BME Test 2 Notes

Peptide bond – Link of carboxyl group of one amino acid to the amino group of another amino acid
-Requires 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 structure

2) Contain a wide variety of functional groups – results for broad spectrum of activities
3) Can interact to form complex assemblies
4) 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 ISOMERS ARE CONSTIUENTS OF PROTEINS

Amino acids in neutral pH = dipolar ions (ZWITTER IONS)
Almost all peptide bonds in proteins are trans
Hydrophobic amino acid side chains – cluster, rather than contact water
-Hydrophobicity effect
Hydroxyl group on a R-side chain = more hydrophilic and reactive

Thiol group ( -SH) is more reactive than –OH groups

-come together to from disfulfide bonds
Positive charge = hydrophilic
Histidine is in active sites of enzymes, where its imidazole ring can bind and release protons with
enzymatic 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
 Determines: Mechanism of action (ie: enzymatic), 3-D structure, evolutionary history (common
ancestor)
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 conformation
fo the peptide backbone), uncharged (allows for polymers of amino acids linked by peptide bonds to
form 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
-Ramachandran 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 φ
-Angle between alpha carbon and carbonyl group is ψ
Flexible polymers with a large number of conformations do not fold into unique structures due to the
unfavorable entropy

Thus, rigidity and ψ/φ 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 acids
that are near each oteh rin linear sequence

Alpha Helix
-Rod-like structure
-Stabilized by hydrogen bonds between NH and CO groups
-1.5A
-3.6 amino acid residues per turn
-Right-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 ionic
interactions
 -Collagen-3 alpha helices (Gly in every 3 rd sequence since collagen is so tightly packed and gly is
the only amino acid small enough to fit)
-No hydrogen bonds – stabilized by steric repulsion of pyrrolidine rings of proline and
dyroxyproline residues

Tertiary structure – overall course of the polypeptide chain

Motif/supersecondary structure – specialcombos of helices/sheets with loops/turns that produce a

specific 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
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
 Like carbohydrates, proteins are polymers of smaller units
 The building blocks of proteins are amino acids
 
Amino Acids
 There are 20 amino acids that built up a protein
 An  amino acid consists of a central carbon atom, called the  carbon to which an
amino group, a carboxylic acid group, a hydrogen atom and a distinctive R group (also
referred to as side chain) is attached.
 Because of the asymmetric  carbon amino acids are chiral and may exist as an L
isomer or D isomer.
 Both the L isomer and D isomer are mirror image of one another and are
nonsuperimposable
 
 
 

 
 The enantiomers can be distinguished by the ability to rotate a plane polarized light either
clockwise or counterclockwise
 Almost all naturally occurring amino acids are L-amino acids except in some bacteria
 Only L-amino acids are incorporated into proteins
 The specific configuration of amino acid and hence the proteins are important for
recognition by cellular enzymes
 
 
Zwitterions
 
 The amino and carboxylic acid groups of amino acids readily ionize
 The ionization state of an amino acid varies with pH
 At neutral pH, amino acids in solution exist predominantly as zwitterions or dipolar ions
when the amino group is protonated (-NH3+) and the carboxyl group is deprotonated (-
COO-)
 

 In acidic pH (e.g., pH1) both the amino (-NH3+) and carboxyl (-COOH) groups are
protonated
 As pH increases, nearing the pKa of carboxylic acid ~2, the carboxyl group starts giving
up its proton (-COO-)
 The zwitterionic form persists until pH approaches pKa of the amino group ~9 when the
protonated amino group loses a proton (-NH2)
 The zwitterionic property of amino acid makes it a good candidate for a buffer. Glycine is
an excellent buffer in the human blood stream
 
R group
 
 The 20 amino acids are distinguished by their R group or side chains
 The side chains vary in size, shape charge, hydrogen-bonding capacity, hydrophobic
character and chemical reactivity
 These properties give rise to the various functions of a protein
 The amino acids can be classified into different groups
Amino acids with aliphatic side chains
 

 
 Glycine is the simplest amino acid, containing a single hydrogen atom as the side chain.
It is achiral due to its symmetric structure
 Alanine is the next simplest amino acid, containing methyl group as the side chain
 Both glycine and alanine made up 40% of silk protein
 Larger hydrocarbon chains are found in valine, leucine and isoleucine
 Methionine aliphatic side chain includes a thioether (-S-) group
 Isoleucine contains an additional chiral center
 The larger aliphatic side chains are hydrophobic and therefore have the tendency to
cluster together in water. This is known as the hydrophobic effect
  Proline is another aliphatic amino acid, which is unique because its side chain is bonded
to both the nitrogen and  carbon.

