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MCB 250 - Exam 1
Terms in this set (246)
Prokaryotes: bacteria and archaea
-DNA is in direct contact with the cytoplasm
-transcription and translation are simultaneous
-DNA is contained in a membrane-bound nucleus
-Transcription occurs in the nucleus
-mRNA is exported to the cytoplasm where translation occurs
-transcription and translation do NOT occur simultaneously
Universal Tree of Life
based on comparisons of ribosomal small subunit RNAs
Relative Sizes of Eukaryotic and Bacterial Cells
-bacteria cell is a lot smaller than eukaryotic
-mitochondria evolved from a bacterial endosymbiont
The Central Dogma
-DNA makes RNA, and RNA makes Protein
-RNA can have enzymatic and regulatory functions
Proteins Dominate Cellular Biochemistry
-many proteins are enzymes --> catalyze chemical reactions that occur in cells
-other proteins allow cells to move, regulate biochemical processes, or function as structural components of the cell
-RNAs can also have enzymatic activity AND are important in regulation
sum total of all of the chemical reactions of the cell
-catabolism vs. anabolism
breakdown food, make energy
synthesize the components of cells so they can grow and divide
Bacterial Molecular Biology
transcription and translation take place simultaneously in the same cellular compartment - the cytoplasm
eukaryotic molcular Biology
-transcription and translation take place at different times in different cellular compartments.
-RNA processing occurs in nucleus, and mRNA is transported to cytosol where it is translated into protein
Why is chemistry important in molecular biology?
-DNA carries cell's genetic information
-Proteins catalyze most of the chemical reactions that allow cells to function
-proteins interact specifically with other molecules
Life is Primarily the Chemistry of Six Elements
-store potential energy
-the stronger the bond, the more external energy required to break it
-strong bonds vs. weak bonds
-resonance and aromaticity, UV absorbance
-stereoisomerism (D- vs. L- isomers)
-van der Waals interactions
Covalent Bonds Result from Shared Electrons
-covalent bonds are strong
-ΔG of formation: -50 to -110 kcal/mol
-Compare weak bonds:: < 10 kcal/mol
-form backbone of a protein molecule; covalent bonds between carbon and nitrogen atoms in successive amino acids
-even if a protein is placed in 100ºC boiling water, the thermal energy is insufficient to break these strong covalent bonds
Properties of Covalent Bonds
-bond angles are fixed, but some bonds can rotate while others cannot
-resonance and aromaticity
-some bonds have high transfer potential: the concept of a "high energy bond"
free rotation around C-C single bonds
no rotation about double bonds
ex. ethylene (ethene) is a planar molecule
cis - trans isomerism
-these are distinct molecules with different physical and chemical properties
-cis --> same side
-trans --> opposite sides
two "resonance" structures with alternating or "conjugated" double bonds
Delocalized Electrons in Aromatic Rings
(aromatic ring stacking)
Nucleic Acids are Aromatic Rings
DNA (deoxyribonucleic acid)
Conjugated Double Bonds
-alternating double and single bonds
-electrons are delocalized over 4 carbons
-results in planar structure
weak non-covalent bonds make a major contribution to all of the following:
-holding together two chains of the DNA double helix
-protein: protein interactions
-3D structure of proteins
-Protein: DNA interactions
which of the following statements is true of hydrogen bonds
in cells, the most biologically significant hydrogen bonds involve O and/or N atoms
which is true of van der waals
Can occur between any two closely spaced atoms
which is true of "hydrophobic bonds"?
occur in aqueous solutions from the tendency of water to exclude non polar groups
Absorption of Light
-electrons move in "quantized" orbitals
-light of appropriate energy can kick electrons into a higher-energy orbital
-the "absorption spectrum" provides information about molecular structure
-all molecules absorb radiation
-aromatics and compounds with conjugated double bonds absorb light in the UV or visible range
-more conjugated double bonds means that light of lower energy will be absorbed
-B-carotene absorbs in the visible blue range (~450 nm) --> colored intensely red-orange
-bases of DNA and RNA absorb light at 260nm, while aromatic amino acid tryptophan absorbs light at 280 nm
-chiral molecule: rotated molecule cannot be superimposed on its mirror image
-achiral molecule: rotated molecule can be superimposed on its mirror image
L- or D- stereoisomers
-19 of 20 amino acids can exist as distinct L- or D- stereoisomers
-Life evolved to use only L-amino acids in proteins
why only one stereoisomer in biological system?
