepiide bond before a proline likes id be cis Hydroxyl groups The OH loves to Hydrogen bond can act as a switch in catalysis Acidic residues Even though they look similar the are not close functionally due to the extra CH2 on Glutamine The COO group clearly likes positive charges which makes these good for metal ion binding Proteins do whatever necessary to dispel charge such as binding to metals Amide Residues Asn Neither is too reactive Have polar ends and are H bond acceptors and donors eg If an Asn and Gly are next to each other a kink could possibly form due to deamidation side chain and backbone reactwhy Gly Basic Residues HIN lf COOH H lylim Normally ionized but if not the side chain becomes very reactive and a potent nucleophile Good Hbond donor Tend to be h dro hobic within chain and very nonhydrophobic at end of chain Interact with DNA nucleic acids Has a very long side chain which likes to interact with water in solution because end is polar Searches for negative species mostly on surface of proteins Imidazole ring Tertiary arnine Good nucieophiie donates eiectronsi In its nonionized form the nitrogen with the hydrogen is an eiectrophiie and donorfor Hbonding The other nitrogen is a nucieophiie and acceptor for hydrogen bonding Very versatiie side ofthe residue The 2 N s within the ring are denoted 5 and 62 Two tautorners can exist where either N can be protonated Predominantiy it appears that the 2 is the protonated one Metals Aromatics 1 KIN IS COOH H Emmin The largest and most least freq uently I phenylalanine The ring can reorient Flipswitch I The 70H group makes it quite reactive Can hydrogen bond Molar absorptivity 40000 20000 l 0000 5000 2pm 1000 llllllll l llllllll lllllllll l q 1 z l l 240 260 Wavelenth am The aromatic residues are responsible for ultraviolet and fluorescence properties of proteins also known as chromophores The spectral properties of the side chains are very sensitive to local environmental changes and are useful probes of structures Sulfur containing residues Very reactive The CH2 can ionize at mild alkaline conditions Nonpolar and unreactive The sulfur can not be protonated Acts as a nucleophile a little Disulfide bonds can form between two cysteine residues in deprotonating conditions The cysteines will lose their H s when in a pH above 7 To break the bond decrease pH below 7 2 00 I Iquot If I i 7 n u Ty I llr IT g m m u f J I i r magmas cystei n
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