structure of biological materials outline

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EBME 303 Exam 1 Review

EBME 303 Exam 1 ReviewLecture 1: Intro

Failure of Biomaterials and Deviceso Thrombosis (clots)o Tissue biocompatibility (inflammation, wound healing)o Infection (bacterial adhesion)

Stents cause injury because they “smash” blood vessels when expanding and this injury restenosis

Vascular grafts: success rate decreases with diameter

Two important considerationso Glycocalyx: controls interfacial adhesiono ECM proteins: mechanisms to regulate cell-surface interactions

The cell membrane is about 50 Å (5 nm) thicko Phospholipid bilayer (assembly)o Glycocalyx (sugar coating)o Membrane proteins (function

Molecular self-assembly: “hydrophobic” driving force = entropically favored reactions o Biomimetic surface mods: artificial glycocalyx + self-assemblingo Small drug delivery particles are cleared in minutes!o Rods and coils: oligosaccharides are helical rods with shape depending on configuration

(cannot rotate)o PEG,PEO: PEG is random coil

Surfactant = “surface active” make surfaces that don’t wash off. o Reduce the surface tension of water

o H20= 72 dynes/cmo Proteins are surfactants.o Critical micelle concentration: interface is filled up (CMC)

Bacteria and cells sometimes change structure/surface due to temperature

Average RBC circulation is 120 days Biomimetic surfactant polymer: self-assembly




Test of protein resistanceo Modify surfaceo Place sample in flow cell

30 min static adsorption with 50% PPP—Platelet-poor plasma

Blood in anticoagulant and then spin out cells in centrifuge sosupernatant is the aqueous media, proteins, and platelets (PRP plateletrich plasma)

5 minute flush with PBS (5 dyn/cm2

)o Remove and dryo Use ATR-IR to detect adsorbed proteino ALTERNATIVE: rotating disk where shear stress varies linearly with radial distance from

disk

Epitaxy: when the organization of a substrate drives a process, also a polymer-polymerinteraction

ECM and fibronectin

o




o o “Cationic cradle”: positive residues on FN interact with negative heparin sulfate on EC

surface

RGD peptide: found in many ECM proteins (FN, laminin, vitronectin, thrombospondin)o Binds to a lot of things!

CRRETAWAC = EC-selectiveo High affinity and specificity for EC integrino Low affinity for platelet integrins

EC adhesion can be controlled with peptide density and polymer compositiono Adhere to RGD (>25%), HBP, CRRETAWAC.o Shear stability based on peptide densityo Cell migration decreases when there’s more peptide density

Lecture 2: Levels of Structure Biological macromolecules are in the range of 103-1012 Da

o Synthetics have Mn, Mw, Mz, PDI instead

Linear polymerso Nucleic acids, proteins, polypeptides

Linear or branched polymerso Polysaccharides (Diverse in structure)

Oligomerso Fatty acids

Monomer unitso Nucleotides, amino acids, sugars

Polypeptide = polymer of peptide group residues

Levels of structure: primary, secondary, tertiary, quaternary




Structure often has a purposeo Proteins in the blood are often globular with random (flexible) structureo DNA has functional groups on the inside to protect them beneath a negatively charged

phosphate backbone

Tobacco Mosaic Viruso Self-assembly of 2130 identical proteins—based on media conditionso The helical assembly of TMV (in the absence of RNA) is favored by an acidic pH and high

ionic strength. The pH of the solution mostly dominates whether TMV assembles in a helical or

disk shape The ionic strength causes higher degrees of polymerization but does not greatly

affect the mode of polymerization (disk, helix)

o o Important: electrostatic interactions




Increasing salt concentration increases mobile ions in media so they can interact with things“charge shielding”

Svedberg = sedimentation rate which is proportional to molar mass Long-range bonds are used to organize

o In nanometerso

Larger than atomic interactions in rangeo Ionic, electrostatic, hydrophobic

Short-range bonds are used to stabilizeo Hydrogen bondso Range of Å

Configuration is a primary thing and conformation is secondary/tertiary

Levels of Structureo Primary

Sequential order of the covalent residues of a biopolymer

Sequence = order

Configuration = arrangement In a protein, the primary structure is the sequence of amino acids which makes

up the protein Describes the sequence and configuration of a biopolymer.

