Biochem 1

structural biology

study of three-dimensional structures of biomolecules, including proteins, nucleic acids, lipid membranes, and oligosaccharides; employs biochemical approaches, physical tools, and computational methods

What we can learn from amino acid sequence:

-3D structure -function -cellular location -evolution

True or false: Proline and Glycine Occur Infrequently in an alpha Helix

True: they generally cause a break in alpha helix structures --proline introduces destabilizing kink in helix: nitrogen atom is part of rigid ring thus rotation about N-alpha carbon bond not possible --glycine has high conformational flexibility which takes up coiled structures

free-energy funnel

The Thermodynamics of Protein Folding: -unfolded states = high degree of conformational entropy, high free energy -N represents native state

Affinity Chromatography

-separates based on binding affinity -specific tag is added to protein of interest which recognizes something in resin (ligand) -eluted by high concentration of salt or ligand

Protein Secondary Structure (beta conformation)

- beta conformation is the backbone that extends into a zigzag -can be just a beta strand which is a single protein segment or a beta sheet which is several strands in a beta conformation side by side --Adjacent Polypeptide Chains in a beta Sheet Can Be Antiparallel or Parallel: -antiparallel = opposite orientation (occurs more frequently due more hydrogen bonds) -parallel = same orientation (less frequent) -Hydrogen bonds form between backbone atoms of adjacent segments which primarily stabilizes the beta sheet; the h bonds occurs between the amide nitrogen and the carbonyl carbon --beta turns -connect ends of two adjacent segments of an antiparallel beta sheet -180° turn -involves 4 residues -hydrogen bond forms between first and fourth residue -Gly (residue 2) and Pro (residue 3) often occur in beta turns -turns should "occur" at the Pro (according to practice exam) --Common Secondary Structures Have Characteristic Dihedral Angles: -dihedral angles φ(phi) and ψ (psi) associated with each residue completely dictate what type of secondary structure is made -all the angles are visualized by Ramachandran plots

How polypeptides fold

--Most polypeptides fold rapidly by a stepwise process: -local secondary structures fold first (ionic interactions play an important role) -longer range interactions follow (hydrophobic effect plays a significant role) -process continues until the entire polypeptide folds *see chp slide 118*

Folding Patterns of Proteins

--motif (aka fold): recognizable folding pattern involving 2+ elements of secondary structures and the connection(s) -can be simple, such as in a β-α-β loop -can be elaborate, such as in a β (beta) barrel -A motif could include a β turn, a β sheet, an alpha helix, disulfide bonds, and more --Complex Motifs Are Built from Simple Motifs: -i.e.- α/βbarrel = series of β-α-βloops arranged such that the βstrands form a barrel --Protein Motifs Are the Basis for Protein Structural Classification: -Protein Data Bank (PDB) = 150,000+ structures archived -Structural Classification of Proteins database (SCOP2) = searches protein information in the PDB

circular dichroism (CD) spectroscopy

-A method used to characterize the degree of folding in a protein, based on differences in the absorption of right-handed versus left-handed circularly polarized light. -measures differences in the molar absorption of left-handed vs. right-handed circularly polarized light -chromophore = peptide bond *Different Secondary Structures Have Different Circular Dichroism Spectra

Fibrous Proteins

-Adapted for a Structural Function -arranged in long strands (fibers) or sheets -give strength and/or flexibility to structures -simple repeating element of secondary structure -H2O insoluble due to high concentrations of hydrophobic residues *see chp 4 slide 75 for several important examples*

Neurodegenerative Conditions

-Alzheimer disease = associated with extracellular amyloid deposition by neurons, involving the amyloid-βpeptide -Parkinson disease = misfolded form α-synuclein aggregates into spherical filamentous masses called Lewy bodies -Huntington disease = involves the intracellular aggregation of huntingtin, a protein with long polyglutamine repeat

Peptide bond length and its affect on conformation

-CO double bond length is usually 1.2 and CN single bond is usually 1.46. But in peptides it's 1.24 and 1.32 respectivley. This is because the peptide bond actually demonstrates resonance -Because of the resonance the CN peptide bond demonstrates a bit of double bond characteristics which causes the 6 atoms of a peptide bond to be rigid and planar which limits the range of conformations -this resonance also causes a partial negative charge and partial positive charge that sets up a small electric dipole within the peptide bond (see chp 4 slide 21) --there are 2 dihedral angles: -phi (φ) angle: angle around the alpha carbon-amide nitrogen bond (between −180 and +180 degrees) -psi (ψ) angle: angle around the alpha carbon-carbonyl carbon bond (between −180 and +180 degrees) -the limitations of these angles define the conformations that a peptide bond can make -many φ (phi) and ψ (psi) values are prohibited by steric interference (i.e. φ and ψ cannot both = 0 degrees)

consensus sequence

-Comprises the most commonly encountered nucleotides found at a specific location in DNA or RNA; reflects most common amino acid at each position This term is applied to such sequences in DNA, RNA, or protein. When a series of related nucleic acid sequences or protein sequences are compared, a consensus sequence is the one that reflects the most common base or amino acid at each position. Parts of the sequence that have particularly good agreement often represent evolutionarily conserved functional domains. Mathematical tools available online can generate consensus sequences or identify them in sequence databases.

