Biochem exam 1

Predicting 3d structure

1 AA sequence will have 1 structure, but it is often hard to predict if 2 sequences have high similarity in aa sequence, you have a good chance at predicting the 3d structure based on one another.

A:T and C:G rations

1:1

VDW interactions

2 atoms that are close enough to induce complementary asymmetric sharing of electrons Ability to attract electrons are different, causing VDW Weak, but alot of VDW increase the total strenght 2 atoms close enough so that the transient asymmetry in Electron distribution from 1 atom induces complementary asymmetry in neighboring atoms instant assymetry of electrons sharing between atoms

reverse transcriptase

2 copies of single stranded viral rna molecules in a protein capsid 2) infection of the cell binds viral RNA to reverse transcriptase 3) reverse transcriptase forms the completemtary dna to the rna 4) after separation and annealing, 2 strands of dna bind together and embeds itself into the host DNA, infecting the host

Disulfide bonds

2 cysteines can be cross linked and oxidized to form a disulfide bond, resulting in one cystine example is insulin, that has multiple disulfide bonds formed with both inter chain and intra chain

primary strucure

AA linked by peptide bonds to form polypeptide chains linear polymer contains polarity, always begins with alpha amino group and ends in alpha carbonyl group N=# of AAs #peptide bonds=N-1 polypeptide chain is able to form many h bonds h bond donor is the NH group h bond acceptor is C=O group

Edman's Degradation

AA sequence can be determined by Edman degradation, the protein is exposed to phenyl isothiocyanate (PTH) which reacts with the N terminal AA to form a PTH derivative. the PTH AA can be released without cleaving the remainder of the protein, and the degradation is subsequently repeated, then HPLC is used to identify AAs gene sequence cant express amounth of protein function in different conditions cant just sequence the genes can get expensive for longer proteins

FUnctional groups in proteins

AA's differ by functional r chain different chains have different capabilities and reactivities

Tree of Life

All organisms can be placed in 1 of 3 domains cant take ancestor and turn into another species (monkey cant be turned into a human) 3 groups Eukarya: multicellular organisms, well defined nucleus Bacteria: unicellular, lacks nucleus Archaea: Unity makes this approach possible, but each organisms has needs that are solved in biochemical processes

Immunology investigating proteins

Antibodies to specific proteins can be generated an antibody in the protein synthesized in response to the presence of a foreign substance called an antigen the antibody recognized a particular structural feature on the antigen called the epitope Identify antibody in an Assay

Indirect ELISA

Antigen in a mixture is coated at the bottom Bodily fluid is added, specific antibody binds to antigen (still invisible) Enzyme linked antibody binds to specific antibody(2ndary antibody recognizes part of the first antibody and binds with it with an enzyme linked) SUbstrate is added and converted by enzyme to crease a colored product, rather of color formation is proportional to amount of specific antibody

lactate dehydrogenase assay

Assay for lactate dehydrogenase is based on the fact that a product of the reaction, NADH, can be detected spectrophotometrically the faster that it generates an absorbance at 340, the more enzyme activity examine how much light is absorbed by solution Lactate is transformed into pyruvate measure activity of enzyme by how much of the product is formed

Zwitterion

At a neutral pH, AA's exist in their zwitterion form (dipolat) this means they contain a negative and positive change At a low pH of of 1.0, the carboxylic acid is protonated, this is a positively charged species as pH increases to about 2, the carboxylic acid loses its hydrogen, forming zwitterion at pH 9, low [H] pulls H off amino group, forming negative species

intrinsically unstructured proteins (IUPs)

Can assume diff. structures & interact w/ diff. partners. Important in signalling & regulatory pathways. -LACK discrete tertiary structure under physiological pH inherently unstrcutures and can exist in multiple conformations no defined structure under physiological ph until they combine with molecules metamorphic proteins exist in an ensemble of structures of approximately equal energies in equilibrium

information the AA sequence provides

Can be compared to identify similarities comparison of the sequence of the same protein from different species yields evolutionary information AA sequence searches can reveal the presence of internal repears sequence info can identify signals that determine the location of the protein or processing signal Sequence info can be used to generate antibodies for the protein AA sequence can be used to generate DNA probes specifically for the gene encoding the protein

Ultracentrifugation

Can be used to examine proteins. When subjected to a centrifugal force, the rate of movement of the particle is defined by the sedimentation coefficient, s Centrifugal force>20000g Separation relates to shape

cation exchange vs anion exchange

Cation= beads have negative charge anion= beads have positive charge (used less than 50 % of the time)

determining disulfide bonds

Cleaved by the addition of a reducing agent, reformation is prevented by the alkylation of the cysteine residues separate with dithiolthretiol, reduce with iodoacetate produces separate carboxymethalated chains

Genome

Data base of dna, total dna in a cell heart cell and neuron have same genome, very diff

Importance of thermodynamics

Determine conditions in which processes can or cannot take place system=matter within a defined space surroundings= rest of the universe

Key Characteristics of DNA Synthesis

Four deoxynucleoside triphosphates (activated precursors) and Mg2+ are required. 2. A template strand is used to direct DNA synthesis. 3. A primer from which the new strand grows must be present(contains a 3'-OH that can create the first phosphodiester linkage). 4. Many DNA polymerases have nuclease activity that allows for the removal of mismatched bases.

Producing monoclonal antibodies

Immortal(dont die fast) cell lines producing A monoclonal cell line is isolated by screening for the antibody of interest antibody must be eurkaryotic cell human cells have contact incubation they dont aggregate, cancer cells will grow out of comtrol

B form DNA

In the cell, the most commonly seen form of the DNA double helix is called the B form or the Watson-Crick helix

Key properties of proteins

Linear polymers composed of Amino acids will end up being folded into all kinds of 3d figures large macromolecules Wide variety of functional groups that carry out functions some proteins are rigid and other flexible catalyst, transporters, structural, mobility, immunity, and communication roles in the body

Ionic interactions

Occurs between fully charged atoms or molecules E=Kq1q2/Dr E is electrostatic attraction given by coulomb energy q= charge of ataom D=dielectric constant of solveent k=constant r=distance attraction can change drastically in different environments

Protein folding cooperative process

Proteins fold by progressive stabilization rather than random search Cumulative selection: partly correct folding intermediates are retained because they are slight more stable than unfolded regions

gel electrophoresis

Proteins migrate in an electrical field w/ velocity directly proportional to electric field strengh, charge of protein, and inversely proportional to friction separation by size, not purification but characterization. treat protein by denaturings, code for protein and check for charges. charge to mass ratio becomes constant when unfolded overcome what charges you have buring into affect. difference by ionizable groups wont count

Example of 2D electrophoresis

Raw tissue from healthy and cancer patient compare pattern and identify differences patters of proteins have been altered by caner cells if this changes can help diagnose things cancer cells alters enzymes in cells to sustain cancer

High Performance Liquid Chromatography (HPLC)

Resolving power of any chromatographic techniques related to the number of potential sites of interaction between the protein and the colum beads. very fine beads allow more interactions and thus a great resolving power, but flow rates through such column are slow. HPLC uses very fine beats in metal colums and high pressure pumps to move the liquid through the column. because of the high number of interaction sites, the resolving power of HPLC is greater than normal columns each protein represents protein that has been separated not good to use high conc of urea to denature or an organic solvent. must be aqueous and suitable ph (6-8)

Why is DNA in the form of a double Helix?