o Secondary Describes the way the primary structure of a biopolymer is locally ordered and

oriented in 3-D space. Can also be defined as a list of all 3-D regions with ordered, locally symmetric

backbone structures E.g. α-helix and β-sheet Describes conformation

Conformation = knowledge of a molecule’s secondary structuresincluding random (non-helical) portions

o Tertiary “Long-range spatial order” Complete three-dimensional arrangement of biopolymer “units” that are

effectively indivisible The “unit” of a tertiary structure is covalently bonded; for example, tertiary

structures of proteins may consist of more than one polypeptide but these arecovalently crosslinked

Often the bioactive conformation of a molecule Describes the position of every monomer unit (primary structure) as well as the

location of all symmetric backbone structures (secondary)o Quaternary

Non-covalent assembly of tertiary subunits May or may not be symmetric Subunits may or may not be identical

o Common structures Linear

Polystyrene, polyamides, polypeptides, polysaccharides,polynucleotides

Helix




Depends on the pitch

8-1 helix:

A 2-1 helix repeats in the x,y position every 2 so it looks like β-sheet

Atoms 2 to 10 = simple translation along z-axis or “pitch”

The z axis is the screw axis of the helix




Zo is 1.5 Å for a α helix and 3.4 Å for a β-sheet When given the position of one residue in a α-helix you can deduce

every residue, not true for random configo Myoglobin is mostly α-helices. Can deliver 02 to tissues, like a compact hemoglobin.

About 75% of Mb is ordered secondary structureo α-Chymotrpysin: less ordered but has some β-sheets.

Protease: digests proteins Made of three chains Disulfide bonding Two sistines (sulfhydryl) and oxidizing environment

o Collagen is typically very ordered: triple helicalo Deoxyhemoglobin 180 degree rotation (C2)

Central Questions for Biopolymerso Is it pure/homogeneous?

Monodispersity: ultracentrifugation, electrophoresis, chromatography, lightscattering

Single biopolymer type or a mixtureo Is it native and complete?

Remember that structure often changes when in vivo v. in vitroo Is it consistent?

Reproducible? What is the origin of the sample (species, cell type) Recombinant techniques and fxnal assays

Nucleosides: sugar and base

Nucleotides: phosphate, sugar, and baseo –OH causes steric constraints that makes RNA and DNA differo Bases are derivatives of pyrimidines and purines (adenine, guanine, cytosine, thymine,

uracil)o Sugars are carbohydrate saccharides

How do you find structure of materials?




o Primary structure Biochemical sequencing

o Secondary structure Optical, NMR spectroscopies

o Tertiary structure X-Ray Crystallography Electron microscopy Scanning force microscopy Hydrodynamic techniques

o Quaternary The above + disruptive and associative techniques

Is structure predictable?o Certain forms like α-helix: you know every residue’s position o Are there changes in structure between active and latent forms?

Prothrombin v. thrombino Similar functional properties to a known molecule = homology

Serine proteases

o Location of residues = based on properties (globular proteins have hydrophobic groupsinside and charged groups on outside, so finding a charged group inside will beimportant)

Charged groups inside = active center

Is it rigid (C=C) or flexible (-CH2-CH2-)?

o Structure depends on pH, salt concentration, solvent (e.g. amphiphile), reducing agent.o Calculate ΔH, ΔS

How do residues affect function?

o RGD v. RGE—even minute differences add up




IgG: disulfide bonds connect heavy and light chains, lead to agglutination

Structure tells us function but we usually know the function before structure

What info can be obtained from structure?o Kinetic (affinity/rate) and thermodynamic (specificity/feasibility) studies are usually

neededo

Sometimes you need the entire structure for fxn, other times not (like RGD) Structure of active sites

o Lysosomes cause cleavage because a polysaccharide doesn’t quite fit so it changesconformation, making bonds easier to break

o Abnormality: sickle cell anemia has one Glu Val substitution and Val is hydrophobic RBCs are not flexible and are destroyed in smaller vessels Forms elongated crystals in RBC S-hemoglobin is a self-assembling fiber of deoxyHb, due to hydrophobic

interactions (“elongated crystals”) Val must be on the outside of structure so that it can interact with other sickle

cell residuesLecture 3: Intra- and Inter-Molecular Forces

SUMMARYo Quantum Mechanical Force (covalent bonding)—1 to 2 Åo Coulombic force: force between charges—function of distanceo Polarization forces: DIRECTIONAL

