Quiz #2 Biochemistry: Chapters 5, 6, and 10

give some examples of roles of proteins in biological systems

structure, catalysts (enzymes), signal transduction, regulatory, mobility, transport

what determines a proteins specific role

their distinctive structures derived from their amino acid sequence (primary structure)

how do amino acids join?

• Join with a peptide bond (covalent bond) between the carboxyl group and the amino group

• water is lost within the process

peptide

• short polymer of amino acids

• dipeptide, tripeptide, 12-20: oligopeptide, many: polypeptide

proteins

• a molecule composed of one or more polypeptide chains

• homomultimer: one kind of chain, heteromultimer: two or more diff types of chains

simple proteins

proteins that consist of normal amino acids only for their biological function

conjugated proteins

proteins that utilize accessory molecules or chemical groups to carry out their function

prosthetic group

non protein part that is essential to the proteins function

orthologous

proteins from different species with similar sequence and function (common ancestor, for example, hemoglobin in human and cow)

paralogous

proteins from the same species with similar sequence (gene duplication, example: alpha and beta subunits of hemoglobin)

cytochrome c

• the electron transport protein found in the mitochondria of all eukaryotic organisms

• varies between 103 and 112 amino acids in different species

• variation in amino acids between species increases as the phylogenetic difference increases

how do you calculate the net charge and pI of peptides/proteins

consider the n terminus (1st amino acid), and the c terminus (last amino acid) and the side chains of all amino acids between n and c termini

name some common purification techniques

salting in and out, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration, etc.

explain salting in and out

• salting in: a low concentration of metal salt generally increases protein solubility

• salting out: high metal salt concentrations reduce protein solubility

• proteins are most insoluble at their isoelectric point

• different proteins have different solubilities and therefore require different concentrations of salt to precipitate

• when it is no longer soluble in the high salt solution, to precipitates (salting out)

explain dialysis of proteins

• allows small molecules and ions to pass through a semipermeable membrane

• the macromolecular solution is placed in a semipermeable membrane bag and immersed in a bathing solution

• diffusible solutes in the dialysis bag equilibrate across the membrane

explain the chromatographic techniques of protein purification

• takes advantage of relative differences in the physical and chemical characteristics of an amino acid, peptide, or protein

• the molecule of interest flows through a medium with two phases (solid-liquid, liquid-liquid, or gas-liquid) and partitions between them

explain ion-exchange chromatography

• depends on the net charge of the protein

• the proteins net charge will depend on the pI of the protein and the pH of the solution in which the protein is (pI of a protein is the pH at which its net charge is 0)

• remember: pH> pI overall charge is neg. , pH< pI overall charge is positive

explain the types of protein chromatography

• ion-exchange: postivie or negative charged ions

• hydrophobic interaction: polarity or hydrophobicity of molecules

• affinity: differential affinity of one molecule for others

• gel filtration, permeation or size exclusion: molecular size of the molecules

explain gel filtration or size exclusion chromatography

• proteins are separated based on their size

• column media are composed of beads on varying pore sizes

• larger molecules are excluded from the gel beads and emerge from the column sooner than smaller molecules, whose migration is retarded because they can enter the beads

explain affinity chromatography

• based on the ability of the protein to bind to a ligand

• a small molecule (ligand) is immobilized on a matrix

• the protein of interest is then passed through the column, which binds to the target ligand, while other proteins pass through without binding

explain proteins analysis by SDS- PAGE, specifically SDS

• SDS molecules disrupt non-covalent interactions that stabilize the tertiary ( or quaternary) structure and denature the protein

• SDS wraps itself around the protein backbone and proteins become rod-like

• SDS binds at a constant weight ratio of 1.4 g SDS/g of polypeptide through hydrophobic interactions

• this way, proteins will have a uniform charge

• larger proteins bind more SDS than smaller molecules

explain protein analysis by SDS- PAGE, specifically PAGE

• the electrophoretic mobility of shift assay (EMSA) is performed on a polyacrylamide gel (PAGE)

• an electric field is applied to the gel to separate proteins

• anions = anode (+), cations= cathode (-)

• migration is a function of size (smaller go farther than bigger)

how do you measure peptide and protein concentration

• proteins and peptides absorb at 215 nm (peptide backbone) amd at 280 nm (specific aromatic side chains)

• calculate measuring absorbance at 280 nm and using Beer- Lambert law

• A= ecl

explain analysis of peptides and proteins using mass spectrometry (MS)

• exploit differences in the mass-to-charge ratio (m/z) of ionized atoms or molecules to separate them from each other