most biological interactions depend on precise interactions between chiral molecules; only one form will fit, the other won't
high energy bonds
-does not refer to bonds that are "strong" but rather those that release significant energy when hydrolyzed
-ex. ATP hydrolysis
weak chemical interactions important in biology
-van der Waals interactions
Water is a polar molecule
water has a dipole moment as a result of unequal sharing of electrons
-account for properties of water, which forms a lattice structure
-strength of hydrogen bonds are affected by orientations straight is better
despite its lattice structure, liquid water can flow because
hydrogen bonds break and re-form rapidly
Biology Happens in Water
-H2O is polar solvent --> makes hydrogen bonds; both a H-bond donor and H-bond acceptor
-two properties make interactions that underlie biological processes possible
-h-bond donor is a hydrogen covalently bonded to electronegative elements such as O and N in biological systems --> can interact with another electronegative element such as O or N (H-bond acceptors)
-h-bonds are directional: stronger interaction if linear
-not all H-bonds are equally strong but generally ΔG is approx. 3-7 kcal/mol
Orientation of Hydrogen-Bonds
-ex. h-bond between two peptide backbones
-orientation matters: straight = stronger, bent = weaker
-all things being equal, they'll line up to maximize (straighten) the hydrogen bond
Ionic bonds - electrostatic interactions
-opposite charges attract
-ΔG approx. 5 kcal/mol in water, but stronger interactions are possible inside protein molecules
-these interactions are not directional
-like charges repel
Van der Waals Forces
-electron clouds of all molecule attract each other early (attraction is weaker than ionic or H-bond)
-effect is highly dependent on distance between two molecules; if molecules are too close, electron clouds repel
-individual van der Waals interactions are weak, but the sum of a large number of van der Waals interactions can result in significant force
-van der Waals forces are most significant when two large surfaces fit together
-bond strength is maximized as a particular distance, when the participating electrons overlap to a specific degree
Stacking of Aromatic Rings
-stacking interactions - van der Waals
-overlapping pi-bonds stabilize interaction of stacked aromatics. very important in stabilizing DNA structure
-hydrophobic = "water hating"
-hydrophilic = "water loving". --> have dipole moments and can form hydrogen bonds
-molecules without dipole moment do not interact well with H2O
-hydrophobic molecules are excluded by H2O
Fatty Acids are Examples of Hydrophobic Biomolecules
-chain composed of hydrogen and oxygens
have both hydrophobic and hydrophilic regions
hydrophobic interactions drive formation of lipid bilayers in biological membranes
Hydrophobic interactions stabilize the folded structures of proteins
-important for interactions between protein molecules
-forces holding 4-helix bundle together involve hydrophobic interactions between hydrophobic amino acid side chains
Importance of Weak Interactions in Biology
-many interactions in biology are dynamic: molecules come together and then come apart - e.g. enzyme and substrate
-if interactions were too strong, interacting molecules would be too stable and wouldn't come apart
-allow and define specific interactions between biological molecules
Many Biological Molecules are Charged
-5 of the 20 amino acids found in proteins have side chains that are charged at neutral pH
-phosphate residues in the sugar-phosphate backbone of DNA and RNA are charged at neutral pH
-phospholipids are charged at neutral pH
-biological molecules frequently interact via electrostatic (charge-charge) forces
-charge of biological molecules depends on the pH of their environment
pH: a measure of H+ concentration
pH = -log[H+]
keq = ([H+][OH-])/[H2O]
Kw = [H+][OH] = 1/0 x 10^-14
in pure water: [H+] = [OH-] = 1.0 x 10^-7
Weak Acids and Bases in Biology
carboxylic acid group - weak acid
amino acid - weak base
Ionization of Weak Acids
Ha <--> H+ + A-
Ka - ([H+][A-])/[HA]
Applying the Henderson-Hasselbalch Equation
when [HA] = [A-], the [A-]/[HA] = 1 and log [A]/[HA] = 0
this means that:
-ph=pka, then [A-] = [HA[
-when pH is one unit above the pea, then [A-]/[HA] = 10
-when pH is one unit below the pea, then [A-]/[HA] = 0.1
pka & pH
weak acids and bases in biology
-the pKa is affected by the local environment
-the pKa of an amino acid side chain in the interior of a protein may differ from the pKa of the same side chain on the surface of a protein
aspartate - pKa = 3.9
lysine - pKa = 10.5
Net Charge of protein or peptide at neutral pH
determined largely by the number of glutamic acid, aspartic acid, lysine and arginine residues
Glu, Asp (pKa ~4)
-essentially 100% negatively charged at neutral pH
Lys (pKa ~10.5), Arg (pKa ~12)
-essentially 100% positively charged at neutral pH
ionic bonds in proteins are affected by pH
In linear DNA molecules, the two ends of a single strand are designated as
5' and 3'
what of the following statements about DNA structure is correct?
-in double helix, complementary base pairs are stabilized by hydrogen bonds
-radius of double helix is relatively constant because A=T and C=G base pairs are the same length
-ration of A-T to G-C varies in different organisms
-bases of DNA are aromatic rings contains both carbon and nitrogen atoms
-pyrimidines have one ring; purines have two
in 1928, Frederic Griffith showed that
a chemical derived from dead bacteria can produce heritable changes in the traits of living cells
in 1953, James Watson and Francis Crick accurately predicted the structure of DNA. they based their production on experimental data obtained by which technique?
History of Genetics
-mendel's work leads to the idea that there are genetic elements (genes) that can exist in different forms (alleles) that somehow determine observable traits (phenotypes)
-T.H. Morgon (1910) -- genes are located on chromosomes. Chromosomes can be observed directly by microscopy
-Beadle and Tatum (1941) -- genes somehow determine individual proteins. "one gene, one enzyme"
state of genetics (ca. 1945)
-genes passed from parents to offspring
-genetic material associated with chromosomes
-chromosomes composed primarily of proteins & DNA, plus a small amount of RNA
was ''transformed" by substance from the dead, wild type bacteria
-something is taken up that transforms the rough strain to smooth, and this smooth trait is then inherited by future generations of bacteria
What is the "something" that transforms rough pneumococci to smooth?
-Avery carefully purified the "transforming principle" and found that it did not contain protein, carbohydrates, RNA, or lipids. Instead it contained, as far as they could determine, only DNA
-use term "transformation" to indicate living cells taking up & incorporating external DNA
Transforming Material is DNA
-Pure DNA transforms rough to smooth
-transforming substance was inactivated by digesting with DNase, but not RNase or protease
DNA is the Hereditary Material
-Hershey and Chase radioactively labeled the E.coli virus (bacteriophage), T2
-Protein coat was labeled with 35S
-DNA was labeled with 32P
-they showed that after infection, they could strip off the protein
-only DNA entered the cells
-infected cells gave rise to progeny phage that contained detectable 32P, but not 35S
There are several types of Strep. pneumonia that have chemically different polysaccharide capsule (Averys previous work) called S1, S2, S3, etc.
Experiment: Dead S1 cells + Live Rough (S2) cells
What is the capsule the bacteria recovered from the mice?
Discovery that Genes are DNA was Surprising
-chemical composition (but not the structure) of DNA was known
-Proteins are made of 20 different amino acids with an enormous variety of possible structures.