The function of DNA is to store and pass on info, so they have to be able to copy itself which can only be done with a double helix Due to their chemical natures, complimentary helices will spontaneously form double helix

coimmunoprecipitation assay

When a monoclonal antibody against a specific protein is available, it is also possible to determine the binding partners of that protein under cellular conditions. • In co-immunoprecipitation, a cell extract is incubated with the antibody; then agarose beads coated with an antibody-binding protein (e.g., Protein A) are added to the mixture. • The antibody-bound protein complex can be separated from all other cellular components. • The identity of other bound protein partners can then be probed by other methods.

Protein purification

essential first step in understanding the their function can be purified on the basis of size, charge, functions to bind, affinity to certain molecules

twisted Beta sheets

h bonds conformational angle beta sheets usually arent flat they are never a single arrow have to show h bonds between arrows A protein rich in Bets sheets will have many arrows representing the beta sheets they can form a barrel called beta sheets because they are stabilized by this type compact nature of proteins is in part due to polypeptide ability to make turns allow to make abrupt turns called beta turns, usually found on the surface of a protein beta turns are typical of Gly and Prol because Gly is easy to fit in anywhere and Pro for steric reasons r chains on outside get to interact with polar solvents and macromolecules on the outside surface

Main purpose for synthesizing peptides

1. As antigen to produce antibodies 2. to isolate receptors (synthetic peptides containing formyl methionine or fMet) have been used to identify receptors on white blood cells 3. as drugs 4. to understand protein folding

zonal centrifugation

A density gradient is formed in a centrifuge tube and a mixture of proteins in solution is placed on top after centrifugation is complete, a small hole is made in the bottom of the tube and portions of the gradients are collected. These portions are analyzed for protein conc. enzyme activity, or other biochemical characteristics they get separated by sedimentation coefficient use a gradient rather than a homogenized buffer low salt and high salt mixture if there is no color difference, collect in separate tubes and test via assays

DNA

A fundamental biochemical feature common to all cellular organisms is the use of DNA for the storage of genetic information Directionality of DNA combined with base variation creates sequences

Gel filtration chromatography

A glass column is filled with porous beads. When a protein solution is passed over the beads, large proteins cannot enter the beads, and exit the column first. Small proteins can enter the beads and, thus, have a longer path and exit the column last requires that 100-200mM ammonium sulfate salt to prevent nonspecific charge to charge interaction even though resin is pretty much inert collected via fraction collection separation based on size beads are insoluble and pourous

Assay

A test for determining whether the protein of interest is present test that can be used to identify the protein of interest based on its unique function and properties 2 questions answered: is the protein present( done by measuring protein activity) and whats the concentration of the protein if its present (helped calculate specific activity which helps purity

Watson crick base pairs

A-t: 2 H bonds C-G: 3 H bonds sugar-phosphate backbone intertwined together h bonds are strong enough to stabilize structures such as double helix but weak enough to be broken reversibly

Protein folding

AA have different propensities for forming alpha helices, beta sheets and turns preferences can be difficult to determine from sequence alone same sequence can be folding in multiple ways

Protein misfolding diseases

Amyloidoses are diseases that result from the formation of protein aggregates, called amyroid fibrils or plaques protein misfolding and aggregation are associated with some neurological diseases normal protein conformations can exist in forms rich in beta sheets, which are prone to aggregate an abnormally folded aggregate serves as a nuclear to recruit more proteins forming amyloids show when brain cells start dying(Multiple different proteins)

AA have different propensities for forming alpha helices, beta sheets, and turns

Analysis shows some AA's are more common in alpha helices, beta sheets or as turn, however these preferences arent extremely strong, making it difficult to predict secondary structure from primary.

process of creating antibodies

Antigen goes into animal, polyethylene glycol with myeloma cells then select and grow hybrid cells, then slect calls with antibody of desired specificity, then propagate, grow in mass culture or in mouse to produce antibody step 1) inject antigen into mouse wait for it to produce antibody producing plasma cells and then extract spleen cells (high conc of plasma cells) 2) cancer cells are mixed with spleen cells in polyethylene glycol solution and creates hybridoma cell 3) hybridoma cell are screen many times with assays to determine and isolate those cells that produce that antibody of interest

Generating antibodies to specific proteins

Any anibody-producing cell syntehsizes antibodies that recognize only one epitore. each antibody producing cell thus synthesizes a monoclonal entibody any antigen may have multiple epitopes. each immune cells selects one clone of genes to mass produce antibodies to just 1 epitope each cell can make antibodies to attack 1 specific part of the antigen

transmission of hereditary info

Because of the base-pairing rules, the sequence of one strand determines the sequence of the partner strand. • The two strands can be separated and complementary sequences synthesized to generate two identical daughter strands. • Because the two daughter helices have one parent strand and one newly synthesized strand, the replication process is called semiconservative replication.

2 classes of molecules

Biological molecules and metabolites Metabolites are low molecular weight molecules like glucose and glycerol and get chemically transformed in biological processes biological molecules are larger molecules like proteins or nucleic acids members of both of these classes of molecules are common in all living organisms with minor variations EX: proteins that play similar roles in different organisms typically have the same 3-D structure

Peptide bonds

Bonds between amino acids loses water forms amide/carboxamide bond dehydrolysis peptide bonds are thermodynamically unfavored (requires ATP) peptide bonds are kinetically stable peptide bonds are resonance stablizied, meaning they have double bond character which makes it planar and prevents rotation about peptide bond

Christian Anfinsen experiment

Christian anfinsen placed the enzyme ribonuclease (degrades rna) inna solution with either urea or guanidinium and beta mercaptoethanol. urea or guanidinium chloride destroyed its noncovalent bonds such as ionic interactions and h bondsand b mercaptoethanol destroyed its disulfide bonds- denaturing the protein ultimately showed that the information needed to form the 3d active protein lies in its AA sequence. Later experiments generalize this idea that the primary structure determines the confirmation of the proteins

Alpha helix

Coiled structure stabilized by H-bonds TIghly coiled, rodlike structures with the r groups emanating out from the axis all of the backbone CO and NH groups form H bonds except for those at the enx of the helix all found in proteins are right handed H bond scheme- CO bonds with the NH 4 AAs away on Ramachandran plot: left handed helix is very rare, small dot in first quadrant right handed helix is much more common, 3rd quadrant backbone on the inner portion of helix, side chains outer right handed rotates clockwise, left handed rotakes counter clockwise right handed predominates for steric reasons and side chains

Human microbiome

Complex bacteria live in and on our bodies 10x more microbial cells than a human cell they play a key role in health and disease

