bài giảng protein (english)

Amino acids in a protein are determined by the genetic code, wherein a three base sequence of nucleotides called a codon calls for a specific amino acid to be added to the growing chain.

Protein Structure and Function - An Overview

Pharmaceutical Biochemistry I Instructor: Patrick M Woster, Ph.D.

Reading Assignment: Berg, Chapter 2,3 and 7

Protein Structure and Function

Proteins play crucial roles in almost every biological process They are responsible in one form or another for a

variety of physiological functions including:

Enzymatic catalysis - almost all biological reactions are enzyme catalyzed Enzymes are known to increase the rate of a biological reaction by a factor of 10 to the 6th power! There are several thousand

enzymes which have been identified to date

Binding, transport and storage - small molecules are often carried by proteins in the physiological

setting (for example, the protein hemoglobin is responsible for the transport of oxygen to tissues) Many drug molecules are partially bound to serum albumins in the plasma

Molecular switching - conformational changes in response to pH or ligand binding can be used to control cellular processes

Coordinated motion - muscle is mostly protein, and muscle contraction is mediated by the sliding

motion of two protein filaments, actin and myosin

Structural support - skin and bone are strengthened by the protein collagen

Immune protection - antibodies are protein structures that are responsible for reacting with specific

foreign substances in the body.

Generation and transmission of nerve impulses - some amino acids act as neurotransmitters, which transmit electrical signals from one nerve cell to another In addition, receptors for neurotransmitters, drugs, etc are protein in nature An example of this is the acetylcholine receptor, which is a protein

structure that is embedded in postsynaptic neurons

Control of growth and differentiation - proteins can be critical to the control of growth, cell

differentiation and expression of DNA For example, repressor proteins may bind to specific segments

of DNA, preventing expression and thus the formation of the product of that DNA segment Also, many

hormones and growth factors that regulate cell function, such as insulin or thyroid stimulating

hormone are proteins

Like most biological macromolecules, proteins are made up of simple building blocks; in the case of proteins, these building blocks are called amino acids As shown below, the amino and carboxyl moieties in an amino acid are alpha to one another; also located on the alpha carbon is an "R" group The nature of this R-group (called the side chain) determines the identity of a particular amino acid There are a total of 20 amino acids

which are used to make up proteins (some modified or otherwise unusual amino acids exist that we will discuss

later in the course) In solution at physiological pH (7.4), amino acids undergo an acid-base reaction to form

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zwitterions In a zwitterion, the + and - charges cancel to give a molecule with a net charge of zero However, the pKa values for a typical amino acid (glycine for example) are 9.6 and 2.3 for the amino and carboxyl

groups, respectively If the pH of an amino acid solution is lowered significantly from 7.4, a species results in

which the amine group has a positive charge, while the carboxyl is neutral Likewise, If the pH is raised from 7.4, a species results in which the amine group is neutral, while the carboxyl has a negative charge

Thus, the ionization state of amino acids is pH dependent

All amino acids except glycine (R = H) are chiral Every amino acid in mammalian systems exists in the configuration, where "L" signifies that the amino acid in Fischer projection is similar to L-glyceraldehyde

L-This description of stereochemistry is outdated, and is seldom used except in trivial names However, all natural

amino acids are also in the S-configuration, which is determined by assigning priorities based on the

Cahn-Ingold-Prelog rules

As was mentioned above, there are 20 amino acids which are used to make up proteins in mammalian

biological systems The amino acids are amphipathic molecules, meaning that they contain both polar and

non-polar functional groups, and thus have a tendency to form interfaces between hydrophilic and hydrophobic

molecules The properties of each amino acid are dictated by the side chain, which can vary in size, shape, charge, reactivity and ability to hydrogen bond The amino acids are grouped according to the properties of their sidechains, as shown in the figure below Each amino acid has a standard three letter abbreviation

which is used in lieu of a full structure, as seen in the figure

The first six amino acids, glycine (GLY), alanine (ALA), leucine (LEU), isoleucine (ILE), proline (PRO) and valine (VAL) are aliphatic in nature Glycine and alanine are too small to have a hydrophobic effect in

proteins, but they are considered aliphatic amino acids Proline is also aliphatic, and because of its cyclic

structure, it can often be found in the bend portion of a protein chain Valine, leucine and isoleucine are

hydrophobic aliphatic, and although they can be found anywhere in the chain, they prefer to cluster in the

inside region of a protein, away from water This effect causes a significant stabilization of the protein structure.

There are three aromatic amino acids, phenylalanine (PHE), tyrosine (TYR) and tryptophan (TRP) These

amino acids have sidechains which contain delocalized pi electrons that can interact with other pi systems in

biomolecules In addition, the phenolic hydroxyl of TYR can ionize under physiological conditions, and thus increase water solubility Two of the amino acids are sulfur-containing, namely cysteine (CYS) and

methionine (MET) These amino acids have special properties that will be covered at a later time Finally, there are two hydroxyl-containing amino acids, serine (SER) and threonine (THR) These two amino acids have

sidechains which can hydrogen bond to water or to other groups on neighboring macromolecules

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Five of the 20 amino acids are considered hydrophilic, in that they are able to ionize at physiological pH The amino acids lysine (LYS), arginine (ARG) and histidine (HIS) are considered basic hydrophilic, since they contain basic sidechain groups that will have a positive charge at pH 7.4 The amino acids aspartic acid (ASP) and glutamic acid (GLU) are considered acidic hydrophilic, since they contain acidic sidechain groups that will have a negative charge at pH 7.4 These two amino acids also have amide counterparts, asparagine (ASN) and glutamine (GLN)

