BMEtest2notes

8/7/2019 BMEtest2notes

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BME Test 2 Notes

Peptide bond Link of carboxyl group of one amino acid to the amino group of another amino acid


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-R equires input of free energy, however, the peptide bonds are quite stable kinetically stable

Polypeptide series of amino acids joined by peptide bonds

-Each unit is called a residue

Properties of proteins:

1) Linear Polymers made of amino acids function is dependent on 3-D structure2) Contain a wide variety of functional groups results for broad spectrum of activities3) Can interact to form complex assemblies4) Can be rigid/flexible rigid cytoskeleton, flexible act as hinges/levers

Amino acids central carbon = alpha carbon, 4-diff groups connected = chiral molecule

ONLY L ISOMER S AR E CONSTIUENTS OF PR OTEINSAmino acids in neutral pH = dipolar ions (ZWITTER IONS)Almost all peptide bonds in proteins are transHydrophobic amino acid side chains cluster, rather than contact water

-Hydrophobicity effectHydroxyl group on aR- side chain = more hydrophilic and reactive

Thiol group (-SH) is more reactive than OH groups-come together to from disfulfide bonds

Positive charge = hydrophilicHistidine is in active sites of enzymes, where its imidazole ring can bind and release protons withenzymatic reactions-2 acidic side chains = negatively charged (do not accept protons)

Polypeptide chain has polarity because end are different.-written starting with terminal amino acid-backbone is rich in hydrogen bonding potential- in side chains, C=O is a good hydrogen bond acceptor, and NH is good hydrogen bond donor-When chain is cross linked, disulfide bonds occur fromed by the oxidation of cycteine residues

-occurs in extracellular, but not intracellular-on rare occasions, nondisulfide cross links can occur from other side chains

Each protein has a unique, defined amino acid sequence


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D etermines: Mechanism of action (ie: enzymatic), 3-D structure, evolutionary history (commonancestor)Folding occurs due to reactions between amino acid sequences and 3-D protein interactions

Peptide bond is planar, has double bond character (prevents rotations, thus contrains the conformationfo the peptide backbone), uncharged (allows for polymers of amino acids linked by peptide bonds toform tightly-packed globular structures)

-Almost all are trans due to steric hinderance of alpha carbons-X- Pro are the only cis peptide bonds (due to steric hindreance of N of prolene to 2 tetrahedral

carbon atoms)

Freedom of rotation in amino acids allows the protein to fold in many different ways-R amachandran diagram demonstrates the possible values due to steric hinderance and steric

exclusion (no two atoms can be in the same place at the same time)-Angle between N and Alpha carbon is -Angle between alpha carbon and carbonyl group is

Flexible polymers with a large number of conformations do not fold into unique structures due to theunfavorable entropy

Thus, rigidity and / angles limit the number of structures and unfolded form of a protein can fold to

Alpha helices and beta pleated sheets form via reations between N -H and C=O hroups of amino acidsthat are near each oteh rin linear sequence

Alpha Helix-R od- like structure-Stabilized by hydrogen bonds between NH and CO groups-1.5A-3.6 amino acid residues per turn-R ight-handed are favorable due to steric hinderance

Beta Sheets-composed of 2 Beta stranda-3.5A-Sheets linked through hydrogen bonds-Antiparallel NH are bonded to CO of each amindo acid are hydrogen bonded-Parallel Same as antiparallel, but 2 amino acids are linked to 1 amino acid

More elaborate structure = loop (interaction taken to stabilize the chain)

Coiled-coil protein 2 Alpha helices intertwined. Cross-Linked by van der waals forces and ionicinteractions


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-Collagen-3 alpha helices (Gly in every 3rd sequence since collagen is so tightly packed and gly isthe only amino acid small enough to fit)

-No hydrogen bonds stabilized by steric repulsion of pyrrolidine rings of proline anddyroxyproline residues

Tertiary structure overall course of the polypeptide chain

Motif/supersecondary structure specialcombos of helices/sheets with loops/turns that produce aspecific function

Purification-Homogeneate is formed by disrupting cell membrane, and the mixture is fractionized by centrifugation,then again a a higher force. (differential centrifugation).

Isoelectric- isoelectric point pH where the net change is 0


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Lecture4: 08/28/06

Proteins

Proteins play a major role in essentially all biological processes such as catalysts,regulation, transport, providing mechanical support and various other functions

Proteins are diverse in terms of structure and function L ike carbohydrates, proteins are polymers of smaller units T he building blocks of proteins are amino acids

Amino Acids T here are 20 amino acids that built up a protein A n E amino acid consists of a central carbon atom, called the E carbon to which an

amino group, a carboxylic acid group, a hydrogen atom and a distinctive R group (alsoreferred to as side chain) is attached.

B ecause of the asymmetric E carbon Eamino acids are chiral and may exist as an L isomer or D isomer.