DNA base pairs with VDW forces

DNA base pairs are optimal VDW distance apart, allowing bases to stack and interact w one another through VDW Allow for bases to be mostly hydrophobic and aromatic

DNA polymerase

DNA polymerase catalyzes phosphodiester-bridge formation. takes instructions from templates nucleophilic attack of 3-OH to innnermost phosphate, releases pyrophosphate and forms phosphodiester bond

folding funnel

Depicts thermodynamics of protein folding maximum entropy on top with minimal structure folded protein exist on the bottom

Genomics and proteomic methods are complementary

Determining AA sequence is difficult genomic techninques that reveal the DNA sequence of the gene encoding a protein also allow determination of AA sequence Knowing the DNA sequence knows the AA sequence

Antibodies

Different domains heterotetramer domains are globular different functions loops are the antigen binding regions almost any cell can generate anitbodies molecular recognition spots only recognize epitope domains held by disulfide bonds heavy chains and light chains Any pathogenic molecule, like sugars, nucleic acids, proteins can serve as antigens

Double helix example of rules of chemistry

Double helix forms because Charges between negatively charged phosphates of the backbone repel each other (repulsion forces are reduced by the high dielectric constant of water and interaction of positively charged ions W/ phosphate groups) water can form h bonds with the negatively charged backbone Double helix keeps a distance of phosphates at an appropriate length in single stranded- h bond donor and h bond acceptor are exposed to solution and can form h bonds with water in double stranded h bonds w water break and h bond with other bases base pairs are stacked and parallel in vdw forces minimize nonpolar exposure to outside surface and water thermodynamically, heat is released to the surroundings so that reaction is spontaneous hydrophobic interactions at play as well ph of solution

Reversibly melted double helix

During replication or transcription, the two strands of the DNA double helix must be separated. • In the laboratory, DNA strands can be separated by heating a solution of DNA, a process called denaturation or melting. The temperature at which half of the DNA molecules are denatured is called the melting temperature (Tm). • Melting can be observed because bases stacked in a double helix absorb less ultraviolet light than bases in a singlestranded molecule, a phenomenon called hypochromism because the bases are able to interact with light when not bonded. • Upon cooling, the two strands can bind to each other to reform the double helix, a process called reannealing in a cell T remained constant, cell uses helicases that use atp molecules to break bonds between bases and melt structure

Quantifying protein purification

Effectiveness can be calculated by specific activity after each technique enzymes catalyze very specifically in a certain type of reaction, so an assay can be performed Specific activity should increase with each step total activity decreases as Yield decreases must account for lost product and errors SDS PAGE can give a visual evaluation of purification scheme

termination sites

Elongation continues until a termination signal is detected; these are better understood in prokaryotes than in eukaryotes. - The simplest stop signal is the transcribed product of a segment of palindromic DNA. - The RNA complement of the DNA stop signal forms a hairpin structure, followed by several uracil residues. - Upon synthesis of the hairpin, the polymerase stalls, the RNA product is released, and the DNA double helix reforms. - In other cases, the protein rho( protein that will interact and follow the machinery to make it dissociate)is required for transcription termination.

Co-immunoprecipitation

Enables the identification of binding patterns of protein whne a monoclonal antibody against a specific protein is vailable, it is possible to determin eht binding partners of that protein under cellular conditions in co-immunoprecipitation, a cell extract is incubated with antibody, then agarose beads coated with an antibody binding protein are added to the mixture the antibody bound protein complex can be separated from all other cellular components. the Identification of other bound protein partners can then be probed by other methods not every protein can do this as there isn't always an affinity to a molecule convenient to you

Most Eukaryotic Genes Are Mosaics of Introns and Exons

Eukaryotic genes are discontinuous, with coding regions called exons, interrupted by noncoding regions called introns. • Introns were initially detected by electron microscopy studies. • The average human gene has 8 introns, while some have more than 100. - Intron sizes range from 50-10,000 nucleotides

RNA Processing Generates Mature RNA

Eukaryotic pre-messenger RNA contains exons and introns. It is first modified by the addition of a 5' cap and a poly(A) tail at the 3' end. • The introns are next spliced out by large complexes called spliceosomes to generate mature mRNA

Hydrophobic AAs

Glycine, Alanine, Proline, Valine, Leucine, Isoleucine, Methionine, Tryptophan, Phenylalanine Side chains lack ability to interact w water and polar molecules Tryptophan is the bulkiest and contains an indole group these are usually buried inside proteins more hydrocarbons makes the side chains more nonpolar methionine is nonpolar because there isn't a large electronegativity difference between S and the carbon atoms the N on tryptophan makes it slightly more reactive, but still hydrophobic These tend to pack together on the inside to avoid interacting w water and display the hydrophobic effect

use of hybridoma cells

Grow to mass produce antibody of interest injected into organism to studt cancer frozen for later use

Polypeptide directionality

Has directionality/Polarity Amino terminal is the beginning, carboxyl terminal the end all other alpha amino and alpha carboxyl groups are gone and have fused int peptide bonds the 2 determine hydrophobicity, charge potentia

Dialysis

How we remove salts from protein solutions protein is placed in a cellophane bag with pores too small to allow proteins to diffuse out but big enough to allow the salt to equilibrate w/ the solution outside the dialysis bag different pore size allows for a molecular weight cutoff 2nd law of thermodynaics tells us salt will disperse to fill greater region of space purify a protein mixture that contains non protein structures 1,2L of buffer

interactions within tertiary structure

Hydrophobic interaction: most proteins will exist in polar environments, and so the nonpolar side chains will aggregate together because it is more thermodynalically stable VDW: nonpolar AA's of the protein core interact w/ one another via their instantaneous dipole moments. the aggregate affect of many nonpolar AA's creates substantial binding effects disulfide bridges: in some proteins usually the ones destined to be extracellular, the polypeptide chains can be cross linked via disulfide bonds between cysteine residues to form cystine h bonds: the polar and hydrophilic side chains on the surface interact with water molecules via h bonds, h bonds also stabilize the protein ionic interactions: 2 oppositely charges side chains can interact via ionic bonds

mass spectrometry

Identifies peptides and proteins allows the highly accurate and sensitive detection of the mass of the molecule of interest/analyte convers analty to gas phase ion, mass to charge ration determines 3 componets, ion source, mass analyzer and detector matrix assisted laser desorption (MALDI) is often combinator TOF (time of flight) better than electron spray proteins sometimes can be purified by function, mass spec then becomes a viable option High res and fragmatizationafter ionization can be sequences at the end for some proteins

Modifications of Eukaryotic mRNA

In eukaryotes, after transcription, the 5' end of mRNA is modified by the attachment of a cap (chemical modification) structure while the 3' end acquires a poly(Adenosine) tail

Prions

Infections agents that consist entirely of protein aggregates. these agents are responsible for mad cow disease in cattle, scrapie in sheep, and CJD in humans prions are aggregates of proteins that are nrmally found in the body that have misfolded they are transmissible the generally cannot be broken down or denatured by typical methods (unsoluable, not able to be broken down)

creating polyclonal anitbodies

Inject antigen into mouse to produce polyclonal antibodies draw a sample blood. it will contain the antibodies against the specific antigen purify the product to isolate the polyclonal antibodies

equilibrium constant of water

Keq = [H+][OH-]/[H2O] Kw=[H+][OH-]=10^-14 [H+]=[OH-]

Why is knowing AA sequence important?