Original Model of the Double Helix (1953)
Rosalind Franklin's X-ray diffraction pattern of DNA from which Watson and Crick deduced that DNA was a helix
Deoxyadenosine Monophosphate (dAMP)
Sugars in DNA and RNA
Bases in DNA and RNA
Purines vs. Pyrimidines
Purines - adenine, guanine
pyrimidines - cytosine, thymine, uracil
Adenosine Nucleoside (A)
Nucleoside Monophosphate (AMP)
nucleoside diphosphate (ADP)
Nucleoside Triphosphate (ATP)
DNA: Bases, Nucleosides, and Nucleotides
Deoxyribonucleotides (deoxyadenosine, deoxyguanosine, deoxythymidine, deoxycytidine)
RNA: Bases, Nucleosides, Nucleotides
Ribonucleotides (adenosine, guanosine, uridine, cytidine)
strands of DNA and RNA are formed by joining nucleotides together with phosphodiester bonds
-5' end usually has a free phosphate group
-3' end usually has free hydroxyl
Chargaff's Rules (1951-'52)
-the % GC or AT varies between organisms
-but always, A=T and G=C
DNA is a Double Helix
-A=T and G=C because DNA is a double helix and A in one strand pairs with T in the other strand via H-bonds. same goes for G and C
-strands are antiparallel. one runs 5'-3', the other 3'-5'
Hydrogen bonding defines the specificity of Base Pairing
-excellent relative alignment of hydrogen bond donors and acceptors
-non-complementary bases do not align and cannot form hydrogen bonds or even fit in the DNA helix
Conventions in righting DNA/RNA sequences
-5' (left) --> 3' (right)
-indicate polarity explicitly
B-form DNA (a right-handed helix)
-one helical turn = 10.5 base pairs
-2nm in width
-minor vs. major groove
Major and Minor Grooves
B-DNA Structural Features
-right handed helix
-strands are antiparallel
-hydrogen-bonded bases lie in the same plane
-20Å in diameter
-34Å per turn
-10.5 base pairs per turn
-bases rotated 36º with respect to each other
-Major Groove (12Å), Minor Groove (6Å)
What forces are responsible for DNA structure?
-base pairing: H-bonds
-Base stacking: stacking interactions between aromatic rings
-hydrophobic interactions of bases
-repulsion of negatively charged phosphate groups - cations (such as Mg+2) are required to neutralize
Stacked Base Pairs
-in addition to van der waals (stacking) forces, non polar surfaces of stacked aromatic rings undergo hydrophobic interactions, thus excluding H2O from interior region of helix
-negatively charged phosphate groups are repelling each other and are on the outside of the helix
Proteins recognize the nucleotide of double-stranded DNAs primarily by forming non-covalent bounds with
chemical groups on the edges of paired bases within the major groove
a solution of dsDNA is placed in a spectrophotometer cuvette and its absorption of UV light is recorded. As the cuvette is heated to above the Tm for this DNA sample, what will happen to its absorption of UV light?
the A260 will increase
-called "hyperchromaticity" of DNA when it melts (denatures). Separation of the two DNA strands changes the local environment of the bases, which in turn changes their ability to absorb UV light. Single-stranded DNA absorbs more UV light at 260 nm than does double-stranded DNA
Chemical information in the Major and Minor Grooves
-proteins interact with DNA in a sequence-specific manner.
-exposed edges of paired basses can make different types of chemical bonds
A: H-bond acceptors
D: H-bond donors
H: Nonpolar Hydrogen
M: Methyl groups
Some proteins specifically bind to DNA of a given sequence. In other words, protein can "read" the sequence.
If you were designing a DNA-binding protein that binds to a specific DNA sequence, how would you make it work?
From hydrogen bonds with "spare" groups in the Major groove
what a protein sees inside a major groove...
Sequence-Specific DNA Binding Proteins
-interactions with H-bond donors and acceptors - and van der Waals surfaces in the grooves - allow proteins to recognize specific sequences of base pairs in DNA without disrupting the double helix
-major groove contains more information. (ex. C-G and G-C look different in the major groove, but not in the minor groove)
DNA Structure is Not Uniform Along the Helix
-most DNA in cells is believed to exist more or less in the B form, but its structure is not uniform & can vary somewhat
-different base sequences can induce local structural variations
-binding of proteins to DNA can also induce bending
DNA is a dynamic molecule
-some regions may be transiently single-stranded (melted)
-loops and cruciforms may form transiently
-water and ions are continuously coming and going
-some regions may from alternative structures transiently (ex. Z-DNA)
Sequence-Dependednt Bends in DNA
runs of A-T rich base pairs spaced about 10 bp apart cause bending
Protein Binding Can Bend DNA
-histone binding bends the double helix
-proteins package DNA into small compartments
normal B-form base pairs (a)
Twisted base pairs (b) --> left handed helix
Different DNA Structures Exist
Chemical and Physical Properties of DNA
-DNA & RNA bases are aromatic. They absorb UV light with a maximum at 260 nm
-Absorption spectrum of DNA& RNA is dependent on the environment of the chromophore (chromophores are the bases)
-chromophore environment is different in single-stranded DNA (ssDNA) vs. double stranded DNA (dsDNA), so the bases absorb light differently
Schematic of a UV-Vis Spectrophotometer
UV Absorption Spectrum of DNA
-Beers Law --> amount of light that is absorbed at a specific wavelength
Spectrophotometry of DNA
measuring the UV absorption of a sample allow:
-Quantitation of DNA
At a DNA concentration of 50 ug/ml
-dsDNA: A260 = 1
-ssDNA: A260 = 1.37
-free bases A260 = 1.6
-Purity check of a preparation of DNA (or RNA):
-A260/A280 = 1.8 highly pure dsDNA
= 2.0 for pure RNA
=.6 for a typical mixture of proteins
Absorption Spectra of Aromatic Amino Acids
-Trp predominates absorption at 280 nm for most proteins
-proteins absorb some light at 260 nm
Trp>>Tyr>>Phe at E280
Assessing DNA Purity
-DNA contaminated with protein can't be quantified using A260
-Purity of a DNA sample can be assessed form A260/A280 ratio
-In a DNA sample that is contaminated with protein (very common), the spectrum will result from absorption of UV light by BOTH the DNA and protein