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Note that 8 of the 20 amino acids have ionizable sidechains Arginine, lysine and histidine can have a positive charge, while aspartic acid and glutamic acid can possess a negative charge under physiological conditions It

is also possible for serine, tyrosine and cysteine to ionize to a negatively charged species during certain

biological processes

Protein chains are held together by peptide bonds, which are simply amide linkages between neighboring

amino acids When two amino acids interact, an equilibrium is set up between unbound amino acids and a

species in which two amino acids are linked, called a dipeptide Since this equilibrium favors the unlinked

forms of the amino acids, it is clear that formation of a peptide bond requires energy When a few amino acids

become linked, the protein species is called an oligopeptide, and when many are linked, the species is called a polypeptide Polypeptides are generally between 50 and 2000 amino acids Their molecular weights are expressed in Daltons, where 1 Dalton is equal to 1 atomic mass unit (the weight of one hydrogen atom) 1000 Daltons is called a kilodalton (kD) Most proteins weigh in between 5500 and 220,000 Daltons

Each peptide chain has two free ends, the amino terminus, which is always drawn on the left by convention, and the carboxyl terminus, which is always drawn on the right This convention extends to peptide chains expressed using three letter abbreviations Thus, the oligopeptide ALA-GLY-TRP-SER-GLU has an alanine at

the amino terminus, and a glutamic acid at the carboxyl terminus

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Amino acids in a protein are determined by the genetic code, wherein a three base sequence of nucleotides called a codon calls for a specific amino acid to be added to the growing chain The process of converting the sequence of codons into a sequence of amino acids entails transcription (the conversion of a segment of DNA into complimentary mRNA) and translation (the conversion of the mRNA code into protein) You will learn a

great deal more about protein synthesis later in the semester

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As shown below, amino acids can participate in reactions that occur after they are positioned in a peptide chain

These reactions are called post-translational modifications, and can be of enormous biological significance One example of a post-translational modification is the crosslinking of two cysteines to form a new amino acid, called cystine This modification most often occurs in extracellular proteins, and can contribute to their

three-dimensional structure

There are other post-translational modifications of biological significance, three of which are shown below In

some proteins, acetylation of the amino terminus occurs This modification greatly decreases protein

degradation, since many proteases require an amino terminus to act In structural protein such as collagen, hydroxylation of proline occurs to afford hydroxyproline (HPRO) Since hydroxyproline has a hydrogen-

bonding sidechain, it is used to lend additional strength to the collagen structure, and hence to tendons and other

like tissues Finally, the amino acids serine, threonine and tyrosine can be phosphorylated within a protein chain This modification is often used by the cell to turn on or off a critical biological process

In addition to the post-translational modifications mentioned above, some proteins are synthesized in inactive

forms called pro forms For example, some enzymes are synthesized as inactive proenzymes, and are trimmed

by a peptidase to form the active enzyme The portion of the enzyme chain that is cleaved is then hydrolyzed,

and the amino acids are reused

A bit of history:

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In 1953, Sanger performed a critical series of experiments in which he demonstrated several facets of protein structure His experiments showed that proteins have a unique amino acid sequence; all molecules of a given

protein are identical, and the sequence of each different protein is unique He also showed for the first time that

all amino acids in mammalian proteins are in the S-configuration, that the peptide bond is an amide bond, and that amino acids have alpha amino groups and alpha carboxyl groups We now know that proteins are made when a section of DNA is read (a process called called transcription) and a complimentary molecule

of RNA is formed This RNA is then used to specifiy the structure of a given protein through a process called translation Thus, the sequence of a protein is encoded in DNA

The sequence of a peptide is important for other reasons including these:

Knowledge of a peptide sequence can aid in the determination of the mechanism of action of the

protein For example, binding areas of a protein often contain hydrogen-bonding amino acids such

as serine

Relationships between amino acids in a protein chain can help to dictate 3 dimensional structure For

example, when two cysteins crosslink to form a cystine (as described above) a loop is formed in the

peptide chain

Variations in the amino acid sequence of certain proteins can cause disease For example,

substitution of a VAL for a GLU at a certain residue of hemoglobin results in a mutant hemoglobin called hemoglobin S This defect, caused by a genetic mutation, results in the disease sickle cell anemia,

since hemoglobin S cannot carry oxygen as well as regular hemoglobin Since hemoglobin has 574 amino acids and a molecular weight of 63 kD, one can conclude that very small variations in struture can have a great effect on biological activity!

The peptide bond has unique characteristics which contribute to the overall structure of proteins The peptide

bond itself is rigid, and thus is not free to rotate This rigidity arises because the amide bond is involved in a tautomerization that gives it considerable double bond character The other bonds in a peptide ar not rigid, and

can freely rotate, giving the protein chain many degrees of rotational freedom The amide bond, together with

the bonds on either side of it that connect to the alpha carbons, are called the backbone of the protein chain

Proteins have a total of four levels of structure, as defined below:

Primary structure - this term refers to the amino acid sequence of a protein, including cystines that

are formed during crosslinking Sequence can dictate three dimensional structure, since amino acid residues need to be in a specific order to foster proper protein folding, and since disulfides must be formed from properly positioned cysteines to afford an active protein An example of primary structure

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is the hypertensive octapeptide angiotensin II, which has the sequence

Another type of secondary structure, the beta pleated sheet is composed of two or more straight chains that are

hydrogen bonded side by side If the amino termini are on the same end of each chain, the sheet is termed

parallel, and if the chains run in the opposite direction (amino termini on opposite ends), the sheet is termed

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antiparallel (see below left) All of the amides are hydrogen bonded except those on the outer strands Pleated sheets may be formed from a single chain if it contains a beta turn, which forms a hairpin loop structure Often

a proline can be found in a beta turn, since it places a "kink" in the chain When the beta sheet curves around itself and the outer edges on either side hydrogen bond to one another, it forms a structure called a beta barrel, which is a common structural motif in proteins