Knowledge of the sequence of a protein is usually essential to elucidating its function AA sequences determine the 3D structure of proteins alterations in the AA sequence can lead to abnormal function of the protein the sequence of a protein reveals much about its evolutionary history does not mean knowing sequence is knowing structure, but 1 predetermined structure for 1 polymer structure determines function aa sequence is primary structure

Using recombinant DNA tehcnology for protein purification

Large quantities of proteins can be obtained this way protein can be modified with affinity tagets that allow purification of the portein or visualization of the protein in the cell proteins with modified primary structure can be generated rare for a biochemist to get samples of human dna and smash them overexposes a protein of interest in a suitable host system such as ecoli or yeast use something other than ecoli when the protein cannot survive in prokaryote

DNA structure

Linear polymer made of 4 types of monomer fixed backbone w protruding bases all bases are planar sequences of bases acts as a mean of storing info each helix can be pulled apart and act as a template for more dna

Fluorescent markers

Make the visualization of proteins in the cell possible Fluorescently labeled antibodies to particular proteins, coupled with flouresence microscopy, allow the cellular locations of proteins to be determined GFP can also be used to take cellular proteins to follow their movement in cells Antibodies labeled w clusters of electron dense metal can be used in electron microscopy GFP cn be expressed and added to detect because they fold into a small fluorescence domain that typically wont disrupt proteins

semiconservative replication

Meselson and Stahl elegantly demonstrated that replication is semiconservative by growing bacteria in growth media supplemented with 15N. • The bacteria were then shifted to growth media with 14N as the nitrogen source. • Density gradient centrifugation established that upon the shift to 14N medium, newly synthesized DNA consisted of DNA with equal parts 15N-DNA and 14N-DNA, a result consistent with semiconservative replication.The 15N isotope is heavier than the 14N isotope. • Density-gradient centrifugation can be used to distinguish between DNA that contains 15N and DNA that contains 14N.

Start codon

Messenger RNA is translated on ribosomes. • The first codon is almost always AUG, which codes for methionine. - In prokaryotes, the AUG is preceded by a purine-rich sequence called the Shine-Dalgarno sequence. FormylMet-tRNA binds to the initiator codon once fMet binds,. - In eukaryotes, the AUG nearest the 5¢ end is the initiator codon special protein called release factor binds onto the stop codon and releases newly synthesized polypeptide. • Location of the initiator codon establishes the reading frame.

universal genetic code

Most organisms use the same genetic code. However, some organisms have slight modifications. - Example: in ciliated protozoa, codons that are stop signals in most organisms encode amino acids. - Mitochondria also use variations of the genetic code

Analysis of protein purification

Must quantify results to see if our processes are getting us anywhere total protein: how much protein is left in mixture enzyme activity: describes the ability of the enzyme to promote a speecific reaction in that sample specific activity: ration of enzyme activity to total protein, tells how pure a sample is yield: enzyme activity now/original eznyme activity *100 purification level: how effective each step is in purifying the protein specific activity of that step/ spec act of original mixture

hydrophobic effect

Non-polar Molecules driver together by the increase of entropy in water A manifestation of the properties of water H bonds hold the NP molecules apart, but this becomes less favorable in entropy, so they bring the 2 NP molecules together, releasing some of the water molecules from their H bonds to go interact with one another high polarity of water makes it dissolve polar substances readily when NP gets added to water, water forms a cage around the NP molecule, which is not favorable thermodynamically because now they cant interact as much with other water molecules

directionality of nucleic acid chains

Nucleic acid chains have directionality since the two ends are different. One end has a phosphoryl group attached to the 5' carbon atom of the sugar, and one end has a free hydroxyl attached to the 3' carbon of the sugar. • By convention, nucleic acid sequences are typically written from left to right in the 5'-to-3' direction.

nucleic acids

Nucleic acids are long, linear polymers constructed from four types of monomers. • Each monomer consists of a sugar, a phosphate, and a nitrogenous base. • The sequence of the bases is the information content of the nucleic acid.

Protein flexibility and function

On binding iron, lactoferrin, undergoes a substantial change in conformation that allows other molecules to distinguish between the iron-free and the ironbound forms.

Polypeptide chains flexibiliy

Peptide bonds cant rotate, but the other bonds in the backbone can rotation is permitted about the N-Alpha carbon bond (phi) and the alpha carbon-carbonyl bond (psi) from -180 to +180 Rotation about the phi and psi is called torsion angle, determines the path of the polypeptide chains, not all torsion angles are permitted in most cases, the trans formation is more favored than the cis because there is no steric hindrance (proline excluded)

Western blotting

Permits the detection of proteins separated by Gel Electrophoresis Immunoblotting is when proteins are separated in SDS PAGE, then transfers to a polymer, then stained with antibody specific to the protein called the primary antibody secondary antibody, specific to first is added 2nd is attached to an enzyme that generates a fluorescently labeled tag allowing for detection of primary antibody and revealing location of protein of interest Mass separation plus antibody very common way to detect protein because it gets rid of most of the pollutant and antigen can recognize transfer proteins via a cellulose membrane to gen and apply horizontal way to make each band stick to polymer sheet block all regions with milk and immobilize the antigen

Buffers regulating pH in organisms

Ph can disrupt molecular structure by protonating and deprotonating key groups substantial change in pH within our body can cause harm to biological molecules by disrupting their molecular structure buffers are solutions that resist ph changes by slwing changing ph due to dilution ph changes gradually in a buffer solution because there is an acid present in equal concentration to its conjugate base, so when h iions are added, the conjugate base consumes some of those ions so the ph doesn't change drastically

Properties of water

Polar molecule Highly cohesive(# of H bonds goes up as it goes from gas to solid) Both an H donor and H acceptor bent shape leads to asymmetric distribution of electron density mall size of hydrogen allow it to get close to negtive oxygen, incrreasing cohesivenest

quarternary structure

Polypeptide chains can assemble into multi subunit structures Many proteins are composed of multiple polypeptide chains called subunits, such proteins are said to display Quaternary structure Quaternary structure can be as simple as 2 identicle polypeptide chains or as complex as dozens of different polypeptide chains multiple molecules tetramer can be a dimer of dimers others exist, but dimers and tetramers are most common sequence of polypeptide chain can be what forms quarternary strucutre way in which polypeptides interact w one another

Reverses and turns

Polypeptide chains can change directionality bymaking reverses turns and loops can make Beta turns(hairpin) or omega loops sometimes they need to turn to fold properly by not forming with the 4th amino acid from the itself, it doesn't because an alpha helix EX: antibodies have variable regions that are determined separately. Dont fall into alpha helix or beta strands