Denaturation of DNA
-two strands in a DNA double helix are held together by a large number of weak, non-covalent interactions
-two stands will separate or "denature" if these interactions are disrupted
DNA denaturation can be caused by:
-Tm = melting temp
-Tm depends on base composition: high GC has a higher Tm
-Choatropic agents like urea
-pH > 11
-Organic solvents (e.g. methanol
-Decreased salt concentration
Base stacking is cooperative
-ends of the DNA "breathe" (not annealed)
-ssDNA-binding proteins can denature DNA
-ssDNA better at absorbing light
DNA Melting Curve
for any given DNA molecule, Tm is defined by 3 factors:
-Length --> shorter DNA molecules have fewer H bonds: lower Tm
-G/C content --> higher G/C conent means more H-bond & more base-stacking (higher Tm)
-Chemical envrionemt --> salt concentration, etc. affects Tm
-pop of identical DNA mol in same enviro exhibits Tm at 1/2 mol have denatured & 1/2 still double-stranded
DNA Renaturation (Annealing)
denaturation is reversible
Sequences Do Not have to be Identical to Renature
-duplex DNA in which one strand differs from the other are called heteroduplexes
-heteroduplexes are hybrid molecules, and the process of forming them is called hybridization
-the application of renaturation of ssDNA and ssRNA is a widely used and important tool in the experiment analysis of nucleic acids
-stable heteroduplexes at high temps
-at low temp, can form imperfect heteroduplexes with C and E strands
DNA denaturation can be caused by
-Tm - melting temp
-Tm depends on base composition:: high GC has higher Tm
-Decreased Salt Concentration
enzymes that cut nucleic acids by hydrolyzing backbone phosphodiester bonds
-DNases cut DNA
-RNases cut RNA
-Some cut both
products may be oligonucleotides or nucleoside phosphates
most nucleases are relatively nonspecific but some have sequence specificity
starte at end to cut
-recognize specific sequences in DNA
-many cut specifically within the recognition sequence (or at a site nearby)
-products are relatively large polynucleotides
-some restriction enzymes produce products with "sticky ends"
-recognition sites vary in length
-short recognition sequences occur frequently in DNA, so enzymes with short recognition sequences produce a large number of shorter fragments
-Long recognition sequences occur less frequently, so enzymes with long recognition sequences produce fewer, longer fragments
-bacterial defense against viral infection by restriction-modification complexes
Restriction Enzyme recognition sites are often palindromes
-a palindrome or palindromic DNA sequence is the same sequence forward on the one strand and backward on the other (both read 5'-3')
Frequency of Restriction Enzyme Sites in DNA
-there are 4 possible bases that occur at any given position
-recognition site for "6-cutter" like EcoRI is found 1 in 4^6 bp or 1 in 4096 bp, ON AVERAGE
frequency = 1/4^n where n is # of bp in recognition sequence
Examples of Restriction Enzymes
-each of these enzymes recognize a specific, 6 bp-long palindromic sequence
-Hpal produces blunt-ended fragments
-EcoRI, HindIII, and PstI produce fragments with sticky ends
Why "Restriction" Endonuclease?
-Bacteria possess enzymes that "restrict" incoming "foreign" DNA (like bacteriophage DNA) by cleaving it
-nearly all bacterial genomes encode one or more "restriction modification" systems
If a bacterial cell contains a "restriction endonuclease" to inactivate invading phage DNA, how does it avoid its own DNA?
Modification: it modifies (by DNA methylation) every recognition site in its own DNA. the restriction endonuclease can't recognize, and therefore won't cut, the modified (methylated) sites
Uses of Restriction Enzymes
-any two pieces of DNA with compatible ends can be easily ligated together
-Modern techniques make it easy to add restriction sites to any piece of DNA
-DNA fragment encoding a protein can thereby be cloned into a plasmid vector & expressed as a recombinant protein in E.coli or another host cell
-the relative position of restriction sites provides information about the sequence of the DNA
-Restriction analysis allows easy comparison of two different DNAs, or confirmation of cloning events
Restriction enzymes play a big role in DNA cloning:
-restriction enzymes cleave large, chromosomal DNA molecules into smaller pieces that are ideal for cloning genes
-single-stranded overhang produced by restriction enzyme can still base-pair with a complementary overhang in another piece of DNA cut with the same restriction enzyme
-In presence of enzyme, DNA ligase, the complementary sticky ends are covalently attached to each other (i.e. the phosphodiester bonds are restored). this typically restores the restriction site sequence
-restriction fragments with blunt ends can also be re-ligated to one another by DNA ligase
-any two blunt ends can be re-ligated, However when two different restriction enzymes are used to generate the fragments, the relegated DNA junction may not be cleavable by either enzyme
Separating (Resolving) DNA by Gel Electrophoresis
-Analytical tool: to identify or learn something about a particular piece of DNA: size, topology, restriction pattern,, purity, concentration
-Preparative tool: to separate fragment of interest from a complex mixture of DNA fragments
Agarose Gel Electrophoresis
-gel sieves the molecules: smaller DNA fragments move further through the gel than larger fragments
-charged molecules will move in an electric field
-gel matrix is used to "sieve" molecules
-agarose: loose gel; broad separation range
-Acrylamide: tighter gel; narrow separation range
-movement of a molecule through the gel is dependent on its charge and shape
-in order for movement to be proportional to mass (molecular weight), the the molecule must have a constant charge-to-mass ratio
-DNA has 2 negative charged (phosphates) per base pair
-all linear, double-stranded DNA molecules have (essentially) the same shape
-movement in electric field in the gel is inversely related to dNA length (MW)
-DNA molecules of same size migrate as discrete bands
Ethidium Bromide Can be used to visualize DNA fragments after Electrophoresis