Tertiary structure - tertiary structure refers to the arrangement of amino acids that are far apart in the chain Each protein ultimately folds into a three dimensional shape with a distinct inside and outside The interior of a protein molecule contains a preponderance of hydrophobic amino acids, which tend to

cluster and exclude water The core is stabilized by Van der Waals forces and hydrophobic bonding By contrast, the exterior of a protein molecule is largely composed of hydrophilic amino acids, which are

charged or able to H-bond with water This allows a protein to have greater water solubility A protein will spontaneously fold to preserve the relationships outlined above

Quaternary structure - protein chains can associate with other chains to form dimers, trimers, and other higher orders of oligomers Generally they contain between 2 and 6 subunits which may be chains with the same sequence (homodimers) or different chains (heterodimers)

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Proteins can be associated with membranes, and in fact carry out almost every membrane function

Interestingly, membrane proteins have special characteristics that allow them to exist in this lipid environment

Proteins that sit on the inner or outer surface of the membrane are called extrinsic or peripheral, and have a

large percentage of hydrophobic amino acids in the portion of the molecule that is close to the hydrophobic membrane structure The amino acids on the outer portion of the protein (facing the aqueous environment of the cytoplasm or extracellular fluid) are mostly hydrophilic, allowing the protein to be compatable with water

Proteins can also traverse the membrane, and in this case they are called intrinsic or integral The portion of

the protein that passes through the membrane is composed of hydrophobic amino acid residues, while the inner and outer portions exposed to water are largely hydrophilic Transmembrane proteins can move laterally in the membrane, but cannot flip-flop

Proteins are a unique class of biomolecules, in that they can recognize and interact with diverse substances The

contain complimentary clefts and surfaces which are designed to bind to specific molecules Often only a

single molecule or even a single stereoisomer can bind to a complimentary protein surface Once this binding

takes place, a complex is formed This induces a conformational change which may act as a signal within the

cell, or may serve to activate an enzyme

Methods for Protein Isolation and Purification

There are a number of experimental procedures which may be used to characterize peptides and larger protein molecules Six of these methods are discussed below:

1 Enzymatic cleavage - A peptide chain may be cleaved at specific peptide bonds using enzymes known as peptidases One of the most common peptidases is trypsin, which cleaves a peptide chain on the carboxyl side of a LYS or ARG Thus the sequence PRO-HIS-ARG-GLY-GLY is cleaved to PRO- HIS-ARG and GLY-GLY Another common peptidase is chymotrypsin, which cleaves the chain on the carboxyl side of each aromatic amino acid (TRP, TYR, PHE) Thus the sequence GLN-SER-PHE-

ASP-GLY-TYR-THR is cleaved to GLN-SER-PHE, ASP-GLY-TYR and THR

2 Electrophoreisis Electrophoreisis refers to the separation of proteins by causing them to move in

an electric field This is usually done on a gel made of polyacrylamide A current is passed through the gel, and the proteins migrate from the cathode to the anode In sodium dodecyl sulfate (SDS)

electrophoreisis, proteins are treated with the detergent SDS and mercaptoethanol to denature them

and disrupt disulfide bonds, and are then loaded onto the gel When the electric field is passed through, smaller peptides migrate fastest, as shown in the diagram below:

A second common electrophoreisis procedure is known as isoelectric focusing, because proteins

migrate until they reach electroneutrality Consider a protein that has 50 ionizable sidechains, 25 that can

be positive and 25 that can be negative The isoelectric point pI is the pH at which the number of

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positive and negative charges equals zero At this point, the net charge is zero In isoelectric focusing, a

polyacrylamide gel is treated with ampholines, which set up a pH gradient across the length of the gel

As shown above, each protein will "focus" at the point on the gel where the pH equals its isoelectric

point, at which time it stops moving Since isoelectric focusing is non-denaturing, it can be used to

isolate active proteins in their native form

3 The Edman Degradation The Edman degradation refers to a reaction that is used to determine the sequence of a given peptide The amino terminus of the peptide is treated with phenyl

isothiocyanate, forming a complex, as shown below Upon acid treatment, the terminal amino acid is removed by cleavage of the first peptide bond, forming a phenylthiohydantoin Note that the R group

of the phenylthiohydantoin is the same as the R group of the terminal amino acid Thus, there are 20 phenylthiohydantoins that can form during the Edman degradation, one for each of the 20 amino acids Repeated cycling allows for each amino acid in the chain to be identified by isolating its

phenylthiohydantoin This procedure is carried out rapidly and efficiently by an automated sequencer

Peptides can also be synthesized by an automated process These peptides are constructed on beads

made of polystyrene or some other solid support in a process known as solid phase synthesis As shown below the bead is reacted with the carboxyl end of an amino acid in which a protecting group such as N-Boc is in place to keep the amine from reacting prematurely Once the amino acid is attached

to the bead, the amino terminus is by treating with acid, and a peptide bond is formed with a second protected amino acid The coupling of these two amino acids is done in the presence of , which fosters the formation of the amide

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The cycle of removal of the protecting group and addition of amino acids is continued until the desired peptide has been formed, and then the peptide is released from the bead using HF

4 Ion Exchange Chromatography Ion exchange chromatography seperates proteins based on their charge, as shown below There are two methods, known as anion exchange (shown below) and cation exchange In anion exchange chromatography, a protein is added to a column packed with beads which bear a positively charged group such as diethylaminoethyl The negative charges on the protein

displace the counterion (chloride is shown) and stick to the bead After washing the coulnm, the protein

is eluted using another negative ion Sodium chloride in a concentration gradient is commonly used,

and the more negative charges on a protein, the better it sticks, and the more NaCl is needed to displace

it A cation exchange column works the same, except that the charge on the bead is negative, and

proteins stick by their positively charged residues

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5 Affinity Chromatography Affinity chromatography is used to isolate one particular protein from

a mixture, as shown in the figure below An epoxysepharose gel is allowed to react with a ligand that has an affinity for the protein of interest, and the protein mixture is then added to the column Only

the protein that binds to the ligand will stick After washing the column to remove the rest of the protein, the protein of interest is eluted using a salt gradient

6 Enzyme-Linked Immunosorbent Assay (ELISA) Enzyme-linked immunosorbent assay, or ELISA, depends on the reaction of a predetermined protein with a specific antibody to form a complex.