B oth the L isomer and D isomer are mirror image of one another and arenonsuperimposable

T he enantiomers can be distinguished by the ability to rotate a plane polarized light either clockwise or counterclockwise

A lmost all naturally occurring amino acids are L- amino acids except in some bacteria O nly L- amino acids are incorporated into proteins T he specific configuration of amino acid and hence the proteins are important for

recognition by cellular enzymes


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Zw itterions

T he amino and carboxylic acid groups of amino acids readily ionize T he ionization state of an amino acid varies with pH

A t neutral pH, amino acids in solution exist predominantly as zwitterions or dipolar ionswhen the amino group is protonated ( -N H3+) and the carboxyl group is deprotonated ( -C OO -)

I n acidic pH (e.g., pH1) both the amino ( -N H3+) and carboxyl ( -C OO H) groups are protonated

A s pH increases, nearing the pKa of carboxylic acid ~2, the carboxyl group starts givingup its proton ( -C OO -)

T he zwitterionic form persists until pH approaches pKa of the amino group ~9 when the protonated amino group loses a proton ( -N H2)

T he zwitterionic property of amino acid makes it a good candidate for a buffer. Glycine isan excellent buffer in the human blood stream

R group

T he 20 amino acids are distinguished by their R group or side chains T he side chains vary in size, shape charge, hydrogen - bonding capacity, hydrophobic

character and chemical reactivity T hese properties give rise to the various functions of a protein T he amino acids can be classified into different groups


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Amino acids with aliphatic side chains

Glycine is the simplest amino acid, containing a single hydrogen atom as the side chain.I t is achiral due to its symmetric structure

A lanine is the next simplest amino acid, containing methyl group as the side chain B oth glycine and alanine made up 40% of silk protein L arger hydrocarbon chains are found in valine, leucine and isoleucine M ethionine aliphatic side chain includes a thioether ( -S- ) group I soleucine contains an additional chiral center T he larger aliphatic side chains are hydrophobic and therefore have the tendency to

cluster together in water. T his is known as the hydrophobic effect


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Proline is another aliphatic amino acid, which is unique because its side chain is bondedto both the nitrogen and E carbon.

Amino acids with aromatic side chains

Phenylalanine contains a phenyl ring in place of one of the hydrogens of alanine I n tyrosine, the aromatic ring contains a hydroxyl group, which is reactive

T

ryptophan contains an indole group joined to a methylene (-C

H2-).

The indole group istwo fused rings containing an N H group

T he hydroxyl group of tyrosine and the N H group of tryptophan make these two aminoacid less hydrophobic than phenylalanine, which is purely hydrophobic

Amino acids containing hydroxyl, amide or thiol groups


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S erine and threonine contain hydroxyl groups ( - O H) attached to an aliphatic sidechain

T he hydroxyl groups make these amino acids much more hydrophilic and reactivethan alanine and valine

T hreonine, like isoleucine contains an additional chiral center


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A sparagine and glutamine are uncharged derivaties of the acidic amino acids aspartateand glutamate. B oth have a terminal carboxamide in place of a carboxylic acid

T he side chain of glutamine is one methylene group longer than that of asparagines C ysteine in structurally similar to serine but contains a sulfhydryl or thiol group ( -S H)

instead of a hydroxyl ( - O H) group.

T

he thiol group is much more reactive than hydroxyl group T wo thiol group may come together to form disulfide bonds

Amino acids containing basic side chains

A mino acids belonging to this group are hydrophilic L ysine and arginine have relatively long side chains that terminate with groups that are

positively charged at neutral pH Histidine contains as imidazole group, an aromatic ring that also can be positively

charged


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A mino A cids (continued)

Proline

Proline contains a secondary amine group, which differs from the other members of the aminoacids

Its side chain is bonded to both the nitrogen and the E -carbon atoms, which makes it moreconformationally restricted than other amino acids

The rigidity of the proline ring greatly influences the structure of a protein For example, compare silk and cartilage Silk, which is made up of mostly alanine and glycine is

very flexible whereas cartilage, which contains a considerable amount of proline is more rigid

www.rejuvenation-science.com/.../joint-knee.jpg

T he structure and function of a protein is determined by properties the amino acids that madeup the protein

Protein StructurePrimary structure Refers to the amino acid sequence of the polypeptide chain or chains


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Secondary StructurePolypeptide chain can fold into regular structures. The secondary structure refers to the spatialarrangement of polypeptide backbone atoms without regard to the conformation of the side chains. Thepolypeptide can fold into any one of the following structures: Alpha (E) helix, beta ( F) sheets, and turnsand loops