Protein Domains

-Domain is a part of a polypeptide chain that is independently stable or could undergo movements as a single entity -domains may appear as distinct or be difficult to discern -small proteins usually have only one domain

protein disulfide isomerase (PDI)

-Isomerization Reaction -catalyzes interchange, or shuffling, of disulfide bonds

peptide prolyl cis-trans isomerase (PPI)

-Isomerization Reaction -catalyzes the interconversion of the cis and trans isomers of peptide bonds formed by Pro residues

peptides and peptide bonds

-The chemical bond that forms between the carboxyl group of one amino acid and the amino group of another amino acid -formed through condensation (lose H2O) -broken through hydrolysis (add H2O) -The 𝛼α‑amino group of one amino acid donates electrons to displace the hydroxyl group from the 𝛼α‑carboxyl of the other amino acid dipeptide = 2 amino acids, 1 peptide bond tripeptide = 3 amino acids, 2 peptide bonds oligopeptide = a few amino acids (cutoff is prolly around a dozen) polypeptide = many amino acids, molecular weight < 10 kDa protein = thousands of amino acids, molecular weight > 10 kDa Peptide terminals: N-terminal- amino end C-terminal- carboxyl end *naming starts from N-terminal (amino-terminal residue); 1st enzyme r-group is called residue number 1, next is residue number 2, etc. Naming peptides example: full amino acid names: serylglycyltyrosylalanylleucine three-letter code abbreviations: Ser-Gly-Tyr-Ala-Leu one-letter code abbreviation: SGYAL Protein types: multisubunit protein = 2 or more polypeptides associated noncovalently oligomeric protein = at least 2 identical subunits, which are called protomers (i.e. hemoglobin)

Anfinsen experiment

-The hypothesis that "protein amino acid sequence determines the final shape a protein assumes in a water solution" was proven to be correct when Christian B. Anfinsen showed that if the enzyme ribonuclease was opened out into a linear chain and then allowed to reform, it reassumed the correct catalytic shape. -The Anfinsen experiment provided the first evidence that the amino acid sequence of a polypeptide chain contains all the information required to fold the chain into its native, three-dimensional structure.

zwitterion

-a molecule or ion having separate positively and negatively charged groups, but is neutral overall -Amphoteric (reacts as acid or base) compound with no net electric charge. -Most often used to describe amino acids -when on a titration curve the amino acid is at the isoelectric point (pI) (vertical point)

amino acids

-a simple organic compound containing both a carboxyl (—COOH) and an amino (—NH2) group. - -can act as acids or bases

Size-Exclusion Chromatography

-also called gel filtration chromatography -separates based on size -large proteins emerge from the column before small proteins do

Protein-Folding Rules

-burial of hydrophobic R groups requires 2+ layers of secondary structure -αhelices and βsheets are found in different layers -adjacent amino acid segments are usually stacked adjacent -the βconformation is most stable with right-handed connections

nuclear magnetic resonance (NMR)

-carried out on molecules in solution --captures dynamics of protein structure: -conformational changes -protein folding -interactions with other molecules -NMRmeasures nuclear spin angular momentum, a quantum mechanical property of atomic nuclei; nuclear spin of 1H, 13C, 15N, 19F, and 31P gives rise to an NMR signal; nuclear spin generates a magnetic dipole -magnetic field causes magnetic dipoles to align in the field in one of two orientations: parallel (low energy) or antiparallel (high energy) -pulse of electromagnetic energy generates an NMR spectrum; spectra can even be translated into a 3D image of protein --Two-Dimensional NMR Techniques: -NOESY = nuclear Overhauser effect (NOE) spectroscopy = measures distance-dependent coupling of nuclear spins in nearby atoms through space -TOCSY = total correlation spectroscopy = measures the coupling of nuclear spins in atoms connected by covalent bonds

cystic fibrosis

-caused by defects in the membrane-bound protein cystic fibrosis transmembrane conductance regulator (CFTR); deletion of a Phe residue causes improper protein folding which leads to CFTR protein