This method is extremely sensitive, and can distinguish between two proteins that differ by only one amino acid A serum or blood sample is added to the specific antibody which has been bound to a

polymer support, and the first complex forms A second antibody, specific for the protein of interest but linked to an enzyme is then added, forming a complex that is bound to an active enzyme The enzyme

carries out the conversion of a non-colored or non-fluorescent substrate to a colored or fluorescent product, which is measured The more color that is produced, the more of the protein of interest that is

present This technique is the basis for many diagnostic tests, including pregnancy tests where human chorionic gonadotropin is measured.

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Specific Examples of Protein Structure and Function

1 The Renin-Angiotensin-Aldosterone System The renin-angiotensin-aldosterone system is used by the

body to regulate blood pressure (see the figure below) In response to lowered blood pressure, the kidney

releases the protease renin, which cleaves the inactive, 14 amino acid peptide angiotensinogen to another inactive peptide, the decapeptide angiotensin I A second enzyme, angiotensin converting enzyme (ACE), converts this decapeptide to its active form, the octapeptide angiotensin II Angiotensin II is a potent

vasoconstrictor that is about 40 times more potent than norepinephrine at raising vascular pressure In addition,

angiotensin II stimulates the release of aldosterone, a steroid hormone that causes the kidney to reabsorb

sodium and water, thus raising blood pressure by an osmotic effect Angiotensin II is ultimately inactivated by a

third peptidase called angiotensinase, which renders the hormone inactive

The renin-angiotensin-aldosterone system is of great importance in the development of a common disease

known as essential hypertension When the renin-angiotensin-aldosterone system is overactive, the basal blood

pressure is elevated, putting increased stress on the cardiovascular system A group of compounds have been

developed known as ACE inhibitors which are used quite effectively to treat hypertension Since they prevent

the conversion of angiotensin I to angiotensin II, they prevent the elevation of blood pressure seen in essential hypertension

2 Oxytocin and Vasopressin Oxytocin and vasopressin are two peptide hormones with very similar

structure, but with very different biological activities Their primary sequences are shown below Interestingly, their structures only differ by one amino acid residue (the hydrophobic LEU number 8 in oxytocin is replaced

by a hydrophilic ARG residue in vasopressin) Oxytocin is a potent stimulator of uterine smooth muscle, and also stimulates lactation However, vasopressin, also know as antidiuretic hormone (ADH), has no effect on uterine smooth muscle, but causes reabsorbtion of water by the kidney, thus increasing blood pressure

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3 Insulin and Glucagon Insulin is an extremely important peptide hormone that is produced by the beta cells of the Islet of Langerhans in the pancreas It has 51 amino acids, three disulfide crosslinks, and is comprised of two seperate chains, termed A and B Insulin has a number of important effects on cells in the

body including:

1 Stimulation of glycolysis (glucose breakdown)

2 Stimulation of glycogen formation (a storage form for glucose)

3 Enhancement of the rate of fatty acid biosynthesis

4 Stimulation of the entry of glucose into cells

5 Overall reduction of blood glucose levels

Insulin is not synthesized in active form, but is first made as a single inactive peptide chain called

preproinsulin (see the figure below) Preproinsulin has no crosslinks, and in addition to the A and B chain, has two additional portions called the signal sequence and the connecting (C) peptide The signal sequence

informs the cell that insulin is being made, and that the finished preproinsulin should be deposited outside the cell The C-peptide is necessary to allow preproinsulin to fold in the correct conformation to ultimately produce

active insulin Preproinsulin is processed by a two step procedure; in the first step, the signal sequence is

cleaved by a peptidase, and two of the three crosslinks are formed to give a new but still inactive peptide called

proinsulin A second peptidase then cleaves the C-peptide, and an internal disulfide forms to produce insulin

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Glucagon is a peptide hormone that is formed in the alpha cells of the Islets of Langerhans in the pancreas It

is a single chain peptide consisting of 29 amino acid residues, and has effects which oppose insulin, including:

Down regulation of glycolysis

Enhancement of the rate of glycogenolysis (glycogen breakdown)

Reduction in the rate of fatty acid synthesis

Enhancement of blood glucose levels

4 Hemoglobin Hemoglobin A (HbA) is a tetrameric protein which consists of two alpha chains and two beta chains, and comprises 98% of human hemoglobin A There is a heme group and an oxygen binding site

on each subunit; therefore, each molecule of HbA can carry 4 molecules of oxygen There are other forms of human hemoglobin A, the most common being HbA2, which has two alpha chains and two delta chains, and accounts for 2% of HbA

Hemoglobin is an example of an allosteric protein, i.e its function can be altered by the binding of some external substance (called the effector) at a site on the molecule other than the active site (the allosteric site) When an allosteric effector binds to a protein, it induces a conformational change which turns the function of the protein either on (positive allosterism) or off (negative allosterism) In the case of hemoglobin, the

allosteric effector is 2,3-diphosphoglycerate (2,3-DPG), which causes hemoglobin to have 1/26th of its normal

affinity for oxygen This is an important issue, since 2,3-DPG in the tissues triggers the release of oxygen at the correct location

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Hemoglobin also exhibits cooperativity, which is a phenomenon wherin the binding of one molecule to a

protein with more that one active site influences the ease of binding of subsequent molecules Cooperativity can

be positive (the second molecule binds more easily), or negative (the second molecule binds less easily) In the

case of hemoglobin, the binding of oxygen to the four sites of hemoglobin is an example of positive

cooperativity

As shown in the figure below, hemoglobin can also exist in a glycosylated form known as HbA1C HbA1C is formed when the amino terminus of HbA reacts with glucose, first reversibly forming an aldimin or Schiff's Base, and then undergoing an irreversible Amadori rearrangement to afford the ketamine form HbA1C In

normal patients, HbA1C accounts for about 3-5% of HbA, but in diabetics who have elevated blood glucose for extended periods, this number can reach 6 to 15% Physicians can measure HbA1C, and are using it as a reliable

way to monitor how well diabetic patients are complying with their insulin therapy