Alpha Helix

TheE helix has a coiled backbone, with side chains sticking outward TheE helix is stabilized by hydrogen bonds between NH and CO groups of the main chain Except for the first NH and the last CO group, all NH and CO groups are H-bonded such that CO

of residue n is H-

bonded to NH of residue n+4 TheE helix has 3.6 residues per turn with a rise (distance between two adjacent amino acids) of 1.5

The pitch of the E helix is 5.4 (the distance of one full turn along the length of axis)


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Hydrogen-bonding scheme for an E helix

B eta Sheets

Compose of 2 or more polypeptide chains called F strand The F strand is almost fully extended with the side chains of adjacent amino acid pointing in

opposite directions

F sheet is formed when two or more Fstrands adjacent to each other are linked throughhydrogen bond between CO groups of one F strand and NH group of an adjacent F strand andvice versa


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The F strands in a b sheet can all run in the same direction (from amino to carboxyl end), and iscalled parallel sheet or in opposite directions and is called antiparallel sheet

The antiparallel and parallel F sheet has distinctive pattern of H-bonding

An antiparallel F sheet

A parallel F sheet


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Reverse Turns and Loops

Proteins are made up from a combination of E helices and F sheets, connected by loops orreverse turns

UnlikeE helices and F sheets, loops do not have regular, periodic structure The main chain CO and NH groups do not hydrogen bond with each other but are expose to the

solvent and can form hydrogen bonds with water molecules

Loops on a protein

Loops that connect adjacent antiparallel b strands are called hairpin loops. Short hairpin loopsare usually called reverse turns (also known as b turns)

In many reverse turns, the CO group of residue i of a polypeptide is H-bonded to NH group of residue i + 3


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Structure of reverse turn

T ertiary StructureR efers to the three -dimensional structure of an entire polypeptide

In Aqueous Environment

The polypeptide chains folds such that hydrophobic side chains are buried and its polar chargedchains are on the surface

Nonpolar residues such as leucine, valine, methionine and phenylalanine are found in theinterior of a protein whereas charged residues such as aspartate, glutamate, lysine and arginineare found on the surface

In Biological Membranes

In biological membranes where the environment is hydrophobic, proteins have reversedistribution of hydrophobic and hydrophilic amino acids

Porins found in the outer membranes of many bacteria, are covered on the outside mainly withhydrophobic residues that interact with hydrophobic alkane chains of the lipid bilayer

In the outer membrane, porins serves as a channel for solutes to get in and out of the cell


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R everse amino acid distribution in porin

Some organisms take up nutrients by a process called endocytosis, through internalization of part of the membrane. This process requires the co -ordination of a number proteins


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www.bact.wisc.edu/.../book_4/chapter_2/2-60.gif

Quaternary StructureMany proteins are composed of two or more polypeptide chains, referred to as subunits. Quaternarystructure refers to the spatial arrangement of its subunit. The simplest quaternary structure is a dimerconsisting of two subunits, such as the Cro protein of bacteriophageP , which is a dimer of two identical

subunits.

Cro protein of bacteriophage P


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Lecture 9: 09-11-06

A mino A cids (continued)

Hydrophilic ( Acidic and Basic) amino acids:

The Amino acids containing dicarboxylic acids are Aspartic acid and Glutamic

acid


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y y Aspartic acid and glutamic acid are the two acidic amino acids that containsd icarboxylic aci d group.

y y These are often called as aspartate and glutamate to emphasize that at physiological pHtheir side chains usually get deprotonated and hence are negatively charged .

It is noteworthy that these side chains do accept protons and this ability to lose and accept protonsmakes these two amino acids functionally important especially where form an important part of theactive site of an enzyme for eg: aspartic proteases.

y The ability to lose and accept protons facilitates reactions as well as to form ionic bonds witheither the neighboring amino acid side chain or with the substrate.

y The carboxyl group of the aspartic acid and glutamic acid ionize only whenthe pH >2 becausethe pK a is around 2-3

y Aspartic and glutamic acid prefers to be on the outside of the protein but can also reside insideif they form an important part of the catalytic site as said before.

y The main difference between aspartic acid and glutamic acid is the extra CH2 group in glutamicacid to which the carboxylic acid group is attached.

y The above scenario shows that these amino acids have a net negative charge at physiologicalpH of 7.0.

y Amino acids asparagines and glutamine are the pseudo-dicarboxylic acids. They have an amidegroup and cannot ionize.

Basic Amino acids: Histidine (His, H), Lysine (Lys, K) and Arginine (Arg, R):


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y Lysine, Histidine and Arginine are the three basic amino acids that have amino groups in theirside chain.