 
Amino acids with aromatic side chains
 

 Phenylalanine contains a phenyl ring in place of one of the hydrogens of alanine

 In tyrosine, the aromatic ring contains a hydroxyl group, which is reactive
 Tryptophan contains an indole group joined to a methylene (-CH2-). The indole group is
two fused rings containing an NH group
 The hydroxyl group of tyrosine and the NH group of tryptophan make these two amino
acid less hydrophobic than phenylalanine, which is purely hydrophobic
 
Amino acids containing hydroxyl, amide or thiol groups
 

 
 
       Serine and threonine contain hydroxyl groups (-OH) attached to an aliphatic side
chain
       The hydroxyl groups make these amino acids much more hydrophilic and reactive
than alanine and valine
       Threonine, like isoleucine contains an additional chiral center
  Asparagine and glutamine are uncharged derivaties of the acidic amino acids aspartate
and glutamate. Both have a terminal carboxamide in place of a carboxylic acid
 The side chain of glutamine is one methylene group longer than that of asparagines
 Cysteine in structurally similar to serine but contains a sulfhydryl or thiol group (-SH)
instead of a hydroxyl (-OH) group.
 The thiol group is much more reactive than hydroxyl group
 Two thiol group may come together to form disulfide bonds
 
Amino acids containing basic side chains
 

 Amino acids belonging to this group are hydrophilic

 Lysine 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
  The imidazole group, which has pKa value near 6 can be uncharged or positively charged
near neutral pH depending on the local environment.
 
Amino acids containing acidic side chains
 

 
       Both aspartate and glutamate, also referred to as aspartic acid and glutamic acid
contain carboxylic acid side chains
       At physiological pH, the acid side chains are usually deprotonated and hence are
negatively charged
 However, in some proteins, these side chains do accept protons and are functionally
important

Lecture 8:  09-08-06

 
Amino Acids (continued)
 
Proline
 
 Proline contains a secondary amine group, which differs from the other members of the amino
acids
 Its side chain is bonded to both the nitrogen and the -carbon atoms, which makes it more
conformationally 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

 
The structure and function of a protein is determined by properties the amino acids that made
up the protein
 
 
Protein Structure
 
Primary structure
Refers to the amino acid sequence of the polypeptide chain or chains
 
Secondary Structure
Polypeptide chain can fold into regular structures. The secondary structure refers to the spatial
arrangement of polypeptide backbone atoms without regard to the conformation of the side chains. The
polypeptide can fold into any one of the following structures: Alpha () helix, beta () sheets, and turns
and loops

Alpha Helix
 

 The  helix has a coiled backbone, with side chains sticking outward
 The  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
 The  helix has 3.6 residues per turn with a rise (distance between two adjacent amino acids) of
1.5Å
 The pitch of the  helix is 5.4Å (the distance of one full turn along the length of axis)
 

 
 

Hydrogen-bonding scheme for an  helix

Beta Sheets
 

 Compose of 2 or more polypeptide chains called  strand

 The  strand is almost fully extended with the side chains of adjacent amino acid pointing in
opposite directions
 

  sheet is formed when two or more strands adjacent to each other are linked through
hydrogen bond between CO groups of one  strand and NH group of an adjacent  strand and
vice versa
  The  strands in a b sheet can all run in the same direction (from amino to carboxyl end), and is
called parallel sheet or in opposite directions and is called antiparallel sheet
 The antiparallel and parallel  sheet has distinctive pattern of H-bonding
 

An antiparallel  sheet

A parallel  sheet
Reverse Turns and Loops
 

 Proteins are made up from a combination of  helices and  sheets, connected by loops or
reverse turns
 Unlike  helices and  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 loops
are 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
 
 

Structure of reverse turn

Tertiary Structure
Refers 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 charged
chains are on the surface
 Nonpolar residues such as leucine, valine, methionine and phenylalanine are found in the
interior of a protein whereas charged residues such as aspartate, glutamate, lysine and arginine
are found on the surface
 

In Biological Membranes
 

 In biological membranes where the environment is hydrophobic, proteins have reverse

distribution of hydrophobic and hydrophilic amino acids
 Porins found in the outer membranes of many bacteria, are covered on the outside mainly with
hydrophobic 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
 
 

Reverse 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
 

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

 
 

Quaternary Structure
Many proteins are composed of two or more polypeptide chains, referred to as subunits. Quaternary
structure refers to the spatial arrangement of its subunit. The simplest quaternary structure is a dimer
consisting of two subunits, such as the Cro protein of bacteriophage l, which is a dimer of two identical
subunits.