5 Collagen Collagen is a connective tissue protein that is found in skin, bone, tendons, cartilage, the cornea, etc It is quite insoluble in water, and is composed of two types of chain termed alpha-1 and alpha-2

In the amino acid sequence of collagen, about every 3rd amino acid is a GLY residue, and there are many

prolines which are hydroxylated to form hydroxyproline (HPRO) LYS residues are also hydroxylated in

collagen to form HLYS These additional sidechain OH residues allow for extra strength due to H-bonding, and the GLY residues allow the protein to coil more tightly, since they fit on the inside of the helix In a collagen

fiber, three of these helices are coiled together to form a rope-like structure called a superhelical coil It is this structure that gives collagen its great strength Collagen structure can be disrupted in diseases such as scurvy,

which is a lack of ascorbic acid, a cofactor in the hydroxylation of proline In addition, collagen structure is

disrupted in rheumatoid arthritis

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Protein Structure

Different Levels of Protein Structure

The wide variety of 3-dimensional protein structures corresponds to the diversity of functions proteins fulfill

Proteins fold in three dimensions Protein structure is organized hierarchically from so-called primary structure

to quaternary structure Higher-level structures are motifs and domains

Above all the wide variety of conformations is due to the huge amount of different sequences of amino acid

residues The primary structure is the sequence of residues in the polypedptide chain

Secondary structure is a local regulary occuring structure in proteins and is mainly formed through hydrogen

bonds between backbone atoms So-called random coils, loops or turns don't have a stable secondary structure

There are two types of stable secondary structures: Alpha helices and beta-sheets (see Figure 3 and Figure 4)

Alpha-helices and beta-sheets are preferably located at the core of the protein, whereat loops prefer to reside in outer regions

Figure 3: An alpha helix:

The backbone is formed as a helix

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An ideal alpha helix consists

of 3.6 residues per complete turn

The side chains stick out.

There are hydrogen bonds between the carboxy group of amino acid n and the amino group of another amino acid n+4 [1][2].

The mean phi angle is -62 degrees and the mean psi angle is -41 degrees [3]

(see also section on Helical Wheels)

Figure 4: An antiparallel beta sheet.

Beta sheets are created, when atoms of beta strands are hydrogen bound

Beta sheets may consist of parallel strands, antiparallel strands or out of a mixture

of parallel and antiparallel strands [4]

Tertiary structure describes the packing of alpha-helices, beta-sheets and random coils with respect to each

other on the level of one whole polypeptide chain Figure 5 shows the tertiary structure of Chain B of Protein Kinase C Interacting Protein

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Figure 5: Chain B of Protein Kinase C Interacting Protein

Helices are visualized as ribbons and extended strands of betasheets by broad arrows

(the figure was obtained by using rasmol and the PDB-file corresponding to PDB-ID 1AV5 stored at PDB, the Brookhaven Protein Data Bank)

Quaternary structure only exists, if there is more than one polypeptide chain present in a complex protein

Then quaternary structure describes the spatial organization of the chains Figure 6 shows both, Chain A and Chain B of Protein Kinase C Interacting Protein forming the quaternary structure

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Figure 6: Quaternary structure of

Protein Kinase C Interacting Protein

(the figure was obtained by using rasmol and the PDB-file corresponding to PDB-ID 1AV5 stored at PDB, the Brookhaven Protein Data Bank)

exercise 1

Motifs and domains are combinations of secondary structures Motifs only consist out of few secondary

structures They may but need not have a function A domain is more complex It is usually defined as a

modular functional unit folding independently

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Bacteriology at UW- Madison

Ken Todar's Microbial World

University of Wisconsin - Madison

Chemical and Molecular Composition of Microbes

From atoms to elements to molecules to macromolecules to life

Chemistry is essential to the study of living things Not to sound irreverent, but much of life is based on

chemical reactions This article will address a few principles of chemistry and biochemistry to prepare you for

these topics which will inevitably come up in The Microbial World.

All matter in the Universe is composed of elements It is the elements that are identified and described in the

Periodic Table of the Elements (Table 1), familiar to all beginning chemistry students Elements are made up

of atoms which consist of a variety of subatomic particles, the most important of which in biology are the negatively-charged electron (e - ) and the positively-charged proton (H + ) Each element has distinct properties

due to the distinct nature of its atom and the behavior of electrons, protons and other subatomic particles in its make-up

The atom is the fundamental unit of an elements and cannot be broken down further without changing the

properties of the element If an atom loses or gains one or more electrons, it will acquire an electrical charge

Such atoms are referred to as ions Thus, if a sodium (Na) atom were to lose an electron it would acquire a

positive charge and be symbolized Na + If a chlorine (Cl) atom were to gain an electron it would symbolized Cl -

Positively charged ions are called cations; negatively charged ions are anions.