Secondary structure

Polypeptide chains can fold into regular structures such as alpha helix, beta sheets, and turns and loops Secondary strcuture is a 3D structure formed by H-bonds between peptide NH and Co groups of AAs that are near one another in the primary structure only bonds between backbone chains forms once the primary structure is formed

Describing proteins

Polypeptide consisting of a repeating main chain or backbone and a variable part consisting of a side chain Backbone has H-bond potential due to carbonyl groups and the H atoms that are bonded to the nitrogen of the amine Most proteins are 50-2000 AA long mean molar mass is 110g/mol can also be measured in daltons which = the mass of an H atom net charge of 0 on a polypeptide chain is the point where proteins aggregate and percipitate

Promotor sites

Promoters are specific DNA sequences that direct RNA polymerase to the proper initiation site. • There are often variations in the sequence of a promoter for different genes. The average of such variation is called the consensus sequence. wont get expressed without promotor site not a coding region, doesn't contain info, just increases affinity to protein machinery to start transcription eukaryotic have enhancer site that is not a promo. simply binds to it and moved it to form a complex and increase efficiency of rna polymerase

Membrane embedded proteins

Protein has many hydrophobic AAs in contact w the hydrophobic membrane and many polar and charged AA in contact with the polar substance it is assisting cross the membrane

two-dimensional gel electrophoresis

Proteins are separated via isoelectric focusing, then attached to SDS page and electrophoresis is performed at a 90 degree angle to the direction of isoelectric focusing Can be used to detect differences in protein expression under different physiological circumstances provides as easy way to visualize protein mixture and helps create a more effective means of purifying a mixture of proteins

tertiary structure

Proteins fold into globular or fibrous structures distribution of AAs is related to a proteins folding and function surface is usually charged AAs, interior is tightly packed hydrophobic AAs wont have bulk solvent in interior of tightly packed hydrophobic AA myoglobin is an example of a highly compact globular mainly helical protein with a heme prosthetic group formed local regions, regular structure, stabilized by h bonds in backbone certain residues just favor alpha helix or beta sheets regidness of the protein is suitable for its function. very ridig proteins might be involved in cell was structure, while not so rigid proteins might be involved in functions that require flexibility. 3d shape the polypeptide will take in its local environment spatial arrangement of AA's that are found far away fro one another along the peptide chain

steps of anfinsen experiment

RIbonuclease consist of 124 AA's and 4 disulfide bonds step 1: all disulfide and H bonds break and potein is denatured step 2: when beta mecaptoethanol is removed first, the inactive enzyme is formed because the imporper disulfide bonds were formed step 3: when trace Beta mercaptoethanol was added, the scrambled enzyme form correcty into the tertiary structure for activity

Unity

Reason for unity is pointing to evolution organisms that have macromolecules with similar shape and function suggests common ancestor

genomic revolution

Sequencing human genome has made sequence determination cheaper and faster ID sequence variation among individuals can be used to understand various diseases

Ramachandran plot

Shows favorable phi-psi angle combinations. 3 main "wells" for α-helices, ß-sheets, and left-handed α-helices.

Cooperative process of protein folding

Similar curve to titration around the pka of the protein when the denaturation begins it falls apart quickly at one point proteins fold by progressive stabilization of intermediates rather than random search partly correct folding intermediates are retained because they are slightly more stable than unfolded regions does not go from 1 to 5, 2-4 will still show up, it can fall back lower stages occasionally, but can stay correct longer

Protein domains

Some proteins have domains, which are independently folding regions within a polypeptide, connected by short flexible linker segments doesn't need rest of the sequence will maintain biological function can be cut, but regions still intact from h bonds

Protein motifs

Some proteins have motifs (super secondary) Which are common combinations of secondary structures that are present in many proteins and frequently exhibit similar functions (helix-turn-helix) represents a biological function

Beta sheets

Stabilized by H bonds between polypeptide strands or different parts of the same stand just folded over polypeptides are fully extended unlike alpha helix Ramachandran plot shows big probability on the 2nd quadrant 7 angstrom difference between AAs can be parallel or anti parralel Antiparallel matches each Nh with each CO on every AA involved parallel shows 1 amino acid binding with 2 on the other chain/side mixed is slightly more stable than parallel can be flat or adopt twisted conformation

Staining proteins after SDS-PAGE

Stain the gel with coomassie blue if a proteins structure and function is defined by sequence, then the sequence will provide characteristic mass, then identify chemical formula and MM Compare to a standards on the left side

Covalent bonds

Strongest bonds Sharing of electrons resonance of bonds increase stablity more bonds means shorter and harder to split can form resonance structures intramolecular

What is biochemistry

Study of the chemistry of life has applications in medicine, dentistry and many other science field most reactions rake place in water, which acts as an important solvent and plays a role in determining the pathway taken by the rxn

differential centrifugation

Supernatant is centrifuged at a higher force to yield another pellet and supernatant this is repeated several more times to yield a series of pellets enriched in various cellular materials and a final supernatant the soluble portion of the cytoplasm assay is conducted to see if the protein activity is high

RNA polymerases

Synthesis of RNA from a DNA template (transcription) is catalyzed by RNA polymerase, which has the following requirements: 1. A DNA template. The sequence of the newly synthesized RNA is complementary to the DNA template. The DNA strand that has the same sequence as the RNA product (with T instead of U) is called the coding strand. 2. Activated precursors in the form of the four ribonucleoside triphosphates 3. Divalent metal ions, usually Mg2+ or Mn2+ • RNA polymerase catalyzes the following reaction: RNA polymerase initiates and elongates the RNA product (acts as an anchor), with the chain growing in the 5'-to-3' direction. • No primer is needed. The 3' OH of the growing chain attacks the innermost phosphoryl (α) group of the incoming ribonucleoside triphosphate. cannot correct its own mistakes Prokaryotic: single RNA polymerase forms all types of RNA molecules humans: 3 types of rna polymerase

Ionizable Amino Acids

Terminal carboxyl= - at 3.1 Asp/Glu: - at 4.1 HIS: neutral at 6 terminal amino: neutral at 8 Cysteine: negative at 8.3 tyrosine: negative at 10.0 lysine: neutral at 10.4 Arginine: neutral at 12.5

A form dna

The double helix can also exist in an A form, which is shorter and wider than the B form with the bases at an angle rather than perpendicular to the helix axis. In the A forms, the sugar is in the C-3'-endo conformation, while the sugar is in C-2'-endo configuration in the B form.The A form is seen in RNA double helices and in RNA- DNA hybrid helices, structures observed in transcription and RNA processin

Z DNA

The double helix can also form Z-DNA. Z-DNA is left-handed, and the backbone is zigzagged, which gives it its name

Nucleic acids strands forming double helix

The double helix is stabilized by hydrogen bonds and van der Waals interactions. • Because of the base pairing rules (A-T and G-C), the sequence of one strand determines the sequence of the partner strand. • The two strands can be separated and complementary sequences synthesized to generate two identical daughter strands.