• evaporate and ionize the molecules in a vacuum

• separate the ions in space and/ or time based on their m/z ratio

• measure the number of ions with specific m/z ratios

explain how we analyze peptides and proteins using sequencing

2 ways:

• direct amino acid sequencing

• sequencing the corresponding DNA in the gene

what are the 6 steps for determining the amino acid sequence of a protein

1. separation of the polypeptide bonds (if a heteromultimer)

2. cleavage of the disulfide bridges

3. analysis of N and C terminals

4. fragmentation of the polypeptide chain

5. reconstruction of the amino acid sequence

explain step 1 : separation of polypeptide chains

• polypeptide separation is achieved with 8 M Urea, 6 M guanidinium HCl, extreme pH or high salt concentration

• if the protein is a heteromultimer, chromatographic methods that can then separate the different chains for individual sequencing

explain step 2: breaking of the disulfide bonds

2 options:

• performic acid oxidation

• reduction with mercaptoethanol (BME) or dithiothreitol (DTT)

followed by treatment with alkylating agents that modify the -SH groups to prevent recombination of disulfides

explain step 3: analysis of the N- terminus

• Edman degradation allows sequential identification of a series of residues starting at the N- terminus

• chromatographic techniques are used to identify the PTH- derivative

explain step 4: analysis of the C- terminus

• carboxypeptidases cleave the amino acid residues from the C- termini of polypeptides

• different carboxypeptidases cleave different amino acid residues

carboxypeptidase A : all residues except Pro, Arg, Asp, Glu, and Lys

carboxypeptidase B : Arg and Lys residues only

carboxypeptidase Y: any residues

• c terminal residue is removed enzymatically with the carboxypeptidase first and then reacted with phenyl isothiocyanate to produce the PTH derivative

explain step 5: fragemnataion of the polypeptide

• enzymatic fragmentation: trypsin, chymotrypsin, clostripain, staphylococcal proteases

• chemical fragmentation: cyanogen bromide

• trypsin class on the carboy side of Arg and Lys

• generates a set of peptide fragments having either Arg or Lys at their C- termini

• chymotrypsin cleaves on the carboxy side of the Phe, Try, and Trp

explain step 5 : fragmentation of the polypeptide

• cyanogen bromide (CNBr) reacts with methionine residues and cleaves the peptide bond between the Met and the next amino acid

explain step 6: construction of the complete sequence

• compare the sequences of fragments obtained from two or more cleavage procedures

• sequence all the peptides produced ( usually by Edman degradation)

• the goal here is to find overlapping sequences

• align peptides from different fragmentations to reveal the overall amino acid sequence

  
  

name some other biomolecules that proteins interact with:

• other proteins

• nucleic acids (DNA and RNA) to form nucleoprotein complexes

• small molecules (metabolites)

• metal ions

explain the strength of protein-ligand interactions: dissociation constant (Kd)

• Kd=[P][L]/[PL] —> v= [L]/ (Kd+ [L]) where v is the fractional saturation of P with L, [PL]/([P]+[PL])

• when v= 0.5, Kd = [L]

• i.e., Kd is equal to the concentration of L when half the protein is bound to L

• smaller kd indicates better affinity of ligand for protein (typical range - 10^-3 M to 10^-12 M)

name some of the overarching principles link the 3 dimensional structures of proteins

• function depends on structure

• structure depends on the sequence and non covalent forces

• number of protein folding patterns is significant

what bonds form the primary structure of a protein?

covalent bonds

explain hydrogen bonding in proteins

• both between atoms in the peptide backbone and between side chains

• usually on the surface of proteins, but can also exist on the interior

• 12-30 kj/mol

explain ionic interactions in proteins

• generally located on the surface of proteins

• 20 kj/mol

explain hydrophobic interactions in proteins

• primarily found in the interior of proteins (drives protein folding)

• <40 kj/mol

explain van der waals interaction

• van der Waals interaction are ubiquitous (appearing everywhere at the same time)

• <0.4 - 4 kj/mol

primary structure

the amino acid sequence

secondary structure

• organization of amino acids into structures through hydrogen bonds

• amide H of one peptide group and the carbonyl O of another

• allows the protein to form regular structures

tertiary structure

3- dimensional organization of secondary structures

quantenary structure

organization of several proteins, subunits

explain the amide plane for higher-level protein structures

• the peptide bond is planar

• rotation is allowed around 2 bonds

• the bond linking the alpha carbon with its carbonyl carbon and the bond linking the alpha carbon with the amide nitrogen

• the angle for the alpha carbon and the carbonyl carbon is denoted by psi, and the alpha carbon and amide is phi

what are some unfavorable combinations of phi and psi?