Dipole-dipole Dipole-induced dipole Induced dipole-induced dipole

Transient dipoles due to movement of e- cloud: lowest attraction

This can be cumulative explaining why polymer properties change withMW

Hydrophobic interactions are a function of increased entropy when in H2O

Distance of interaction decays with distance of atomso Repulsion radius = 1/r12 so decays quickly with distanceo Attractive van der waals = 1/r6 balance the above 2 forces for optimum distance of

bond

Quantum Mechanical Force (covalent bonds)o 1-2 Å or 0.1-0.2 nmo Directional bondso Strong bond energy (200-800 KJ/mol)o Tend to decrease in strength with increase in bond lengtho Look for CONPS: Carbon, oxygen, nitrogen, phosphorus, sulfuro DISULFIDE bonds (-S-S-)

Link between two sulfhydryl bonds (-SH) Stabilize the tertiary structure of proteins Increase the rigidity of globular structures

E.g. fibrinogen and VWF Can be easily reversed to –SH by mild chemical reduction and back to S-S by

mild oxidation, but this is SELECTIVE so it only affects disulfide and not other

covalent bonds

Fibrinogen (*QUIZ/TEST*)




6 chains held together by multiple –S-S- bonds

o 2α, 2β, 2γ chains

Fn is the 3rd most abundant protein (albumin, IgG, Fn)

Rotational and inversion symmetry

Fn binds both to platelets and to the ECM Coulombic (electrostatic) Force: between charges

o Strong bonding energy—similar strength to covalent bondso Not directionalo Long range, up to 20 nm (200 Å)o Strength of bonds decreases as 1/r2 (slow slope)o Mobile ions in solution = “charge shielding” which reduces force

Polarization forces: Van der Waals, dispersive, etc.o May be attractive or repulsiveo Weak in comparison to covalent bondso May be long or short rangeo Dipole-dipole

Weak, decaying with 1/r6 (about 0.1 kJ/mol @ 3 Å) Hydrogen bond is a very strong dipole-dipole interaction (15-20 KJ/mol) Directional and short-range (1.5-3 Å)

You can’t bring a protein together or stabilize structures with these short-rangebonds!

Remember that polarity is based on charge AND direction—some dipoles cancelout

Β-sheet structures held together by H-bondso Dipole-induced dipole

E.g. water Decay at 1/r6 (relatively weak and short-range)




o Induced dipole-induced dipole London dispersion About 0.2 KJ/mol, decaying 1/r6 Effects can be significant out to about 50 Å because they are additive (high MW

polymers)o Van der Waals

Van der Waals radius = 3 Å = 0.3 nm

This is due to attractive forces decaying at 1/r6 and dispersive forcesdecaying at 1/r12

Van der Waals of other atoms

H ---- 0.12

O ---- 0.14 N ---- 0.15

C ---- 0.17 S ---- 0.18

P ---- 0.19 Van der Waals forces: important in biopolymers due to high MW ALWAYS present even in vacuum Important for:

Adhesion

Surface tension

Adsorption

Wetting Colloid flocculation/aggregation

Biopolymer structure




Water

o Dielectric constant = ε = 80o H-O-H angle is 104.5 degrees, O has 4sp3 hybridized orbitals (hydrogen is 1s)o Polarity

Net charge is 0 on molecule Oxygen = negative, hydrogen = positive Polar molecule—dipoles exist

o Bonds O-H covalent bond (keeps it together) is 460 KJ/mol H-bond (bonds H20 together) is 16 KJ/mol

This discrepancy is why H2O dissociates quickly (belly flop breaks lots of H-bonds!)

O-H covalent bond is 1 Å so size of H2O = under 2 Å

Intermolecular H-bond is 1.8 Å Oxygen-oxygen distance is 2.76 Å Ice: each H20 is H-bonded to four others

Hydrogen bondso Symmetry—can form four H-bonds (dynamic, not static)

Two H-bonds : 1-D chain/ring Three H-bonds: 2-D sheet/layer Four H-bonds: 3-D network




o THIS MAY BE ON QUIZ/TEST

o o As the MW increases, the boiling point increases.o Hydrogen bonds in nature

O-H—O-H N-H—O=C O-H—N= N-H—O- N-H—O= N-H—N= S-H—O- (weak H-bond) Protein groups form H-bonds Bonds between base pairs in DNA