-ethidium is an aromatic dye that fluoresces
-when it absorbs UV light, it emits visible light
-when bound to DNA, its fluorescence intensity increases about 20-fold
Flourescence Excitation and Emission Spectra of Ethidium Bromide Bound to DNA
-excitation spectrum - light absorbed from UV lamp
-emission spectrum - the visible light emitted by dye
intercalates into the DNA helix
Restriction Fragment Analysis
the "marker" lane contains DNA fragments of known size, allowing the sizes of the unknown DNA fragments to be determined
-restriction enzyme digestion of DNA, followed by resolving the cleaved DNA by gel electrophoresis
Information from Gel Electrophoresis
-DNA size: distance migrated is inversely proportional to the log MW = or log (# base pairs)
-DNA Quality: binding of ethidium is directly proportional to MASS of DNA
Quantifying DNA by Ethidium Bromide Staining
-marker dna: known size & quantity
-"size" of box is meant to reflect "brightness'' of the band
-density of gel matrix defines resolution of gel
-ethidium bromide stains all DNA fragments --> become visible
-after hybridization to a specific probe and autoradiography - only the DNA fragment containing the sequence complementary to the probe is visible
Labeled DNA ''probe" hybridizes only to complementary DNA sequence
after hybridization to the radioactive probe, membrane is placed on piece of photographic film. Radiation from labeled probe will expose the film - "autoradiography"
Levels of Protein Structure
-Primary Structure --> amino acid sequence
-Secondary Structure --> alpha-helix
-Tertiary Structure --> Folded Polypeptide Chain
-Quaternary Structure --> Assembled Subunits
L-Amino Acids - Building Blocks of Proteins
The Peptide Bond
-Condensation reaction; water is released
-Charges remain on N- and C- temini
-Convention: Protein sequences are written N-terminal --> C-terminal (the N-terminus is on the left)
Primary Structure of a Protein
-a 'polypeptide' is the product of a single gene.
- it is a linear chain of amino acids linked together by peptide bonds
-in order for a polypeptide to become a mature functional protein, it must fold in 3D and adopt a specific conformation
The Peptide Bond is Rigid - and Planar
note resonances structures; peptide bonds have partial double-bond character
Hydrophobic Side Chains
these side chains are often found buried inside globular proteins (buried in hydrophobic core that is kept away form water)
-Proline, Glycine, Leucine, Alanine, Valine, Methionine, Isoleucine
Hydrophilic (Polar) Side Chains
-polar (can make hydrogen bonds with water), uncharged side chains (at pH 7) are often found on the surface of proteins ("solvent-exposed")
-these types of side chains can participate in H-bonds
-cys can participate in disulfide bonds
-cys is active sites of enzymes can be deprotonated & act as a nucleophile
-Serine, Threonine, Cysteine, Asparagine, Glutamine
Aromatic Side Chains
-aromatic, uncharged side chains (at pH 7)
-often found in the interior of proteins
-can participate in aromatic stacking interactions
-Phe & Top are nonpolar
-Tyr is largely hydrophobic, but can participate in H-bonding interaction through its -OH group
-Phenylalanine, Tyrosine, Tryptophan
Acidic Side Chains
-negatively charged at pH7
-often found at surface of proteins (solvent-exposed) or in enzyme active sites
-can make ionic interactions with positvely-charged groups or metal ions
-Aspartate (aka 3.9), Glutamate (pka 4.3)
Basic Side Groups
-positively charged at pH 7 (although His is only partially charged)
-often found at surface of proteins (solvent exposed) or in enzyme active sites
-can make ionic interactions with negatively-charged groups
-Lysine (pka 10.4), Arginine (pka 12), Histidine (pka 6.2)
Amino Acid Side Groups Have Multiple Properties
all of which affect protein folding and properties
disulfide bonds can form spontaneously in oxidizing environments, but interconversion is more likely to be enzyme-mediated
-two cysteine joined together by a disulfide bond (Cystine)
-can be intramolecular or intermolecular
-a salt bridge can form between the oppositely-charged side chains of residues in proteins from a combination of two relatively weak non-covalent interactions:
-salt bridges can make important contributions to overall stability of the folded state of proteins
Forces Affecting Protein Structure
-multiple interactions among side-chain and backbone atoms of amino acid residues and/or 2º structures contribute to the stabilization of the folded state (3º structure) of a protein
-all chemical interactions shown (ionic bonds, h-bonds, hydrophobic cluster, van der waals interactions, stacked aromatic rings) are non-covalent, except for disulfide bonds
Ways of Representing protein structures
** look at notes (Lecture 6, slide 18)
a) van der waals surface
c) alpha-carbon trace
d) ribbon diagram
e) electrostatic surface representation
Peptide Bonds Rotate Freely Around the alpha-carbon
-however some conformations are energetically unfavorable
involves hydrogen bonding between carbonyl c=oxygen (-C=O) and amide nitrogen (-NH) groups that are part of the peptide backbone
This can lead to two compact & stable structures
-compact and stable structures
-easy to pack together
-all backbone H-bonding donors and acceptors are satisfied, so the if the side chains of that protein segment are hydrophobic then the protein segment is totally hydrophobic
-hence, and alpha-helix or beta-strand can be packed into protein core or placed in a phospholipid bilayer
-held together by H-bonds
-3.6 amino acid residues per turn
-H-bonds between the backbone C=O of residue n and the NH of residue n+4
-all backbone H-bond donors and acceptors are joined in H-bonds except the first 4 NH and last 4 C=O
-an amino acid in a protein is called a residue since it has lost water and is no longer an amino acid
-r groups face out, so the chemical properties of an alpha-helix are determined by its amino acid sequence
the properties of helix are defined by the amino acid side chains
-hydrophobic and hydrophilic properties
the alpha helix is the most stable secondary structure known. Are all amino acid sequences equally likely to form an alpha helix?