• 0 and 180 for either

• 0 and 0

explain alpha helices in secondary structures

alpha helices:

• upiquituous component of proteins

• stabilized by H- bonds

• phi: -60, psi: -45

• residue n forms h-bond with the n+4 residue

• residues per turn= 3.6

• each amino acid extends 1.5 Å along the helix axis

• therefore rise per turn= 3.6 * 1.5 Å = 5.4 Å

• each peptides bond possess a dipole, meaning the alpha helix itself has a significant dipole moment

• In a typical 𝛼-helix of n residues, there are n-4 hydrogen bonds

explain beta strands and sheets in secondary structures

• beta strands form when amino acids adopt phi of -120 and psi of 120

• beta strands do not exist standalone because the can’t be stand alone

• to stabilize, another stretch amino acids should form a beta strand with which the first strand can interact

• R groups of consecutive amino acids are on opposite sides of the sheet

• beta strands are usually represented by arrows with the arrowhead indicating the direction from N- terminus to C- terminus

• at least 5 strands in parallel are needed to be stable whereas antiparallel sheets are stable with 2

• parallel rise per residue= 3.25 Å, antiparallel rise per residue= 3.47 Å

what connects alpha helices and beta sheets?

• loops

• can be divided into structured and unstructured

explain beta turn (or bend) formation

• the peptide chain forms a loop with the carbonyl oxygen of one amino acid forming a hydrogen bond with the amide hydrogen of the residue 3 residues down the chain

• 4 resides are required for a beta turn

explain the tertiary structure of proteins

• the arrangement of a single polypeptide chain in 3-dimensional space

• proteins fold to form the most stable structure possible, that arises from: the formation of many intramolecular hydrogen bonds and the reduction in the surface area accessible to the solvent that occurs upon folding

• proteins are usually a mixture of hydrophilic and hydrophobic elements

• the hydrophobic elements usually cluster together in the folded interior of the protein

what are the 3 main classes of biological proteins?

• fibrous proteins: simple, linear structure, insoluble in water

• globular proteins: spherical in structure, soluble in water

• membrane proteins: hydrophobic exterior, insoluble in water

describe fibrous proteins

• consist of polypeptide chains organized along a single axis, producing long fibers

• tend to be mechanically strong and play a structural role in nature

• ex. alpha keratin, fibroin, collagen

explain key characteristics of alpha keratin

• predominant constituents of claws, fingernails, hair, etc. in mammals

• alpha helical segments dominate their structure

• the sequence consists of 311-314- residue- long alpha helical rod segments capped with non-helical N and C termini

• the primary structure of helical rods consists of 7 residue repeats (a-b-c-d-e-f-g)n, where a and d are non polar

• this structure promotes helix association to form coiled coils

explain key characteristics of fibroin

• found in silk fibers and bird feathers

• form extensive beta sheets with an alternating sequence Gly-Ala/Ser-Gly-Ala/Ser...

explain key characteristics of collagen

• principle constituent of connective tissues

• very high proline content

• 1 out of 3 residues is glycine, forming long stretches of the polypeptide chain consisting of Gly-Pro-Pro repeats

• the unusual amino acid composition of collagen is not suitable for alpha helices and beta sheets

• it is ideally suited for the collage triple helix: three intertwined helical strands

explain globular proteins

• exist in an enormous variety of 3D structures

• contain large amounts of alpha helices and beta sheets folded into a compact structure

• both polar and non polar interactions stabilize the tertiary structure

• include enzymes and the proteins involved in immune and signaling responses

describe bovine ribonuclease A

• example of a globular protein

• The space between the helices and sheets in the interior of the protein is tightly filled with residues that have mostly hydrophobic side chains

• most polar side chains face the outside on the structure and interact with water

how does water play a critical role in globular proteins structures?

• there are usually several water molecules per amino acid residue

• the polar backbone and side chain groups on the protein surface make h- bonds with solvent water

• relatively few water molecules are found inside the protein

what are the protein groups based on secondary structure arrangements?

• All alpha groups, in which alpha helices predominate

• all beta groups, in which beta sheets predominate

• alpha/beta proteins, in which alpha helices and beta sheets are intermingled

• alpha + beta proteins, which contain separate alpha helical and beta sheet domains

explain the quantenary structure of proteins

• used in cases where proteins are composed of two or more subunits

• stabilized by hydrophobic interactions, hydrogen bonding, electrostatic interactions, and, in certain cases, disulfide covalent bonds and interactions with metal ions

how does a protein fold into its functional form: randomly or with some pattern?