Water and solvationo Interacts with polar solute to reduce electrostatic and H-bonds between solute

moleculeso Forms “solvation shells” around dissolved ions like NaCl o ENTROPY

Structure-breaking ions

E.g. thiocyanate (SCN-) Attracts water molecules so H20 near SCN- more ordered than in the

bulk Water molecules right outside sphere of attraction = disordered

Overall increase in entropy Structure-forming ions

Phosphate (PO4)-3

o Water molecules are more ordered close to ion




o Not much effect outside of sphere,o Overall decrease in entropy

Entropy is main concern in solvation

Nonpolar solvent into polar solvent = insoluble ΔH-TΔS=ΔG, G<1 = insoluble

o Think about the effect of ΔS between polar and nonpolar Hydrophobic effect:

Water molecules force non-polar molecules together and surround withcages

They clump up to reduce surface area: called the hydrophobic effect Nonpolar molecules increase ordering of water molecules over a long

range

Two nonpolar molecules are attracted over a long distance (up to 50nm)

o “Hydrophobic interaction”

By aggregating nonpolar species in H20 entropy is increased

Single most important factor that drives initial protein

folding/assembly of cell membranes

Hydrophobic amino acid residues cluster in interior of proteins, alsonucleic acid structure

Long range assembly, short range stabilize




o Lecture 4: Lipids

All surfactant molecules are amphiphilic but not all amphiphilic molecules are surfactants

Lipidso Water insolubleo Saturated and unsaturated hydrocarbons




o Ampiphiles = “fatty acids” consisting of polar head group and hydrophobic tail determine cell membrane structure

18 is the magic number!o Below 18 C-C bonds they tend to be soluble

o o This disrupts packing and makes membrane more fluido Note the kink in oleic acid below:

o In aqueous solutions common fatty acids will minimize surface tension and ball up




Phospholipid = fatty acyl (removal of hydroxyl groups) + glycerate + phosphate + polar head

groupo Major constituent of cell membraneso Polar head group linked thru phosphodiester to glycerolo Glycerol linked to 2 fatty acids through ester bondso Know the following:

They are attached to a phosphate (-) so ethanolamine and choline are neutral at

physiological pH Serine has extra (-) so net negative chargemostly on one side of membrane Choline is also known as lecithin




L to R: ethanolamine, serine, choline Choline has steric hindrance due to bulky head group often found on outside

not inside of membrane

Sphingolipidso Based on ceramide (a sugar) which is sphingosine (long chain amino alcohol) coupled

thru amide linkage to another fatty acyl chaino Abundant in neural tissueo Sphingomyelin = neutral at normal pH.

Same head group as phosphatidyl choline but no intermediate glycerol, it’slinked to ceramide instead.

o Cerebrosides Sugar residues directly attached to ceramide—no phosphate

o Gangliosides Sugar residues and acetylneuraminic residues attached to ceramid (no

phosphate) Difference is # of sugars attached Net negative charge at neutral pH

Primarily found in outer membraneo Glycolipids: similar to phospholipids but the phosphate polar head group is replaced by

saccharides o Galactocerebroside = one sugar add to this section if time

Plasmalogenso Fatty acyl chains linked to glycerol-3-phosphate through vinyl ether (rather than ester

link) Ceramide v. ester linkage Found in nervous system, cardiac muscle tissue

Lipids aggregate in water! Drug delivery vesicles liposomes begin with a phospholipid, often stearate

Amphiphilic assemblyo Depends on geometry and compositiono If polar head group is bigger than tails, then there will be a micelle or a curved bilayero If polar head is same as tails, planar bilayero Membrane assembly occurs in presence of water

Polar so weak interactions Change of environment = structural changes Driving force is entropy

Phospholipids have lateral mobility in the membrane but not across membraneo Slow kinetics moving through membrane to other side

Cholesterolo

25% of membraneso Increase nature of the reaction –kink pushes adjacent lipids apart to increase fluidity of

membraneso Amphiphilic but not a surfactanto Extremely hydrophobico Predominantly hydrocarbon—flexible part, rigid part, terminal –OH groupo Associated with the hydrocarbon component of lipid bilayerso 1) Affects physical properties of the membrane




o 2) broadens phase transitions

o

Chemistry and size interactions

Phospholipid bilayers




o Two solvent interfaceso Constraints:

Cell curvature Range of phospholipids Mean that structures are asymmetric

o RBC: choline head groups (phosphatidyl choline and sphingomyelin) are in outer half of bilayer

o Charge asymmetry and more fluid inner layero Glycolipids found only in outer part of membrane

Excess negative charge glycosylated = mostly on outsideo REMEMBER H2O is 2Å: size and symmetry have been on test

Membrane permeabilityo Depend on molecular size, solubilityo Easiest to go through = hydrophobic and small molecules (O2, N2)o Ion channels are gatedo Benzene is carcinogenic because it enters cells easily (hydrophobic) and reacts with

nucleic acids easily to change themo Urea distrupts hydrophobic and forms “pores”—has dipole moment and H-bonds?????