MALEK are favored; P is a helix-breaker; G is disfavored
-not a rule, just a tendency
B-strand and B-pleated sheet
-in beta-sheets, the polypeptide chains can be in the same direction (parallel) or alternating direction (antiparallel)
-by convention the arrow point N-term to C-term
-secondary structures are connected by loops
-loops may be as small as 2 amino acids or much larger, constituting significant portions of the protein
-B-sheet is not flat. it is pleated with alternating R groups extending above & below the plane of the sheet
-possible for a single face to be either hydrophilic or hydrophobic
-possible for entire sheet to be amphipathic, with polar R groups on one side & nonpolar groups on the other face
-note that polar & non polar r groups alternate every 2nd position within a given chain
Side chain of an Arg residue in the middle of an alpha-helix is interacting with the side chains of a Glu residue on an adjacent beta-sheet. How would you best describe this interaction?
Tertiary & Quaternary Structures
-proteins fold into compactly-folded domains (3º structures) made up of polypeptides with multiple 2º structures
-sometimes small molecules are included in the folded protein
-multiple polypeptide chains (subunits) can bind to each other to build up complexes (4º structures)
Tertiary Structure: Domains
-proteins often fold into multiple domains with various combinations of 2º structures
-interactions depend on the nature of the side chains
-typical protein has multiple regions of 2º structure separated by less regular turns or loops
-known structures of transmembrane proteins belong to two classes, based on the secondary structures of their transmembrane regions:
2º, 3º, & 4º Structure: Hemoglobin
-some proteins- such as hemoglobin- are mostly helical
-blue molecules are hemes
-hemoglobin is a heterotetramer
-biologically active polypeptides come in a wide variety of sizes:
-small: thyrotropin-releasing hormone
-3 amino acids - Gln-His-Pro, a peptide, not a protein
-Huge: Titin, a molecular spring in cardiac muscle
- ~30,000 amino acids - 3450 kildaltons (kDa)
- ~260 amino acids about 30 kDa
how many different 260 amino acid proteins are possible?
20^260 = 10^338, so only a tiny fraction of the possible amino acid sequences are actually used
Methods for Determining Protein Structure: X-ray Crystallography
-90% of the structures we have were determined by this method
-rate-limity step is usually getting crystals. some proteins don't crystallize
-provides the structure of a crystallized protein- although this may differ from the structure in solution
-improved instruments and computational methods are leading to a rapid increase in the number of structures determined
Nuclear Magnetic Resonance (NMR)
method for determining protein structure
-provides structure in solution
-currently, feasible for relatively small proteins
Phosphorylation is an important form of postransitional modification for intracellular proteins. Which of the following amino acids is a target for phosphorylation?
threonine, histidine, serine, tyrosine
Which of the following are major conclusions of the Anfinsen Experiment?
-correct folding of a polypeptide chain requires the formation of noncovalent bonds
-amino acid sequence determines the 3D structure of a protein
-correct doling of this polypeptide does not require additional cellular machinary
-experimental results show that when cysteine residues are allowed to re-form disulfide bonds before any noncovalent interactions could take place, the resulting structure was scrambled and nonfictional. when the researchers instead allowed cysteine residues to form together with the noncovalent interactions, the protein regained its native form and function
The Anfinsen experiment proved that some proteins can fold appropriately without any external folding "machinery". but some proteins do indeed need assistance during folding process. the general term used for the proteins that help other proteins fold is:
proteins often change conformation when they bind to their specific substrate. why?
formation of energetically favorable contacts allows the proteins to take on a more stable conformation
Protein - Protein Interactions
-Protein-Protein interactions can lead to assembly to oligomeric complexes
-stability of these complexes depend on the nature of the bonds in the interfaces of the proteins
Domain Interactions: Ligand Binding
-hemoglobin is an α2β2 heterotetramer of globular protein subunits
-binding of O2 to the heme of one subunit influences binding of O2 to the remaining hemes: cooperative binding
Protein-Protein Interactions (2)
-binding interfaces between proteins depend on complementary shapes employ noncovalent interactions, including:
-electrostatic interactions (salt bridges)
-ex. ribonuclease inhibitor forms extensive protein-protein interactions with ribonuclease (RNAse). this restricts the motion of RNAse and blocks its interactions with its substrate, RNA
Structural Motifs in Protein -DNA interactions
few common structural motifs are present in sequence-specific DNA-binding proteins:
-coiled coil (leucine zipper) --> 2 alpha-helices try to wrap around each other
GCN4 - Coiled Coil ("Leucine Zipper") protein
-GCN4 is a transcription activation in yeast
-Leucine Zipper domain holds the two polypeptide chains together (note that large dimerization interface)
-the other domain binds to specific nucleotide sequences via the major groove of DNA
Leucine Zipper Motif
-forms dimerization interface
a coiled-coil protein in muscle
-Helix-Turn-Helix motif in a DNA binding protein
-many different sequence-specfic DNA binding proteins have HTH motifs
Bacteriorphage 434 Repressor:
a helix-turn-helix DNA binding protein
-often found in regulatory proteins that specifically bind to DNA and regulate gene expression
Integral Membrane Proteins
Proteins with hydrophobic interfaces can enter phospholipid membranes
a helical transmembrane protein
a beta-barrel Transmembrane protein
Ions such as Na+ or Cl− cannot pass through a lipid bilayer, but must cross cell membranes by flowing through the pore of an "ion channel protein." Ion channel proteins are typically composed of α-helices. What would you predict about the structure of the pore?
MulJple amphipathic α-helices surround the pore, with nonpolar R groups facing the lipids and polar/charged R groups facing into the pore.
-ion pore surrounded by ring of α-helices.
-ion pore (arrow) lined by polar or charged R groups.
-All the informaJon for proper folding of a protein is present in its amino acid sequence
-Given free rota:on around the α-carbon, proteins can assume an essen:ally infinite number of conforma:ons.