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Table 1 Image PeriodicTable.jpg The Periodic Table of Elements Given in the table are the distinct characteristics of the

atom that comprises the element: 1 atomic number of the element (in the upper right corner of the element symbol) is the number of protons in the atom; atomic weight (below the symbol of the element) is derived from the combined weight of

electrons, neutrons and protons which make up the atom The atomic weight of an element must be known to calculate the

molecular weight of a chemical compound that is formed when elements bond together together into molecules The major

elements of living systems are C, H, O, N, S, and P Minor elements are Na, Mg, K, Ca, Mn, Fe, Co, Ni, Cu and Zn

A cell, the fundamental unit of life on Earth, is composed of organic matter, the exact definition of which will

be given below Organic material is made up of a relatively small handful of elements, cells being composed of over 97% carbon (C), oxygen (O), nitrogen (N), hydrogen (H), phosphorus (P) and sulfur (S)

Table 2 The major elements of bacteria

(O) joined together form O 2 , or molecular oxygen; two atoms of nitrogen (N) joined together form N 2 (nitrogen gas); carbon (C) bonded with two atoms of O forms CO 2 (carbon dioxide), the predominant gases in earth's atmosphere Two atoms of hydrogen (H) joined to an atom of oxygen form a molecule of H 2 O or water, which is the predominant liquid on the planet.

Table 3 Major types of chemical bonds in biological molecules

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Ionic bond: Force that hods ions together in a molecule

Covalent Bond: Force resulting from sharing electrons among the atoms of a molecule

Hydrogen Bond: The force from attractions between oppositely charged poles of adjacent molecules

When a covalent bond is formed between a carbon atom (C) and a hydrogen atom (H), an organic molecule is

born Since carbon atoms can bond to themselves in chains of great length, and since each carbon atom has four bonding sites to other atoms, elements or molecules, it makes sense to think that there are endless possibilities for the structure of different organic molecules First, let's just look at some possibilities for a "one-carbon" organic molecule.

CH 4 is methane, or natural gas

CH 3OH is methanol an alcohol that is an excellent fuel but causes blindness if you drink it

HCHO is formaldehyde, the stuff they will try to embalm you with

HCOOH is formic acid, put into insecticides and used in the dye industry

HCN is cyanide, a powerful respiratory poison

CO(NH 2 ) 2 is urea, a waste product in urine (Actually, urea doesn't contain a carbon to hydrogen bond (C-H),

but it nonetheless should be considered an "organic" molecule.)

Image C-1.jpg Some 1-Carbon organic molecules

As the number of carbon atoms in an organic molecule is increased, correspondingly-different organic

compounds are created Example are given in the table below.

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Image C-2.jpg Some C-2 and C-3 organic molecules

Depending on the occurrence of so-called "functional groups" on an organic molecule, it will have particular

chemical properties and activities Some important functional groups in biological molecules are listed and described below:

-CH 3 methyl group, the beginning group of fatty acids, many amino acids, some vitamins

-NH 3 amino group, seen in amino acids, peptides and proteins

-PO 4 phosphate group that occurs in phospholipids, nucleotides and some vitamins

-SH sulfhydryl group in certain amino acids, vitamins and proteins

-OH hydroxyl group seen in alcohols and sugars

-CHO is an aldehyde group as in acetaldehyde or formaldehyde

-C=O is a ketone group characteristic of key compounds in important metabolic pathways

-COOH is a carboxyl group, as in carboxylic acids and fatty acids

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Image FunctionalGroups.jpg Some important functional groups in molecules encountered in microbiology

In any case, different arrangements of C, H, O, N, P and S atoms comprise the the molecules that make up the structural and functional components of cellular life But usually this requires that these monomeric "small"

molecules be polymerized into polymeric "large" molecules called macromolecules There are four

fundamental types of macromolecules that occur in all forms of cells Polysaccharides are composed of

carbohydrate (sugar) molecules; lipids are composed of fatty acids; proteins are composed of amino acid molecules; and nucleic acids (DNA and RNA) are made up of molecules called nucleotides These are the

molecules of microbes and all other forms of life

Table 4: Macromolecules that make up cell material

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Macromolecule Primary Subunits Where found in a bacterial cell

Proteins amino acids Flagella, pili, cell walls, cytoplasmic membranes, ribosomes,

cytoplasm (as enzymes) Polysaccharides sugars or other carbohydrate

molecules

capsules, inclusions (storage), cell walls

Nucleic Acids

(DNA/RNA)

nucleotides DNA: nucleus, nucleoid (chromosome), plasmids

rRNA: ribosomes; mRNA, tRNA: cytoplasm

How macromolecules run living systems

All microbial cells have various structural and functional components composed of macromolecules that

account for almost every aspect of their existence and behavior as cells All cells have essential structural

components such as a chromosome (DNA), ribosomes, a cell membrane, and some sort of cell wall or surface layer Also, all cells have a self-replicating genome and hundreds of enzymatic proteins that are

responsible for the business reactions of life

As discussed above, these macromolecules are made up of monomeric subunits such as carbohydrates, lipids,

nucleotides or amino acids It is the arrangement or sequence in which the subunits are put together, called the primary structure of the molecule, that often determines the exact properties that the macromolecule will

have Thus, at a molecular level, the primary structure of a macromolecule determines its function or role in the cell, and the functional aspects of cells are related directly to the structure and organization of the

macromolecules in their cell make-up It is the diversity within the primary structure of these molecules that accounts for the diversity that exists among life forms Let us now look briefly at the composition and function

of the macromolecules of microbial cells.

Carbohydrates

Carbohydrates are organic compounds of carbon, hydrogen and oxygen Their empirical formula, (CH2 O) n , is widely used as a symbol for an organic compound Carbohydrates have a vital function as energy sources for many types of cells They are also found in several bacterial structures including capsules and the cell wall The common notion of a carbohydrate is a sugar such as glucose or sucrose, but certain alcohols and aldehydes also

apply Carbohydrate molecules, also called "saccharides", can be polymerized into polysaccharides which most

often occur in microbial cells in the form of starch, glycogen and cellulose Starch and glycogen are stored in microbial cells as reserves of energy; cellulose is a component of cell walls in the protista, and otherwise is the most abundant polymer on the planet because of its occurrence in the Plant Kingdom Complex types of

polysaccharides also occur in the cell surface structures of the bacteria (walls, capsules, etc.).