Non-ionized structure with carbonyl and two amino groups (NH2) Strongly associates water and forms “cages” to trap hydrophobic molecules Assumes more control over water around hydrophobic residues than do the

hydrophobic residues About dehydration between molecules –entropy driven Remove all the water between two objects to lower entropy Urea pulls out water and therefore disrupts kinetic process

o Ion concentrations differ inside and outside of membrane Na+

Intracellular: 5-15 mM

Extracellular: 145 mM K+

Intracellular: 140 mM Extracellular: 5 mM

Red blood cellso 43% lipido 8% carbohydrateo 49% protein

Cell membrane composition depends on the membrane Arrangement of membrane proteins

o Phospholipids = structureo

Proteins = functiono Membrane bound proteins

Peripheral “easy to remove” Integral “difficult to remove”

Anchored or penetrate through bilayer (transmembrane) or formsolvent channels

May interact with a ligand

May be for stability within by interacting w/cytoskeleton etc.




o Type of arrangement can be predicted from amino acid composition of proteins Hydrophobic regions through membrane Hydrophilic regions exposed to water on either side of the membrane

Glycoproteins

oligosaccharide ligands located on external face of membraneo Secondary structure of transmembrane proteins

Often α-helical passing through the membrane Often β-sheet passing multiple times 1.5 Å per residue in α-helix through a 50 Å membrane = about 33 amino acids

o Serine, tyrosine, threonine are O-linked (have –OH groups)o Ser and or Thr clusters= sugar binding region

Glycophorin




This is hard to manufacture because CHO (carbohydrates) sterically hinder unless put as small

CHO and added to things later

Cytosol reduces

Decay Accelerating Factor (DAF)o Complement factors like oposino Complement cascade

Macrophage labeling Attack complex: punches holes in cell membranes

o Almost identical protein subunits inhibit C3bo Ser/Thr region attaches O-linked sugarso Deficiency may cause deatho Glycan is a sugar sequenceo See pic below:




o It is much easier for phospholipids to translate around the lipid membrane than cross through it

to another positiono This is due to alignment of polar heads and hydrophilic tails, it’s already aligned so to

move from one point to another (or different sides of membrane) w/o translation ishigh energy

o “Flipases” are enzymes that aid in moving them from one side to another

Ion channelso “gated” pore-forming integral membrane proteinso To study ion channels, must keep them in a surfactant to protect structureo Present in all cell membraneso >106 ions/sec transporto Ions pass down an electrochemical gradient

Function of ion concentration and membrane potentialo Usually involve a circular arrangement of identical proteins symmetrically packed

around a water-filled pore through membrane planeo Symmetry




o G-proteins

o Guanine nucleotide-binding proteinso These have β and γ subunits o Molecular switches

o They turn GDP into GTP and vice-versa Integrins

o Transmembrane receptors mediating attachment between cell and ECM proteinso Have α and β subunits o Shape changes if active/inactive through cytoskeletal changes

How to view cell membraneo Freeze-etching: freeze it, put in vacuum chamber, remove ice by sublimation, cleave

with cold and sharp knife to image (e- microscopy) Red blood cells

o Concave shape is due to a network of membrane proteins Actin, spectrin, ankyrin (“anchor”), etc.




REVIEW:

Go over symmetry and the error on the packet regarding S36.

Must know the structures of purine and pyrimidine

o Which have 2 rings? Which have 1?