• the amino acid sequence contains all the information required to fold a polypeptide into its native structure

• study of denaturation and renaturation of proteins in the 50’s confirmed this

• solutions of ribonuclease were treated with: urea (unfolded the protein) or BME (reduced the disulfide bridges)

explain levinthals paradox

• a typical protein can adopt so many conformations that it does not have enough time to reach its most stable state by sampling all possible configurations

• therefore proteins must fold by specific folding pathways

explain folding tendencies and patterns in globular proteins

globular proteins adopt the most stable tertiary structure possible by:

1. satisfying the constraints inherent in their own structure

2. folding to bury the hydrophobic side chains

• polypeptide chains tend to twist slightly in a right-handed direction

explain natural proteins being found multimeric

• proteins with 2 or 4 subunits predominate in nature (dimeric or tetrameric forms)

• The typical Kd for two subunits: 10−8 to 10−16 M

• These values correspond to energies of 50 to 100 kJ/mol at 37° C

• entropy loss due to association: unfavorable

• entropy gain due to burying of hydrophobic groups: very favorable!

describe intrinsically unstructured proteins (IUP’s)

• many proteins exist and function normally in a partially unfolded state

• adopt well defined structures in complexes with their target proteins

• are characterized by an abundance of polar residues and a lack of hydrophobic residues

what is an example of an IUP?

• p53 is a tumor supressor gene

• consists of: an N terminal disordered domain of 93 residues, a central DNA binding domain of 200 residues, and a C terminal disordered domain of 100 residues

• n terminal binds to over 40 proteins, c terminal binds to over 50 proteins

denaturation

• the loss of protein structure and function

• weak, non covalent forces maintain the secondary, tertiary, and quantenary structures

• a variety of environmental stressors can disrupt these forces

explain denaturation of proteins by heat

• as a protein is heated, it maintains its native state until it approaches its unique melting temperature (Tm)

• above this temp., the structure unfold and function is lost

explain denaturation of proteins by chemicals

• proteins can be denatured by: acids, bases, detergents

• denaturation involves disruption of the weak forces that stabilize proteins

• the covalent bonds are not affected

explain the central dogma

• the flow of genetic information: DNA to RNA to protein

• unidirectional and irreversible

• replicate, transcribe, translate

nucleotides

• the building blocks for nucleic acids

• nucleic acids are polymers of nucleotides, the nitrogenous base can vary from nucleotide to nucleotide

pyrimidines

• six-membered, heterolytic aromatic rings

• planar

• cytosine, thymine, and uracil

purines

• consists of two heterolytic rings (six and five membered)

• slight pucker between the pyrimidines and imidazole portions

• adenine and guanine

tautomers

• constitutional isomers (two molecules with the same molecular formula) but with different connectivity

• can interconvert in a rapid equilibrium

explain what tautomers are dominant at physiological pH

• keto tautomers of thymine and guanine predominate

• amino tautomers of cytosine and adenine predominate

explain nucleobases UV light absorption

• purines and pyrimidines strongly absorb UV light because of their aromaticity

• at or near 260nm

describe the structure of a nucleoside

• nucleobase plus a pentose sugar

• nucleosides are compounds formed when a base is linked to a sugar via a glycosidic bond (C-N)

• can have a ribose or deoxyribose sugar

• OH vs. H

• 2 OH found in DNA

name some of the most common naturally occurring ribonucleosides

• pyrimidines: cytidine, thymidine, uridine

• purines: adenosine, guanosine

describe the structure of a nucleotide

• a nucleoside plus a phosphate group

• most nucleotides have 5’ phosphate groups

• the nucleotides can have multiple phosphate groups

what role do nucleoside 5’ triphosphates play in cells?

• indispensable agents in metabolism because their phosphoric anhydride bonds are a source of chemical energy

• the cyclic nucleotides are signaling molecules and regulators of cellular metabolism and reproduction

examples

• ATP is central to energy metabolism

• GTP drives proteins synthesis

• CTP drives lipid synthesis

explain what nucleic acids are composed of

• linear polymers of nucleotides linked 3’ to 5’ by phosphodiester bridges

• they are formed as 5’ nucleoside monophosphate are added to the 3’ OH group of the preceding nucleotide

• you read the chains from the 5’ to the 3’ end

explain the structure of DNA

• DNA consists of two polynucleotide strands (helices)

• the strands are antiparallel

• they are held together via the hydrogen bonds of the base pairs

• A=T two h-bonds, g-c three h-bonds

• the polar sugar-phosphate backbones of the chains are on the outside

who found the structure of DNA?