Electrophoresis determining mass

The electrophoretic mobility of many proteins in SDS page gels is inversely proportional to the long of their mass

The Central Dogma

The flow of genetic information is generally from DNA to RNA to proteins, a scheme called the central dogma.

reverse denaturation ribonuclease

The next step in Anfinsen's experiment was to show that denatured enzyme slowly regained its activity if the urea and β-mercaptoethanol were removed. - The most stable, lowest-energy, native conformation forms, which is also the one enzymatically active form. • However, activity is extremely low if the urea is not removed. Favorable noncovalent interactions are prevented from forming, and all 105 mathematically possible combinations of disulfide bond pairs form with the eight cysteines, including the 104 that are not enzymatically active. To regain enzyme activity, the urea must be removed and a trace of βmercaptoethanol must be present. • This allows the slow formation and breakage of disulfide bonds over the course of several hours, until the lowest-energy, most stable (i.e., native, functional) active form is finally regenerated.

How are protein sequences made?

The sequence of every protein fround in our body can be traced back to some gene that is found in our dna DNA->RNA->Proteins

DNA vs RNA

The sugar component of deoxyribonucleic acid (DNA) is deoxyribose( more stable because its more resistant to hydrolysis) , a ribose in which the 2'-hydroxyl is replaced with a hydrogen. • Ribonucleic acid (RNA) contains the sugar ribose. • The backbones of DNA and RNA consist of sugars linked by phosphodiester bridges (less likely to be hydrolyzed) between the 3'-hydroxyl of one sugar and the 5'-hydroxyl of an adjacent sugar. • Bases are attached to carbon atom 1' in the sugar via glycosidic bond. • Two of the bases are purines (adenine and guanine), and two are pyrimidines [cytosine and thymine (DNA) or uracil (RNA) RNA does not form double helix, but can intertwine to form secondary or tertiary structure DNa contains genetic information, RNA transcribes DNA info to a form that is understood by cells and assist in protein synthesis

structure dictates function

The way something is put together determines what it does. If you change the structure, you change the function A protein component of the DNA replication machinery surrounds a section of DNA double helix depicted as a cylinder

Utility of Amino acids

These AA provide chemical versatility They have been available for prebiotic rxns Larger AA may be too reactive

Characteristics of the Genetic Code

Three nucleotides, called a codon, encode an amino acid. 2. The code is nonoverlapping. 3. The code has no punctuation. 4. The code has directionality. It is read from the 5¢ end of the mRNA to the 3¢ end. 5. The code is degenerate in that some amino acids are encoded by more than one codon; this minimizes the deleterious effects of mutations

Circular and supercoiled dna

To fit inside a cell, the DNA molecule must be compacted. In E. coli, the DNA double helix is a circular molecule that is twisted into a superhelix by the process of supercoiling. • The relaxed circular DNA and the superhelix form are topological isomers of each other supercoiling can affect the unwinding process, which stimulates processes like DNA replication

Hydrogen bonds

Total distance between H donor and H acceptor is between 2.6-3.5 easily broken requires H donor and H acceptor largely ionic weaker than covalent tendency to be in a straight line

1st law of thermodynamics

Total energy of the system and environment is consistent

2nd law of thermodynamics

Total entropy of a system and its surrounding always increase No entropy=Perfect crystalline entropy can decrease locally if it increases in the universe deltaG=deltaHsystem-TdeltaS g<0 its spontatnous deltaSsystem >deltaHsystem/T

Features of Watson-Crick Model

Two helical, antiparallel polynucleotide strands are coiled around a common axis in a right-handed helix. 2. The sugar-phosphate backbones are on the outside and the purine and pyrimidine bases lie on the inside of the helix. 3. The bases are nearly perpendicular to the helix axis. 4. The various measurements in Angstroms of the helix could be determined. Bases are separated by 3.4 Angstroms, 10 bases per turn, each turn is about 34 angstroms and diameter is 20 angstroms

Features of peptide bonds

Uncharged planar, all 6 atoms are in the plane (alpha carbon, carbonyl carbon, oxygen, nitrogen, hydrogen on nitrogen, alpha carbon) has partial double bond character due to the carbonyl resonance, which limits rotating about peptide bonds trans form is preferred to cis form due to steric reasons planes can rotate but do not bend forward condensation rxn is thermodynamically unfavored, peptide bond required energy activation energy of the reverse is too high under normal condition the reverse would need a high temperature or special enzyme to happen

how does urea/beta mercaptoethanol disrupt native ribonuclease

Urea/guanidinium present plenty ways to form h bonds differently, therfore they act as competitors for h bonds, other interactions that stabilize the tertiary structure of ribnuclease. beta mercaptoethanol does the same with the cysteine molecules, competing for bonds and breaking the disulfide bonds in the protein.

Sandwich ELISA

Used when there isnt enough antibody or its too expensive follows same process determine what to use based on what can be generated

sedimentation coefficient

Usually expressed by svedberg units smaller s value= slower protein moves in centrifugal field not determined by mass, rather rate of migration not a linear relationship between mass and S value size and shape of particle affect

Proline unique geometry

WHen proline follows another AA, the peptide bond between then is equally likely to be cis or trans due to its geometry (x-pro) Pro-X does not work due to the r group of the x and the large cyclic group on the proline having steric hindrance the 5 membered ring of proline makes it structurly restrictive allowing it to greatly influence the structure of proteins

Globular proteins

Wide range of functions and relatively spherical in shape EX: hemoglobin, insulin, DNA polymerase

determining 3d protein structure (xray crystallography)

Xray crystallography reveals 3D structure in atomic detail(not always but a lot) Crystals of proteins are irradiated w/ xrays (very very strong xrays) Electrons of the atom scattter x rays (cryoprotected sample) The scattered waves recombine the ways in which the scattered xrays recombine reveals atomic arrangement

Steps of xray crystallography

Xrays refract electrons->contains special Electron distribution->electron revolve around nucleus->e density map is created->fit sequence in and find atomic structure-> need large amount of purified proteins and very pure-> chemical condition to form crystals must be found

heterodimer

a chain of two molecules that are different from each other can be more than dimer

isoelectric focusing

allows separation of proteins in a gel on the basis of their relative amounts of acidic and basic amino acids If a mixture of proteins is placed in a gel w a ph gradient and an electrical field(voltage source) is applied, each protein will migrate to its isoelectric point (PI), the PH at which it has no net charge and stops moving you must adjust the ph gradient to make the net charge 0 a horizontal electrical field is applied PI is determined by sequence proteins with the same AA composition and different sequences will have the same pI

ion exchange chromatography

allows separation of proteins on the basis of charge beads in column also have charges when a protein mixture is passed through, proteins w the same charge as beads will exit quickly proteins with opposite charges to beads will bind then release by increasing salt concentration or adjsuting ph proteins vary in charge distribution provides a large amount of cations and anions salt will compete for ions in the column and once a continuous gradient is approached, the desired protein will disconnect only helpful if we know net charges on proteins in the mixture are different

intramolecular bonds

atoms bond with one another nonpolar covalent: 2 atoms same electronegativity polar covalent: pulls E- density because of difference in electronegativity ionic bonds: when one atom is much more electronegative than the other, forming seperation of charges