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y These basic amino acids like their acidic counterparts are hydrophilic and would like to be onthe surface of the globular protein rather than being buried inside.

y However, like acidic amino acids the basic amino acid can reside inside if they are a part of theactive site.

y Normally, the acidic and basic amino acids stabilize each other by electrostatic interactionswhen they are buried inside the protein active site.

y Atphysiological pH both arginine and lysine have a net positive charge.

y As seen from above structures lysine has a primary amino group , arginine has a forkedstructure with positive amino groups and histidine has an imidazole ring which enables it tointeract with the substrate s negatively charged group as well as with aspartic acid or glutamicacid in the active site of the protein itself.

y Histidine has an imidazole ring that offers the characteristicpKb of histidine , which is around 6 .

y With a pKb of 6 histidine can be uncharged or positively charged near neutral pH depending onits local environment.

y Histidine is often found in the active site of enzyme where it can bind and release protonsduring enzymatic reactions.


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It is noteworthy that all other amino acids are charged at physiological pH except histidine.

y By convention always the amino terminus is written on the left hand side and the carboxylterminus is written on the right hand side.

y The amino terminus always carries a positive charge and the carboxyl terminus a negativecharge.


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Le cture 12: 9-18-06 Protein Purification Protein Purification

T he purification of proteins is the first step in understanding their function Proteins vary in size and chemical composition, which makes it possible to separate the

proteins from one another through various method T he protein of interest, which may make up only a fraction of 1% of the starting material

must be purify to ~98% purity

M ethods in protein purification

Homogenization

Proteins must be released from cells to be purified Various methods are used to disrupt cell membranes such as

French press pressure is applied to cells in a close chamber Homogenizer C ell suspensions are forced through a very narrow

channel under high pressure A homogenate is form


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Differential Centrifugation

T he homogenate can be fractionated by centrifugation I n centrifugation, denser material will collect at the bottom of the tube in a pellet whereas

material with lower density will remain in the soluble fraction called the supernatant Higher centrifugal force is required to pellet material of lower density (Fig. 3 - 1) T his method serves to eliminate selectively components of the starting mixture so that

only the required component remains O nly the fraction that contains the protein of interest will be further purified

Salting out

A t high salt concentration, the solubility of protein decreases T he salting out effect is the result of the competition of between the added salt ions and

proteins for molecules of solvent A t very high salt concentration, the bulk solvent is not available to dissolve proteins such

that proteins precipitate Different proteins precipitate at different salt concentrations, so the salt concentration

may be adjusted to precipitate the desired protein

A

mmonium sulfate is the most commonly used reagent for salting out proteins

Column chromatography

Protein mixtures can be separated based on the characteristics of proteins such as size or charge

G el-filtration Chromatography I n this method, proteins are separated on the basis of size S ample is applied to the top of a column containing porous beads (the solid support or

matrix) S mall molecules can make their way through the pores of the beads while larger ones

cannot A s a result, large molecules flow more rapidly through the column and elute first M olecules of intermediate size, which can occasionally enter the beads will elute at an

intermediate position T he small molecules that take a longer path will elute last


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I on-Exchange Chromatography Proteins are separated on the basis of their net charge Depending on the charge of the protein different types of ion - exchange column is used

N et charge of proteins Type of ion-exchange column Counter ion

Positive C ation exchange: C M - cellulose N a+ N egative A nion exchange: DE A E- cellulose C l-

C ation- exchange column I f a protein has a net positive charge, it will bind to a column of beads containing

carboxylate groups (i.e. C M - cellulose), which are negatively charged T he positively charged protein bound to the column can then be eluted by increasing the

concentration of sodium chloride S odium ions compete with the positively charged groups on the protein for binding to the

column Proteins with lower density of positive charge will tend to elute first followed by proteins

having a higher charge density

Anion-exchange column Proteins with a net negative charge can be separated on positively charged (i.e. DE A E-

cellulose) columns T he counter ion is chloride ion


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G el Electrophoresis

O ne way to monitor the progress of protein purification is to ascertain that the number of different proteins decreases with each purification step

A molecule with a net charge will move in an electric field T he electric force caused the charge molecules to move towards the oppositely charged

electrode Electrophoresis separations are carried out in gels, which serves as a molecular sieve


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Polyacrylamide gels are commonly used for electrophoresis U nlike gel - filtration chromatography, all molecules, regardless of size are forced to move

through the same matrix U nder denaturing conditions, proteins are separated largely on the basis of protein mass.

S odium dodecyl sulfate ( S DS ), an anionic detergent disrupts all non - covalent

interactions in native proteins M ercaptoethanol or dithiothreitol (D TT ) is added to reduced disulfide bonds T he S DS anions bind to main chains at a ratio of one S DS anion for every two

amino acids T he S DS bound proteins has a large net negative charge, which is proportional

to the mass of the protein T he negative charge contributed by the S DS is much greater than the charge

on the native protein that the native charge is insignificant T he S DS bound protein will move towards the positively charged electrode

during gel electrophoresis S maller proteins move faster through the gel compared to larger proteins

When electrophoresis is complete, proteins in the gel can be visualized by stainingthem with silver or a dye such as C oomassie blue

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