Purines = “two-rings” & 2 syllables = “A-G”

o Do not have to draw them

o AG = purine

o CUT = pyrimidine

o GC-Three hydrogen bonds

o AT-Two hydrogen bonds

Remember stuff from Watson-Crick movie GO OVER HW

Patterson calculations done to find the lowest energy conformation. Very long calculation

Go over STRUCTURE of amino acids—make flash cards

Think of design

o Amino acid design capabilities

40% of glycocalyx is protein

Sugars:

o Go over Fischer projections vs. ring form

o C5 hydroxyl determines right or left handed form

o Look at dat slide

Rank by platelet permeability Basic differences between A and B form DNA (z-form is syn v. anti, so it’s left-handed)

Puckered conformation of that ring

Lecture 5: Proteins

AMINO ACIDS: 20 α-amino acids are monomers of proteinso Each has a central carbon atom, called the α-carbon

Attached to 4 groups: the α-amino group (basic), an acidic carbonyl group, ahydrogen atom, an R group




The α-carbon is CHIRAL (optically active, asymmetric) in everything but glycine Two isomers are possible: D (dextro) and L (laevo) form Configuration = L-stereoisomer

o o Amino acids are “zwitterions” and have both + and – ionized groups

In proteins they form peptide bonds and become non-zwitter

o o Hydrophilic groups (polar)

Often located on the outside of a protein in contact with water Can stabilize structure through H-bonds Neutral: “squinty”

Serine [S]o Forms H-bonds, e.g. serine proteaseo S and T are often found at the active site of enzymeso S and T often linked to carbohydrateso O-linked carbohydrate bonds




o Has an –OH

Glutamine [Q]o Has amide group in side chain

Asparagine [N]o N-linked carbohydrate bondso Has amide group in side chain

Threonine [T]o S and T are often found at the active site of enzymeso S and T often linked to carbohydrateso T has a second asymmetric center around β-carbono Also O-linkedo Has an –OH

Tyrosine [Y]o Less polar than mosto Also has –OH

CHARGED AMINO ACIDS: HERD-K Basic: R,H,K

Lysine [K]o Flexible: rotational freedomo Strong base @ ph 7o Longer crosslinks + elasticity

Arginine [R]o Most basic amino acid

Histidine [H]o Charge depends on pH: pKa is 6o Has an imidazole ring in side chain

Acidic: ED (remember RGD RGE)

Asparatate/aspartic acid [D]o Side chains ionized at pH 7, so confer a negative charge

Glutamate/glutamic acid [E]o RGD v. RGE: difference is one methyl (1Å) but still changes

specificity

pKa is the Acid Dissociation Equilibrium Constanto Important in considering biopolymer structureo What charge do things have under physiological conditions?o Graphical representation




o o The above image found through titrationo Isoelectric point: pH where a protein is charge neutralo pKa values can shift due to local interactions within a protein

o




o Hydrophobic groups PILGAMVFWC “pill game + fw + cv” Chemically unreactive Establish and maintain the 3-D structure of proteins thru hydrophobic

interactions Often located in the interior Proline [P]

Forces a turn due to ring structure: will NEVER find it in an α-helix or aβ-sheet: DISRUPTIVE

More polar than most hydrophobic amino acids Isoleucine [I]

Has a second asymmetric center around β-carbon Leucine [L]

Chemically unreactive, so it gets 3-D structure through hydrophobicinteractions

Often in the core of the protein

Very common Glycine [G]

Spacer amine

Optically inactive

SIMPLEST its side group is simply a hydrogen atom.

Often found in α-helices Alanine [A]

Chiral and optically active

Very common Methionine [M]

AUG “start” codon for translation

Has sulfur in side chain Relatively rare in proteins

Valine [V] Phenylalanine [F]

Hydrophobic aromatic side chain Tryptophan [W]

Signaling Hydrophobic aromatic side chain Relatively rare in proteins

Cysteine [C]

Has S-S (disulfide) bonds

Chemically reactive -SH group

o Can react with another sulfhydryl to firm cysteine, with adisulfide bond

Weak H-bonds with O, N

Short crosslinks in globular proteins

Intramolecular covalent linkages to stabilize 3-D structure of protein More polar than most hydrophobic residues

o Collagen—has lots of G, P, A




Are proteins internal or external membrane associated?o Predict empirically by “discriminant function” Z

Based on hydrophobicity, ratio of polar:nonpolar residues Z is about 0.52 for internal v. 0.12 external About 0.17 for non-membrane proteins

o o Histone has lots of lysine and arginine (+) because it binds to DNA which is negativeo Purple membrane has lots of leucine, alanine, glycine b/c they are hydrophobic

Other protein moietieso Zymogens: enzyme conversion by cleavage near the active site