NMR spectroscopy

based on the fact that certain atomic nuclei are intrinsically magnetic and can exist in 2 spin states when an external magnetic field is applied Nuclei of sample absorb electromagnetic radiation at different frequencies termed chemical shifts Chem. shifts depend on the environment of the nuclei, and the environment depends on protein structure typically used with 100 or less AAs crystals in solid state forms with 40 % water (comparable to solvent) conserve solvation of protein, preserving its native structure larger protein makes the puzle more complex

Proteins

built of a repertoire of 20 AAs usually chiral, only L (s) isomer found in proteins Proteins are linear polymers composed of amino acids. (*but after folding... so, don't take "linear" too literally here) • Proteins have a wide variety of functional groups. • Proteins can interact with one another and with other macromolecules to form complexes. • Some proteins are rigid while others are flexible

Cleaving proteins

can be cleaved into small peptides to facilitate analysis edmans and Mass spec arent 100% accurate, so we chemically or enzymatically cleave the peptide to yield peptides fewer than 50 AAs the peptired are then ordered by performing a differeny cleavage procedure to general overlap peptides if a protein is big, it cant just go into collision chamber it must go through different parts used to determine specific order of AAs

Releasing protein from cell

cell must be disrupted to form homogenate, a mixture of all the cells components the homogenate is then centrifuged at low speeds to yield a pellet at the bottom consisting of nuclei and supernatant top has the soluble parts including the protein

posttranslational modification

confers new capabilities to proteins Lack of appropriate protein modification can result in pathological conditions like lack of vitamin c preventing hydroxylation of proline in collagen, leading to scurvy lack of vitamin K prevents carboxylation of clotting proteins, which can lead to hemorrhaging modifications can occur after it has reached through central dogma and has folded

Folding funnel

depics thermodynamics of protein folding 3d funnel with varying structures maximum entropy with minimal structure on top folded proteins exists on bottom

Cryoelectron microscopy

determines structure A thin layer of protein solution is prepared in a fine grid then frozen quickly. This traps the molecules in an ensemble of orientations the sample is then placed in a transmission electron microscope under vacuum conditions and exposed to an incident electron beam each protein interacts with the beam to produce a 2D projection on the detector many projections are detected, each capturing a molecule i a different orientation a computer uses these to build 3d model shoot electron and take pics of results gives low resolution see protein protein interactions have to find alignment and orient them

Protein Purification Techniques

dialysis salting out gel filtration ion exchange chromatography affinity chromatography

Identifying individual proteins

done via mass spec specific protein cleavage, follwed by separation of the resulting peptides and mass spec allows the identification of individuals this is called peptide mass fingerprinting

Proteome

entire set of proteins expressed and modified by a cell (tissue, microorganism, etc) under a particular set of biochemical conditions (in milk, muscle, with or without cancer cells) not an unvarying characteristic of the cell rarely does 1 protein cause a disease, usually an entire pattern changes each cell has the same genome, but proteomes can differ

ELISA

enzyme-linked immunosorbent assay Antibodies can be used as a reagent to quantify the amount of a protein or other antigen ELISA quantifies the amount of protein present because the antibody is linked to an enzyme that produces readily identified colored products Dye doesn't usually work for antibodies

monoclonal antibodies with virtually any desires specifically can be readily prepared

formed from a single type of plasma cell and only binds to one epitope collections of the same protien

Green Flourescent Protein

from jellyfish can be attached to cellular proteins using molecular biologist techniques. GFP flouresces green when exposed to blue light, allowing the cellular location of the attached protein to be determined or followed

Separation techniques

gel electrophoresis isoelectric focusing 2D electrophoresis

DNA turning into proteins

gene: fundamental unit of hereditary info codon: ser of 3 bases on dna strands that determine AA genetic code: set of rules that links the DNA sequence to protein sequence

Exons

in prokaryotes, newly synthesized mRNA molecules consists of a continuous sequence of codons that can be used by ribosomes to synthesize proteins, mRNA is in its fully functional form and doesn't need to be mediated, humans do however with exons and introns Encode Protein Domains Many exons encode discrete structural and functional units of proteins. • Some new proteins have evolved by exon shuffling. • Alternative splicing allows the generation of multiple proteins from a single gene.

The Tissue Plasminogen Activator (TPA) Gene was Generated by Exon Shuffling

independently folds into a functional structure human genes bigger and more complex, domains together and exons shuffle more complex than a pure code because they are conveniently coded into a domain portions of ancestral genes are shuffled and mixed into what they are today

Positively charged AAs

lysine, arginine, histidine Arginine has guanidinium group due to histidine being at pka 6, near phisiological ph, it acceps and donates Hs readily

mRNA, tRNA, rRNA

mRNA: 5% of the total rna, serves as template for translation, in eukaryotes: distanct mRNA molecule is produced for each gene. In PRokaryotes: mRNA molecule code for several genes, tRNA: 15%, carry activated AAs to the ribosome mRNA complex during protein synthesis, each AA has 1 tRNA made specifically to carry it to ribosomes rRNA: 80%, major consituent in ribosomes. cell machinery responsible for synthesizing poltpetide chains, prokaryotes have 3 t types

Things that affect its sedimentation rate

mass, size and shape more spherical particles have lower friction coefficient values of less spherical particles of equal mass, therefore more spherical particles of equal mass therefore more spherical particles will move faster down more dense particles move quicker than less dess because there's less resistive force density of fluid: =1, wont move, densities are equal, >1 will float to top, negative s, wont travel through fluid, <1 will sink, travel through fluid to end of tube

nucleotides

monomers of nucleic acids A nitrogenous base bound to a sugar is called a nucleoside. Base lies above the plain, Sugar unit is conalently bonded via beta glycosidic bond to base The nucleosides of DNA are deoxyadenosine, deoxyguanosine, deoxycytidine, and deoxythymidine. By convention, deoxythymidine, which rarely occurs in RNA, is simply called thymidine. • The nucleosides of RNA are adenosine, guanosine, cytidine, and uridine. • The C-1' of the sugar is attached to the N-9 of the purine or the N-1 of the pyrimidine by a β-glycosidic bond. • A nucleotide is a nucleoside with one or more phosphoryl groups. • Nucleoside triphosphates are the building blocks of DNA and RNA.

determination of pI

no ionizable side chains: pI= average of pka values of terminal amino and carboxyl acidic side chains: pI= average of pka values of terminal carboxyl and side chains basic side chains: pI= average of pka values of terminal amino side chains all other: determine middle pKa values and average it with the terminal alpha carboxyl