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Image CH1.jpg (above) Structure of two important carbohydrates, in this case the sugars fructose and glucose Glucose and fructose are isomers because they have identical molecular formulas (C 6 H 12 O 6 ) but different structural formulas Glucose is shown in its chain form and in its more common ring form Glucose and fructose are monosaccharides Two other common sugars, sucrose and lactose, are disaccharides, because they consist of two sugars molecules bonded together Sucrose is made

up of glucose plus fructose, and lactose is composed of glucose and galactose Polysaccharides such as starch, glycogen or cellulose are made up of hundreds of sugar molecules polymerized into a polysaccharide macromolecule.

Image CH2.jpg (below) The structure for two disaccharides and one polysaccharide During the synthesis of the

dissaccharides water is removed from the reactants during the process This is referred to as "dehydration synthesis" The polysaccharide pictured is a very complex arrangement of glucose molecules that have been joined together in a linear and branched fashion.

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Lipids are a broad group of organic molecules that dissolve in organic solvents such as benzene, ether or

alcohol, but generally do not dissolve in water Like carbohydrates, lipids are composed of C, H and O, but the

proportion of O is much lower The best known lipids are fats Fats serve living organisms including some

microbes as important energy sources They are located in storage granules in bacteria and they are structural components of the cell membrane of most organisms.

Fats are made up of a 3-carbon glycerol molecule attached to 2 (or 3) long-chain fatty acids Each fatty acid usually has 16 or 18 carbon atoms in the chain There are two major types of fatty acids The saturated fatty acids contain the maximum number of carbon to hydrogen bonds (C-H), while the unsaturated fatty acids

contain less than the maximum An unsaturated fatty acid molecule reveals itself immediately by the occurrence

of one or more double bonds between adjacent carbon atoms (C=C).

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Image Lip1.jpg The molecular structures of fat components and synthesis of fat Fats consist of glycerol (a 3-carbon alcohol) and fatty acids The fatty acids may be saturated or unsaturated as shown Unsaturated fats contain less hydrogen Fat synthesis occurs when fatty acids are joined to glycerol during a dehydration reaction.

Proteins

Proteins are by far the most abundant organic components of microorganisms and other living things They function as structural materials in bacterial cell walls, cell membranes and ribosomes, and they function as enzymes, a group of biological molecules that regulate the rate of most chemical reactions in biological

systems Denaturation of proteins in an organism as with heat or chemicals, usually leads to cell death.

Proteins are composed of amino acids that are linked to one another by a peptide bond Each free amino acid has a carboxyl (-COOH) and a free amino (-NH2) as part of its molecular structure During protein synthesis two amino acids can be joined together by a dehydration reaction that combines the carboxy group of one amino acid to the amino group of another amino acid via a peptide bond (CO:NH) as shown below There are 20 amino acids used during protein synthesis Since proteins are polypeptides (many amino acids joined together)

consisting of up to hundreds of amino acid molecules, there is unlimited potential in theie primary structure

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Image AA1.jpg Structural formulas of several important amino acids The so called "side groups" (highlighted blue) differ and determine the exact compound, but the "amino acid" portion of the molecule is the same in all molecules consisting of an amino (NH 2 ) group and a carboxyl (acid) (COOH) group.

Image AA2.jpg Amino acids are joined together to form peptides or polypeptides or proteins (depending on how many AA are joined together This cartoon shows how one amino acid is joined to the next by a type of bond called a peptide bond The -OH from the acid group of alanine combines with -H from the amino group of valine to form water (H 2 O) The open bonds then link together to form a peptide bond between the two amino acids forming the dipeptide alanylvaline.

It is the exact sequence of amino acids in a protein, encoded by the genetic material (DNA), that determines the function of the protein in either its structural or enzymatic role in the cell The chain of amino acids in the

protein represents the primary structure of the protein Most proteins have a secondary structure that forms

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when the amino acid chain twists itself into a helical pattern Hydrogen bonds and S-S (disulfide) bonds help

maintain this structure In addition, many proteins including enzymatic proteins, have a tertiary structure that

results from the coiled (secondary) structure folding back on itself Hydrogen bonding maintains the protein in its tertiary structure If subjected to heat or chemicals, the bonds break easily and the protein becomes

denatured, thereby losing its activity

Image lysozyme.gif The primary structure of the enzyme lysozyme The protein is a polypeptide chain of 129 amino acids There are four pairs of cysteines that form disulfide bridges within the molecule Secondary structure of the molecule forms when the amino acid chain twists itself into a helical pattern Tertiary structure of the protein results from the coiled

(secondary) structure folding back on itself Almost all proteins that are enzymes must maintain their tertiary structure in order to be active Therefore, proteins are denatured by chemical or physical events that destroy their tertiary structure Nucleic Acids

The nucleic acids are among the largest macromolecules that occur in cells There are two types of nucleic acids found in all cellular organisms: Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) DNA acts as

the genetic material of the chromosome, and RNA mainly functions during the synthesis and construction of proteins.

Both DNA and RNA are composed of repeating subunits called nucleotides A nucleotide has three molecular components: a carbohydrate molecule (deoxyribose or ribose), a phosphate (PO4) group, and a nitrogen- containing base, which is either a purine or a pyrimidine In DNA and RNA the purine bases are adenine (A) and guanine (G); in DNA the pyrimidine bases are thymine (T) and cytosine (C), but in RNA uracil (U) occurs

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in place of thymine.

DNA occurs in the chromosome of the cell and its function is to pass on genetic information to progeny cells and to direct the synthesis of proteins To form the complete DNA molecule, two single strands of DNA oppose each other in a ladder-type arrangement where opposing bases hydrogen bond to one another G and C line up opposite one another as does A and T This forms the "complementary" double strand of DNA and ensures that one strand of DNA can encode precisely for the opposite strand of DNA which is required for faithful DNA replication

Image Nuc1.jpg The molecular structure of nucleotide components Nucleotides, the components of DNA and RNA are composed of a carbohydrate (sugar), phosphate ions (PO 4 ), and a nitrogenous base The carbohydrates in nucleotides are ribose and deoxyribose Phosphate is formed from phosphoric acid The nitrogenous bases include adenine and guanine, which are purine molecules, and thymine, cytosine and uracil which are pyrimidine molecules Four bases occur in both DNA and RNA However, DNA contains thymine and no uracil and RNA contains uracil and no thymine At the bottom left in the drawing, a DNA nucleotide and an RNA nucleotide have been formed from the component molecules.