Cro protein of bacteriophage l

 

Lecture 9:  09-11-06

 

Amino Acids (continued)

 

Hydrophilic (Acidic and Basic) amino acids:

 
The Amino acids containing dicarboxylic acids are Aspartic acid and Glutamic
acid
 
 

       Aspartic acid and glutamic acid are the two acidic amino acids that contains
dicarboxylic acid group.
 

       These are often called as aspartate and glutamate to emphasize that at physiological pH
their 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 protons
makes these two amino acids functionally important especially where form an important part of the
active site of an enzyme for eg: aspartic proteases.

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

 The carboxyl group of the aspartic acid and glutamic acid ionize only when the pH >2 because
the pKa is around 2-3
 

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

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

 The above scenario shows that these amino acids have a net negative charge at physiological
pH of 7.0.
 

 Amino acids asparagines and glutamine are the pseudo-dicarboxylic acids. They have an amide
group and cannot ionize.
 

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

 
  Lysine, Histidine and Arginine are the three basic amino acids that have amino groups in their
side chain.
 

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

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

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

 At physiological pH both arginine and lysine have a net positive charge.

 

 As seen from above structures lysine has a primary amino group, arginine has a forked
structure with positive amino groups and histidine has an imidazole ring which enables it to
interact with the substrate’s negatively charged group as well as with aspartic acid or glutamic
acid in the active site of the protein itself.
 

 Histidine has an imidazole ring that offers the characteristic pKb of histidine, which is around 6.
 

 With a pKb of 6 histidine can be uncharged or positively charged near neutral pH depending on
its local environment.
 

 Histidine is often found in the active site of enzyme where it can bind and release protons during
enzymatic reactions.
 
 It is noteworthy that all other amino acids are charged at physiological pH except histidine.

 By convention always the amino terminus is written on the left hand side and the carboxyl
terminus is written on the right hand side.
 The amino terminus always carries a positive charge and the carboxyl terminus a negative
charge.
 

 
Lecture 12:  9-18-06
Protein Purification
 
Protein Purification
 
 The 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
 The protein of interest, which may make up only a fraction of 1% of the starting material
must be purify to ~98% purity
 
Methods 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 – Cell suspensions are forced through a very narrow
channel under high pressure
        A homogenate is form
 
 
 
 
 
 
Differential Centrifugation
 
 The homogenate can be fractionated by centrifugation
 In 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)
 This method serves to eliminate selectively components of the starting mixture so that
only the required component remains
 Only the fraction that contains the protein of interest will be further purified
 
 
Salting out
 
 At high salt concentration, the solubility of protein decreases
 The salting out effect is the result of the competition of between the added salt ions and
proteins for molecules of solvent
 At 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
 Ammonium 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
 
Gel-filtration Chromatography
 In this method, proteins are separated on the basis of size
 Sample is applied to the top of a column containing porous beads (the solid support or
matrix)
 Small molecules can make their way through the pores of the beads while larger ones
cannot
 As a result, large molecules flow more rapidly through the column and elute first
 Molecules of intermediate size, which can occasionally enter the beads will elute at an
intermediate position
 The small molecules that take a longer path will elute last
 
 
Ion-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
 
Net charge of proteins Type of ion-exchange column Counter ion
Positive Cation exchange: CM-cellulose Na+
Negative Anion exchange: DEAE-cellulose Cl-
 
Cation- exchange column
 If a protein has a net positive charge, it will bind to a column of beads containing
carboxylate groups (i.e. CM-cellulose), which are negatively charged
 The positively charged protein bound to the column can then be eluted by increasing the
concentration of sodium chloride
 Sodium 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. DEAE-
cellulose) columns
 The counter ion is chloride ion
 
 
 
 
 

 
 
 
Gel Electrophoresis
 
 One 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
 The 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
  Polyacrylamide gels are commonly used for electrophoresis
 Unlike gel-filtration chromatography, all molecules, regardless of size are forced to move
through the same matrix
 Under denaturing conditions, proteins are separated largely on the basis of protein mass.
     Sodium dodecyl sulfate (SDS), an anionic detergent disrupts all non-covalent
interactions in native proteins
     Mercaptoethanol or dithiothreitol (DTT) is added to reduced disulfide bonds
     The SDS anions bind to main chains at a ratio of one SDS anion for every two
amino acids
     The SDS bound proteins has a large net negative charge, which is proportional
to the mass of the protein
     The negative charge contributed by the SDS is much greater than the charge
on the native protein that the native charge is insignificant
     The SDS bound protein will move towards the positively charged electrode
during gel electrophoresis
     Smaller proteins move faster through the gel compared to larger proteins
 
        When electrophoresis is complete, proteins in the gel can be visualized by staining
them with silver or a dye such as Coomassie blue