Converts a single chain protein into a 2-chain active enzyme Often due to a change in conformation to weaken it then cleavage

o Modification of amino acids Dicarboxlation adds an extra carboxylate to a carbon which aids binding to

platelets through a calcium brige

This happens in the liver




This is the basis of rat poison. Blocking inhibits coagulation Addition of hydroxyl groups to amino acids

Proline hydroxyproline Lysine hydroxylysine

Addition of phosphate

Phosphoserine Zwitterization

o Bound carbohydrate residues To Ser, Thr, hydroxylysine

o Bound divalent cations and prosthetic groups Iron in chromatium, myoglobin, Hb

o Bound small organic molecules Most often to lysine E.g. biotin which is used to bind H+

Location of crosslinks e.g. cysteine pairso Disulfides –C-S-S-C— provide crosslinks between different parts of a protein

Structural stabilityo May be intra and or intermolecularo Important for tertiary structureo Indicates that residues far apart in sequence may be in close proximity in tertiary

structure

o Cystine bond

Non-linear Non-planar: carbons are in different planes

Many are present in IgG

o Other crosslinks

Lysine-glutamine in fibrin

Lysine-lysine, lysine-histidine in collagen/elastin

Lysine: in elastin bc it has a long chain so can make materials rubbery Free rotation

Elastin: desmosines (aromatic elastomaeric rings with structural rigidity inmiddle but flexy chains




Sequence structure and function

o Repeates indicate symmetry

Every 3rd residue of collagen is usually Gly (it is small and has structuralflexibility—extra space) but prolines prevent α helices and β sheets fromforming so there’s a triple helix structure

Clusters of positively charged residues in histone bind to DNA

o Transmembrane protein glycophorin =

Lots of O-linked (circle: Ser and Thr) and N-linked (square, asparagine) on theside where glycocalyx would be, then there’s a nonpolar region where it goesthru wall, then more polar on the other side

o SIMILAR FUNCTION = USUALLY SIMILAR HOMOLOGYo Mutations change properties e.g. hemoglobin

One point defection: polar Glutamine nonpolar Valine




Nonpolar Val aggregates on outside and causes it to form fiberso Symmetry shows where helices are locatedo Myoglobin = easy to figure out because it has lots of α-helical parts to it

Secondary structureo Polypeptides

Peptide bond = resonance hybrid and polar Bond length 1-1.5 Å

Notice the resonance

N,C,O are forced into one plane. “plate-like” structure Configurations around peptide bond are either cis or trans

Almost all are in TRANS due to cis steric hindrance

Trans: has 3.8 Å between carbons Polypeptide conformation is determine by rotation around two bond types

Ψ = Cα-CO bonds Φ = N-Cα bonds

No rotation around the O-C-N resonance structure

Will undergo energy minimization

“Ramachandran plots” to predict the two rotation angles For the right-handed α-helix,

o Ψ = -57 degrees

o Φ = -47 degreeso Helices

α-helix

Pitch is 5.4 Å (advance per turn)

Rise is 1.5 Å (advance per amino acid residue) 185: 18 residues over 5 turns

o This is 3.6 residues per turn (3.6 * 1.5 = 5.4)




Ψ = -57 degrees

Φ = -47 degrees

Proline cannot sterically fit Diameter without side chains is 6 Å

Polyproline

Synthetic collagen

Polyproline Io 103 helix, 3.3 residues/turn, 1.9A residue repeato All cis peptide bonds

Polypropiline IIo 31 helix with all trans peptide bondso 3 residues/turno More stable than PP1 in aqueous media

Collageno 3 strands (three PPII twisted around each other)o Interchain H-bondso Proline points to outside, glycines inside

Glycine is not functional but for chargeo Secondary amine leads to crosslinking for stability, elasticity

310 helix

Short parts of some proteins, like α helix but has 3 residues/turn not 3.6 β-sheet

Two antiparralel or parallel polypeptide strands 2 residues/turn

Interstrand hydrogen bonding

3.4A per residue

When there are kinks that make the sheet fold back and forth it’s oftenproline

o If it’s charged you need a +/- interaction between B sheetso Or you can design it to assemble hydrophobically (Val-Leu-Val-

Leu)o X-ray diffraction

Bragg’s Law: calculate angles Scattering occurs due to crystal structure For DNA: put in a salt to induce charge shielding

Tertiary structureo Molecules “breathe”—lots of small movementso Lowest energy conformation