RIbonuclease reforming

protein folding of bovine ribonuclease requires 4 disulfide bonds when urea and beta mercaptoethanol were slowly removed, the enzyme regained its native form slowly remove he simulated a thermodynamic process for the protein to find its most stable way to exist in solution (dialysis them against lower concentration of urea and beta mercaptoethanol) not every protein can do this, some need machinery or chaperones that we might not have access to a polypeptide sequence contains info that permits its folding these results demonstrated that the info required for a polypeptide chain to fold into a functional protein w/ a defined 3d shape inherent in the primary structure

protein interactions

proteins do not usually act by themselves proteins can combine w/ other proteins or with other macromolecules to form longer complexes that carry specific functions EX: transport proteins usually embed themselves into the phospholipid membrane to carry out their functions of ion and molecule transport EX: in DNA replication, large proteins complexes are required to create new dna strands

fibrous proteins

provide structural support for cells and tissues ex: keratin and collagen Alpha keratine- member of superfamily of structural proteins called coiled-coil proteins. THey are both strong and flexible because of thing intertwinning coil design 2 right handed alpha helices intertwine to form a left handed super helix stabilized by ionic interactions and VDW forces and disulfide bonds others include cytoskeleton and muscle proteins super helix: expression of tertiary structure large proteins that form long fibers and play a role structurally the more disulfide bonds= more rigidity of protein

Alternative splicing

provides a means of forming a set of proteins that are variants of a basic motif without requiring a separate gene for each protein use a blueprint to generate different proucts they are generated by splicing 1 gene differently by exon sequencing, proteins products with different locations and same function can have different layers of complications Alternative splicing (AS) therefore is a process by which exons or portions of exons or noncoding regions within a pre-mRNA transcript are differentially joined or skipped, resulting in multiple protein isoforms being encoded by a single geneThis mechanism increases the informational diversity and functional capacity of a gene during post-transcriptional processing and provides an opportunity for gene regulation

Collagen structure

requires many carefully positions glycines collagens strenght makes it useful as a structural protein in skin and bone the consistency of gly in a column gives the collagen flexibility proline in the collagen AA gives it a kink gly every 3rd residue common gly-pro-hyp(hydrolylated proline) and gly-pro-pro sequences consists of 3 intertwined helical polypeptide chains that form a superhelical cable

Polar AA

serine, threonine, asparagine, glutamine, cysteine Asparagine and glutamine have polar carboxamide groups

SDS-PAGE

sodium dodecyl sulfate polyacrylamide (hydrophobic interion polar surface) gel electrophoresis use SDS page and mercaptoethanol to break disulfide bonds allow for accurate mass determination. denatures protein, 1 molecule of sds binds for every 2 AAs side chains Proteins have same charge to mass ration and migrate in the gel on the basis of mass unity smaller size will move to the bottom faster migrates in the same electrical field due to less frictional force

Undesirable reactivity in amino acids

some AA's are unsuitable for proteins because of underisrable cyclization. Homoserine can cyclize to form a stable five membered ring, potentially resulting in peptide bond cleavage. the cyclization of serine would form a strained 4 membered ring, making it unstable

VDW forces in dna

stabilize by base stacking interactions

affinity chromatography

takes advantage of the fact that some proteins have a high affinity for specific chemicals or chemical groups. Beads are made with the specific chemical attached. A protein mixture is passed through the column. Only protein with affinity for the attached group will be retained. The bound protein is then released by passing a solution enriched in the chemical (glucose solution) to which the protein is bound will be left with 2 collection, one with protein of interest that binded to beads, and a collection with another mixture of proteins alot of proteins very specifically bind to certain ligands protein will naturally have a great affinity to the resin only useful if we know the molecule that the enzyme has an affinity to

Salting out

takes advantage of the fact that the solubility of proteins varies with the salt concentration. Most proteins require some salt to dissolve in water, a process called salting in. As the salt concentration is increased, different proteins will precipitate at different salt concentrations, a process called salting out different proteins have different salt concentrations when salt is added, the solubility of the protein decreases because the salt takes up those water molecules and the nonpolar AAs will precipitate out due to the number of h bonds that can stabilizing the protein decreasing, they must aggregate to reach similar stability

Hemoglobin

tetramer heme units make blood red if it wasnt for hemoglobin and its iron units, O2 and CO2 couldnt be transferred without the hetrotetramer

polyclonal antibodies

the antibodies produced to the antigen by difference cells are said to be polyclonal binding of the antibody to the antigen stimulates plasma cells to produce antibodies a mixture of different antibodies that all bind to the a specific antigen but may differ in the epitope its attached to collection of different proteins

3rd law of thermodynamics

the entropy of a system approaches a constant value as the temperature approaches absolute zero

Enthalpy

the heat content of a system,how much heat needed to change system

Alternative confirmations of a peptide sequence

the same few seqeucnes in one proteins can show as an alpha helix and in anothers as beta sheets

What do side chains affect

they affect size of protein, polarity, structure and shape, charge, hydrophobic properties, ability to bond. These variations determine chemical reactivities of AA's

Differences in amino acids

unique side chains that vary in size, shape, charge, h-bond capacity, hydrophobic charector, and chemical reactivity 4 classes- hydrophobic, polar, positively charged, negatively charged (at physiological ph)

Homodimer

when two polypeptides encoded by the same gene bind to each other to form a dimer 2 identical single chains

Structures of single stranded nucleic acids

• A common structural motif seen in nucleic acids, most notably RNA, is the stem-loop, which occurs when complementary sequences in the same strand form a double helix. • Non-Watson-Crick base pairs occur frequently in RNA. • More elaborate structures may form, often stabilized by Mg2+ ions stem loop forms when rna folds onto itself to form a double helical structure that contains 2 completemtary sequences and mismatches pairs mismatched regions destablizes the local structure of the RNA and determines how the rna will fold next nucleotides far away can interact via base pairs to form tertiary structures t rna molecules fold into a specific shape and function to move amino acids to the ribosomes during protein synthesis

The Spliceosome Recognizes Specific Sequences Within the Intron that Specify the Splice Sites

• Introns almost always begin with a GU and end with an AG. 2 specific sequences will specify splice sites spliceosome complex recognizes the introx and catalytically temoves them while splicing exons together common feature i splicing is that exons are added in the same sequence as they appear in the gene of the DNA

Virus genes

• Some viruses, such as the tobacco mosaic virus, have RNA genomes that are replicated by RNA-directed RNA polymerases. • Retroviruses, such as HIV-1, have single-stranded RNA genomes that are converted into DNA double helices by the action of reverse transcriptase. final double helical viral DNA goes into cell can reprogram an entire cells DNA essentially lifeless before it enters the cell not all have DNA

Transfer RNAs are the Adaptor Molecules in Protein Synthesis

• Transfer RNA (single stranded, shorter than mRNA) molecules react with specific amino acids in a reaction catalyzed by aminoacyl-tRNA synthetases. • Transfer RNA molecules also contain a template recognition site, called the anticodon. The anticodon, which consists of three bases, recognizes a complementary 3-base sequence in the mRNA called the codon. complementary interactions with local regions form shape Anticodon loop contains completemtary info of genetic code, modifications to the ened

Polymerization Reaction Catalyzed by DNA Polymerases

• dNTPs are considered to be activated precursors because although only one phosphate will be incorporated into the backbone, the subsequent breakdown of the released pyrophosphate helps to drive the phosphodiester bond formation. based on chemical nature of DNA, directionality, sequence coding, and semiconservative propagation