-Some conforma:ons are much more energe:cally favorable than others
-Usually only one conforma:on—the one with the lowest free energy—is the ac:ve or na:ve state
How Does a Protein Find the EnergeJcally Favorable State?
• Assume each residue can have three possible conformations (a very low estimate).
• Therefore, a 260 amino acid protein has 10^124 possible conformations.
• Assume 10-13 seconds to transition from one state to another.
• Therefore it would take ~10^100 years.
• Age of the universe ≈ 10^10 years!
General Concepts in Protein Folding
• For many proteins there are probably many pathways for folding - most molecules end up at the right place (the na:ve structure) but each one got there a different way.
• Concept of a funnel- shaped energy landscape - lots of non-na:ve states (unfolded or folded wrong) but only one na:ve state (folded right).
• Folding of localized secondary structure is rapid. - Random coil to α-helix takes <10-6 sec
• Folding is coopera:ve.
- Ter:ary structure can stabilize secondary structure
- Zip and Assemble (ZA) - local structures can form quickly and then assemble into global structures
• Domains fold independently.
• Protein folding takes place as the protein is being
- N-terminal regions fold before (in the absence of) the C-terminal regions
More General Concepts for Protein Folding
• A major driving force for folding is the tendency for hydrophobic side chains to get away from water and be on the inside of the folded structure.
• Other interac:ons (e.g, H-bonds) probably determine the exact nature of the fold.
• Most of the amino acid residues with polar and charged side chains end up on the surface, exposed to the solvent (water).
Sometimes Proteins Don't Fold Properly
• Not all proteins fold appropriately.
• If we think of folding as a pathway leading to the most energe:cally favorable state, some:mes proteins go "off-pathway" and fold into the wrong conforma:on.
• These wrong conforma:ons can have a favorable enough energy state that the protein cannot refold into the proper state.
• These misfolded proteins oven have exposed hydrophobic regions. Such proteins will aggregate.
• Consequences of misfolding of proteins:
- Alzheimer's disease, Parkinson's disease, Mad Cow disease, Aging?
• Some proteins will refold into the ative state in vitro
- Usually small proteins with simple structures
• In the cell, folding is assisted by "chaperones"
and other proteins
• Some examples of proteins that assist in folding:
- GroE (an ATP-dependent molecular chaperone)
- Disulfide Bond Isomerases
- Peptidyl Proline Isomerases
Chaperones are Important for Protein Folding, Refolding, and PrevenJng Protein Aggregation
• Help in folding newly synthesized proteins
• Help in refolding proteins that have become partially unfolded (maybe as a result of some stress like exposure to a high temperature - "heat shock")
• Bind to exposed hydrophobic surfaces, preventing protein aggregation
• If refolding fails, misfolded protein is degraded
GroE Helps Proteins Fold
ATP hydrolysis causes conformational (and therefore local environmental) changes in the interior of the "barrel"
PepJde Bonds in Proteins Are Almost Always Trans
A cis peptide bond brings the two Cα's closer together.
Cis-Trans Peptide bonds
• Most pep:de bonds are trans—R groups opposite each other.
• In proline, the side chain is "tied back" to the backbone N atom.
• Cis proline peptide bonds are found in proteins, particularly at
• But, big energy barrier to rotation around the peptide bond, so
cis proline peptide bonds are formed very slowly.
• Thus, spontaneous formation of cis proline peptide bonds can
slow down protein folding.
Peptidyl-Prolyl Isomerases (PPIs)
• PPIs catalyze the interconversion of cis/trans proline peptide bonds and speed up folding of proteins that contain cis-proline bonds
• All organisms contain PPIs. Humans have 16 PPIs in one structural family + 2 in other families.
Disulfide Bond Isomerases
• Protein Disulfide Isomerase (PDI) catalyzes the formation and breaking of disulfide bonds as protein fold
• This allows correct formation of disulfide bonds in the fully folded state
Proteins may be modified post-translationally
• Just making and folding the polypeptide chain does not necessarily produce the active form of a protein. Many additional modifications of amino acids may occur.
• There are more than 200 types of post-translational modifications known, and more are being discovered.
• Post-translational modifications can have major effects on the properties of proteins.
• As a result of post-translational modification, a single gene can produce a family of proteins with very different properties.
Some examples of post-translational modifications of proteins
• Addition of a co-factor or prosthetic group - e.g., heme in hemoglobin
• Glycosylation—covalent attachment of sugar residues
• Many modifications are reversible, and can be regulatory.
-glycosylation is covalent attachment of oligosaccharide (chains of sugars) to amino acid R groups - usually, Asn, Ser, or Thr
-found in eukaryotic proteins; common in secreted or extracellular proteins
-the modifying enzymes for attachment of sugars to proteins are in the endoplasmic reticulum (ER) & Golgi apparatus
-disulfide bonds in extracellular portion of this protein, but reduced cys residues in the cytoplasmic portion
-The 3° (and 2°) structures of proteins are held together by weak, noncovalent interactions. A protein will "denature'' if these interactions are disrupted
-Upon denaturation, hydrophobic regions of the proteins are exposed and most proteins will aggregate & precipitate (e.g. a boiled egg)
-this sort of denaturation is usually irreversible
-Denaturation can be induced by: heat, chaotropic agents (urea), extremes of pH, strong detergents like sodium dodecyl sulfate (SDS), some organic solvents
Gel Electrophoresis (2)
• Proteins are charged molecules
• Charged molecules will move in an electric field
• Polyacrylamide gels provide a convenient matrix to "sieve" the protein molecules as they move in an electric field
- Polyacrylamide Gel Electrophoresis (PAGE)
- Movement through gel depends on charge and
shape of molecule.
• This is "native" or "non-denaturing" PAGE
• For electrophoresis of DNA, we saw that the movement of DNA through the gel during electrophoresis was inversely proportional to the log of its MW
• But, in order for movement of macromolecules to have this relationship to MW, the molecules must have constant charge to mass ratios.
• This is true for DNA, but not for proteins
• Protein charge is dependent on its amino acid sequence.