Hydrophobic effect

Hydrogen bonds Van der Waals Covalent disulfide bonds Electrostatic interactions

o Charged groups on outside and hydrophobic on the insideo H-bonding residues usually find a low-energy partner (often water)

H-bonds between amino acids, and water on surface H-bonds in helices and sheets stabilize folded conformation




o Globular proteins are sensitive to environment changeso Narrow range of stability

Denaturation can be reversibleo Packing density

Ration of total volume from Van der Waals spheres to total volume 0.79 close packed, 0.91 packed cylinders

Quaternary structureo Non-covalent assemblies e.g. Hb

Lect. 6: Nucleic Acids

Nucleoside = sugar and base

Nucleotide = sugar, base, phosphate (es”t”er) o The base is linked at the 1’ position o Phosphates linked at 3’ and 5’

DNA is read from 5’ to 3’

o Sugars

o




o The 5-hydroxyl determines if it’s D or L (D = right L = left)o The C1 aldehyde reacts with the C5 for ring structure

o Bases

o A,T-2 bondso C,G-3 bondso Ionization Equilibria

A: neutral or positive C: neutral or positive G: neutral, positive, negative U: neutral or negative

o TAUTOMERS Prevalent: keto, amine

There is also enol, imine

A,C amine

T,G ketoo Chargraff’s Rules

Pair A/T and C/G based on ratios of bases 40-45% CG in mammals This holds true for double stranded

o Cations will associate with phosphates (-)o DNA usually associated with specific proteins e.g. histone

Primary structureo Covalent chains

Double stranded DNA usually 5 x 103 to 108 residues

RNA is almost always single stranded

-OH causes steric hindrance

tRNA = 75 to 84 residues up to hnRNA with 2 x 105 residues Different structures: sometimes one strand is in pieces or there are loops.

Some structures of DNA/RNA caused by degradation Sequencing: use restriction endonucleases to cleave Genetic code: codons (64 codons)

These are degenerate—more than one codon for an amino acid usually

Stop = UAA, UAG, UGA

Start = AUG (Met) Long continuous stretches of complementary pairs almost always form double

stranded hairpins

tRNA = cloverleaf structure

Unpaired tail at top is “sticky end” pairing to stuff




Predict structure from sequence!

Double stranded RNA: bases resistant to reaction so they are probablyon the interior

Double stranded structure is independent of sequence

Watson and Cricko Keto-amine tautomers selectedo Points of attachments of bases to sugars = virtually identicalo Pseudo C2 axis in plane of base pair




o AT rich sections are SPLICED because 2 bonds weaker than 3o H-bonds between the bases:

Double Helix structure

Right-handed for A and B form Dyad (rotation) symmetry

Antiparallel

Distance between neighboring base pair planes is 3.5 Å, van der Waalsradius distance for planar aromatic compounds (remember that graphof optimal distance)

B-Form DNA

Naturally occurring (frequent in nature)o Aqueous solution form

Planar bases

High humidity (95%)

10.4 residues/turn, 34.6 degrees rotated each nucleotide Pitch = 34Å, diameter is 23 Å (much more than 6 Å α-helix)

IMPOSSIBLE FOR RNA A-form DNA: crystalline; tighter and wider

Bases are much more tilted

26 Å diameter May not exist under physiological conditions

11 residues/turn, 32.6 degrees rotated

Pitch = 24.6Å Z-form DNA: left handed, no grooves, maybe for short sections

Originally synthetic




Often GCGCGC syn/anti/syn/anti

A and B don’t have syn form only anti o Pyranose ring & puckering: A v. B forms

o When C2 is pushed down C3 is pushed up (C3 form) A-formo When C3 pushed down then C2 is pushed up (C2 form) B-form

Other

INTRINSIC PATHWAYINTRINSIC PATHWAY EXTRINSIC PATHWAYEXTRINSIC PATHWAY

XII XIIaXII XIIa

XI XIaXI XIa

IX IXaIX IXa

VIIVII

TF/VIIaTF/VIIa

Xa XXa X

FibrinogenFibrinogen FibrinFibrin

VIIIaVIIIaCa++Ca++

Ca++

HMWK

Kallekrein

Tissue

FactorCa++

IIaIIaIIIIXaXa

VaVa

Ca++Ca++

VaVa



Nucleosomes lead to packing

structure of biological materials outline

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