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Image Nuc2.jpg Formation of a double-stranded molecule of deoxyribonucleic acid (DNA) (a) Two single-stranded DNA molecules line up next to each other to form a double-stranded molecule Adenine (A) molecules always oppose Thymine (T) molecules, and Guanine (G) molecules always oppose Cytosine (C) (b) The double-stranded DNA molecule is is twisted as shown to form the "double helix" that is characteristic of chromosomal DNA in all living organisms.

RNA occurs as a single stranded molecule although there may be regions of the molecule where complementary base pairing can take place as in DNA There are three primary classes of RNA, each of which has a role during the process of protein synthesis Messenger RNA (mRNA) occurs in the cytoplasm of bacterial cells and

functions as a carrier of the genetic message for protein synthesis from the DNA to the ribosome which is the site of protein synthesis Ribosomal RNA (rRNA) is associated with the ribosomes and stabilizes the protein synthesizing machinery during the process Transfer RNAs (tRNA) are relatively small molecules of RNA that transfer specific amino acids from the cytoplasm to the ribosome and the growing polypeptide chain, during the process of protein synthesis.

Topics in Photographic Preservation Vol 4, 1991 pp 124-135

Protein Chemistry of Albumen Photographs

Paul Messier

Post-Graduate Conservation Fellow, Conservation Analytical Laboratory,

Smithsonian Institution

Introduction: Albumen As Protein

The use of albumen as a traditional artist's material is long and well

established Cennino Cennini in his Il Libro del' Arte, mentions the use of egg

white or "glair" as a binder for pigments and as a varnish As early

photographers sought a suitable medium for the fabrication of prints and

negatives, the use of egg white became inevitable The outstanding result of

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this initial experimentation is the albumen print.

In general, the term albumen refers to hen's egg white In the egg,

albumen is a relatively pure mixture of numerous proteins dispersed in water.

Composition of Albumen (Powrie 1973)

87.9-89.4%-As can be seen above, the protein component accounts for the bulk of the solids in albumen As the water component dries off during albumen paper

manufacture, the resulting surface coating is almost entirely protein After

aging, this layer is notably susceptible to a characteristic: discoloration and

cracking As a first step towards understanding the properties and degradation behavior of albumen photographs, an overview of the involved proteins and their chemistry may prove helpful.

Constituent Proteins of Albumen

The major proteins found in egg white (> 1% of the total protein content) are listed below.

MAJOR PROTEINS IN ALBUMEN OF TOTAL PROTEINS

Other protein components include, flavoprotein (.8%), ovoglycoprotein (.5%), ovomacroglobulin (.5%), ovoinhibitor (.l%) and avidin (.05%).

Biological Functions of Albumen Proteins

Proteins are classified by biological functions and properties Ovalbumin1and conalbumin are included under the broad category albumin It is worth

noting that the word albumen ("-men") refers to the protein mixture derived

from egg white while albumin ("-min") refers only to a specific class of

proteins As a group, albumins are generally soluble in water and dilute saline solutions They are coagulated by heat and are found in the interstitial fluids

of animals The biological function of conalbumin is to isolate and sequester metallic contaminants in the egg white.

Ovomucoid and ovomucin are classified as mucoproteins The

mucoproteins are usually stable in heat and are easily soluble in water and

mild saline solutions Generally, they can be coagulated by low pH and

ethanol In nature, they occur in skin, bone, blood and eggs In the egg,

ovomucin is responsible for thickening the white.

Globulins is another general classification for proteins These proteins

resemble albumins in that they are coagulated by heat and are soluble in mild saline solutions As a class they are generally insoluble in water Like

albumins, these proteins occur in the interstitial fluids of animals Lysozyme, globulin G2 and G3 fall into this category Lysozyme, which is also found in

human saliva, is an enzyme that destroys bacteria.

Amino Acids and Peptide Bonds

Proteins are made by joining amino acids by what is called a peptide

bond the peptide bond is formed in a reaction between the amino group

(-NH2) of one amino acid and the carboxyl group (-COOH) of another amino

acid the resulting linkage yields water the R-groups in the equation stand for the functional portion of the amino acid which are known as side chains

Proteins (which can include hundreds of peptide bonds) are often referred to

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as amino acid polymers Amino acids vary only in the orientation and

composition of the R-groups in nature there exists twenty known amino acids which are diagrammed and listed below (Hardy 1985).

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© Chapman & Hall Ltd.

The functional groups of the amino acids are classified by characteristic properties Measured at, or near, neutral pH, there are polar (hydrophilic) and non-polar (hydrophobic) functional groups There are also positively charged (basic) and negatively charged (acidic) groups.

Primary Structure of the Albumen Proteins

The elemental content, amino acid content and amino acid sequence of a protein is known as its primary structure All proteins contain carbon, oxygen, nitrogen and hydrogen Proteins usually contain sulfur and, more infrequently, phosphorous the elemental composition and an amino acid analysis of the

major proteins contained in egg white appear on the next page the first table

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includes protein molecular weights the following amino acid tabulations also indicate which proteins are non-polar (np), polar (p), basic (+), or acidic (-) the amino acids which contain sulfur are also indicated [S].

ELEMENTAL COMPOSITION THE MAJOR ALBUMEN

as measured by grams of amino acid per 100 grams of protein (Tristam 1953)

Ovalbumin Conalbumin Ovomucoid Lysozyme