• In order to create a constant charge-to-mass ratio and a constant "shape," proteins can be dissolved in the strong detergent, sodium dodecyl sulfate (SDS) and treated with a reducing agent to break disulfide bonds (& prevent disulfides from re-forming).
• Most proteins bind 1-2 SDS molecules per amino acid. Proteins with bound SDS are unfolded, denatured, "random coil" molecules that are ~uniformly negaUvely charged
• The protein sample is denatured by heating in the presence of SDS and β-mercaptoethanol (BME).
• BME, a reducing agent, will reduce -S-S- bonds (if present) to -SH, and prevent them from forming if not originally present.
• Multi-subunit proteins are converted to their component peptide chains by this treatment.
Sodium dodecyl sulfate (SDS)
a detergent; an amphipathic molecule. Also known as sodium lauryl sulfate—look on your shampoo bottle or toothpaste tube
Resolving Proteins by SDS- PAGE
Migration of proteins is inversely proportional to log MW
An SDS-PAGE Gel
• The dye, Coomassie Blue, binds tightly to protein.
• SDS-PAGE separates proteins based on their size
• Migration of proteins is inversely proportional to log MW
Isoelectric Focusing (IEF)
• For any given protein, the pH at which its net charge is zero is its isoelectric point (abbreviation: pI).
• At its pI, a protein has a net charge of zero and will not move in an electric field.
• On an isoelectric focusing gel, a protein will migrate up or down the pH gradient until it comes to a location at which the pH equals its pI, and then stop moving
pI Is the pH at Which a Protein Has a Net Charge of Zero
-potential charged groups in proteins (in ~order of pka): C-terminus, Asp, Glu, His, N-terminus, Cys, Lys, Tor, and Arg
• It is difficult to resolve complex mixtures of proteins by SDS-PAGE alone.
• Isoelectric focusing (IEF) and SDS-PAGE can be combined to allow high resolution separation of complex protein mixtures.
• First dimension: IEF (doesn't depend on size of the protein, only its pI).
• Second dimension: SDS-PAGE (depends only on size of the protein, not its pI).
2 Dimensional Polyacrylamide Gel Electrophoresis
-2-D PAGE of proteins associated with the cell membrane of the organism that causes Lyme disease
-Many more individual proteins can be resolved on this 2-D gel than on a single SDS-PAGE or IEF gel alone
the study of large groups of proteins. The "proteome" - all of an organism's proteins
• All the proteins in the individual spots on the previous 2-D gel can be identified:
- Pick spots, then digest with trypsin (a protease that cuts only aper Lys and Arg residues). This can be done roboUcally.
- Mass spectrometry can determine the masses of all the pepUdes produced from each spot very precisely
- The DNA sequence of the genome of the organism is known, so the computer can compare the masses of the pepUdes from each spot with masses of the pepUdes expected from gene's coding region.
- The gene encoding each spot can then be idenUfied.
The complete DNA sequence of an organism. Humans have about 20,000 genes (but much more DNA).
The complete set of RNA molecules transcribed from a genome. The size of the human transcriptome unknown, but very large.
The complete set of proteins. (About 100,000 proteins in the human.)
The complete set of small molecules found in a cell
• To study individual proteins in detail you need relatively large amounts (milligrams) of pure proteins.
• Goal of purification is to obtain a pure, undenatured protein from a mixture that may contain hundreds or thousands of different proteins.
• Various techniques are used to separate proteins based on their properties; usually chromatography, based on one of the following:
- Specific Binding Affinity
• But you must have an assay to identify your protein & follow it during the purification process
Separation Based on Charge: Ion Exchange Chromatography
• In this example (Cation Exchange Chromatography), a positively charged protein sticks to the negatively charged beads, while the negatively charged protein does not.
• The positively charged protein can be eluted from the column by increasing the concentration of salt in the elution solution.
• (Anion Exchange Chromatography uses positively-charged beads and binds negatively charged proteins.)
Separation Based on Size: Size-Exclusion or Gel-Filtration Chromatography
• The beads contain aqueous pores.
• Smaller proteins can "explore" the spaces, slowing their progress through the matrix.
• Larger proteins are excluded from some or all of the spaces; they elute more quickly.
Separation Based on Binding Affinity: Affinity Chromatography
Idea: If you could put a tag on a protein that makes it bind Ughtly to something that proteins do not normally bind to, you could separate the tagged protein from all the other proteins in a mixture of proteins.
Use molecular cloning
SDS-PAGE can be used to follow purification of a protein
Importance of Overexpression in Protein Purification
• Many proteins in the cell are present at very low concentration.
• Important proteins may comprise 0.1% or less of the total
protein in the cell
• By cloning the gene encoding the protein you wish to purify, you can usually greatly increase the level of the protein.
• Plasmids may be present in many copies, amplifying the gene encoding your protein. It's much easier to purify a protein that is 10% of the total protein than one that is 0.1%.
• Using cloning, proteins can be expressed in and purified from organisms that are convenient for purificaUon. A human
protein might be expressed in E. coli, for example, making purification much easier.
In preparation for polyacrylamide gel electrophoresis, proteins are frequently treated with sodium dodecyl sulfate (SDS). Which of the following terms accurately describes this chemical compound?
Strong ionic detergent
Which of the following methods is effective for separating proteins on the basis of their net charge?
- Ion-exchange chromatography
In ion-exchange chromatography, an extract of proteins is poured though a column containing beads coated with charged chemical groups. If the beads are coated with negatively charged groups, then positively charged proteins will bind, while negatively charged proteins will elute from the column. Adding a weak salt solution will shield weaker charges in the column, which releases some of the weaker-bound proteins. Gradually increasing the salt concentration can cause the stronger-bound proteins to elute into different fractions, each of which is defined by charge strength
How do you elute proteins bound to an ion exchange column?
By increasing the salt concentration of the eluting buffer
Which of the following techniques can be used to separate proteins by size and shape?
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