Watson, Crick, Wilkins, Franklin

explain the path to discovery of DNA

• Chargaff examined the base composition of various DNAs but didn’t explain the implications

• the four bases did not occur in equimolar amounts

• number of purines is always equal to the number of pyrimidines

• rosaland franklins x ray fiber diffraction data were crucial

• crick showed that it was a helix

• Watson found that the base bair h-bonding was specific

explain what determines base paring

• large bases pair with small bases

• the A-T base pair and the G-C base pairings have virtually identical dimensions

explain Hoogsteen base pairs

• bases do not always follow Watson-crick base pairnigs

• the purine n7 is a h-bond acceptor and can thus form h-bonds with other donors

• the functional groups of A and G that participate in wanton-crick base paring remain accessible in hoogsteen base pairs

explain non-canonical paring

• bases can form triples such as TAT and CGC

• each purine interacts with one of its pyrimidine partners via a hoogsteen base pairing and the other through a Watson-crick base pairing

what structures can guanine and cytosine nucleobases form?

• four guanine bases can form a quadraplex structure due to hoogsteen h-bonds

• cytosines can form c+:c pairs at lower pH

explain the difference between DNA and RNA building blocks

• DNA contains 2-deoxyribose instead of ribose in RNA

• DNA contains thymine instead of uracil in RNA

• ribo and deoxyribonucleotides form different conformations

explain the structure of naturally occurring RNA

• usually single stranded

• more degrees of freedom per nucleotide meaning they have many more conformational possibilities than DNA

• however, RNA is rich in double stranded regions that form when complementary sequences within the same chain for intrastrand base pairs
  
  

explain why RNA is less chemically stable than DNA

• RNA is susceptible to alkaline hydrolysis

• OH- acts as a nucleophile and removes a proton from the 2’ OH group

• then the 2’O- attacks the phosphodiester bond

• leads to the cleavage of the sugar phosphate backbone

explain nucleases and their role

• enzymes that hydrolyze nucleic acids

• these enzymes are technically phosphodiesterases because they catalyze the cleavage of phosphodiester bonds

• because each phosphate in a nucleotide backbone is involved in two phosphodiester linkages, cleavage can technically occur on either side of the phosphorus

name some of the types of nucleases that cleave DNA and RNA

• endonuclease: cleave within an olionculeeotide chain

• exonucleases: cleave at the end of a polynucleotide chain, can cleave at either the 5’ or 3’ end

• Rnase: nuclease specific for RNA

• DNase: nuclease specific for DNA

explain how restriction enzyme (DNase) cleaves dsDNA

• restriction enzymes are enzymes purified from bacteria that can cleave dsDNA

• type 1: require ATP to hydrolyze DNA, catalyze chemical modification of DNA, cleave DNA randomly

• type 2: do not require ATP but require Mg2+, do not modify the DNA, cleave at specific sites depending on the DNA sequence

• type 3: require ATP, recognize specific nucleotides and cleave at or near the site, catalyze chemical modification of DNA

explain why type 2 restrictive enzymes are very handy in biology

• widespread use as a tool in cloning

• recognizes a specific DNA sequence and cuts the phosphate backbone at specific sites at or near DNA sequence

• recognition site is typically 4-6 bp in length

• about 3000 restriction enzymes have been characterized

how do we name restriction enzymes

• first is a capital letter denoting the genus of the organism of origin

• the next two letters are an abbreviation of the species

• if there is more than one strain, then the next will be a capital letter to denote the strain

• last there is a roman numeral to indicate the order of purification from a given species (strain)

example: E means genus Escherchia, co means species is coli, R means the strain is RY13, I means first identified from this strain

EcoRI

explain agarose gel electrophoresis

• very common DNA analysis method

• the gel is submerged in the electrophoresis buffer

• DNA is loaded into the wells of the agarose gel

• agarose gel separates DNA as a function of size

• larger DNA fragments move more slowly through the gel than the smaller fragments

how do we analyze agarose gel electrophoresis

• markers: commercially available solutions contain DNA of different lengths and amounts to be used as standards

• DNA bands can be visualized by: labeling the DNA ends with 32P (radioactive) and exposing it to photographic film or a phosphor imaging plate, which is highly sensitive, ethidium bromide: intercalates in DNA, and fluoresces when exposed to UV light, DNA is not affected by running on a gel: it can be extracted and used (purification)