Some Proteins Undergo Assisted Folding

-chaperone proteins facilitate correct folding pathways or ideal microenvironments; they fold proteins into a native conformation Examples: -Hsp70 = bind to hydrophobic regions; a type of ATPase -chaperonins = required for the folding of proteins that do not fold spontaneously

proteostasis

-continual maintenance of the active set of cellular proteins required under a given set of conditions -"protein homeostasis" -involves chaperones, degradation, unfolding, disaggregation, aggregation, folding, autophagy (*see image*)

globular proteins

-fold back on each other into a spherical or globular shape -more compact than fibrous proteins -spherical, water-soluble proteins -examples include: enzymes, transport proteins, motor proteins, regulatory proteins, immunoglobulins -each globular protein has a distinct structure, adapted for its biological function; there is much more structural variety when compared to fibrous proteins -These proteins are not classified as either all α (alpha) or all β (beta)

free metabolites

-intermediates when the body synthesizes amino acids -(e.g., ornithine, intermediate in arginine biosynthesis)

intrinsically disordered proteins

-lack definable structure; often changes structure significantly depending on what it's bound to -often lack a hydrophobic core -high densities of charged residues (Lys, Arg, Glu) and Pro -facilitates a protein to interact with multiple binding partners -might only be segments of protein -PONDR score: measures peptides tendency to be disordered (anything above .5 is considered a intrinsically disordered region or segment) -example include p53

denaturation

-loss of three-dimensional structure sufficient to cause loss of function -can occur by heat, pH extremes, miscible organic solvents, certain solutes, detergents -often leads to protein precipitation -can lead to the cause of many diseases

mass spectrometry

-measure molecular mass with high accuracy -can sequence short amino acid sequences (20 to 30 amino acid residues); which is actually rather limited. So we use proteases to cleave polypeptide chains -can document the entire cellular proteome (an organism's complete set of proteins)

Modifications of common amino acids

-modified after protein synthesis (e.g., 4-hydroxyproline, found in collagen) -modified during protein synthesis (e.g., pyrrolysine, contributes to methane biosynthesis) -modified transiently to change protein's function (e.g., phosphorylation. Turns on or off protein activity and may modulate structure)

The Prion Diseases

-prion protein (PrP) = misfolded brain protein; can lead to death

renaturation

-process by which certain denatured globular proteins regain their native structure and biological activity -Anfinsen experiment showed the amino acid sequence contains all the information required to fold the chain -Denaturing followed by renaturing of a protein demonstrates that primary structure dictates tertiary structure.

amyloid fiber

-protein secreted in a misfolded state and converted to an insoluble extracellular fiber -amyloidose diseases include: type 2 diabetes, Alzheimer disease, Huntington disease, and Parkinson disease Formation of Disease-Causing Amyloid Fibrils: -native= high degree of β-sheet structure; misfolded β (beta) amyloid promotes aggregation, forming an amyloid fibril

Protein Families and Superfamilies

-proteins with significant similarity in primary structure and/or tertiary structure and function are in the same protein family - ~4,000 different protein families in the PDB -strong evolutionary relationship within a family superfamilies: 2+ families that have little sequence similarity, but the same major structural motif and have functional similarities (evolutionary relationship is probable)

quaternary structure

-refers to an arrangement of tertiary protein subunits in a three-dimensional complex -assembly of multiple peptide subunits -oligomer (multimer) is a multisubunit protein; a repeating structural unit is called a protomer

cryo-electron microscopy (cryo-EM)

-sample of the structure of interest is quick-frozen in vitreous (or noncrystalline) ice and kept frozen while being observed in two dimensions with the EM -greatly reduces damage to the specimen -useful for determining the molecular structure of large, dynamic, macromolecular complexes and integral membrane proteins

General Steps Involved in Mass Spectrometry

-sample prep: digest samples using proteases (if needed) -first step: ionize analytes (substances being measured) in a vacuum -second step: introduce charged molecules to electric and/or magnetic field -third step: charged molecules move through field as a function of the mass-to-charge ratio, m/z -fourth step: deduce mass (m) of analyte

Protein Secondary Structure (alpha helix)

-secondary structure describes the spatial arrangement of the main-chain atoms in a segment of a polypeptide chain -in a regular secondary structure φ (phi) and ψ (psi) remain the same throughout the segment -common types are alpha helix, beta conformation, beta turn, random coils --The alpha helix is a common protein secondary structure: -it's the simplest arrangement which is stabilized because it has the maximum number of hydrogen bonds that each atom can make in the peptide (hydrogens hydrogen bond to nearby electronegative nitrogen) -the backbone (main chain atoms) is wound around an imaginary longitudinal axis -the R groups protrude out from the backbone -each helical turn = 3.6 residues, ∼5.4 Å **each hydrogen bond in alpha helix occurs between hydrogens and nearby electronegative nitrogen and they occur 4 amino acids away from each other which confers stability --the alpha helixes are also affect by amino acid residues' intrinsic propensity: -interactions between R chains spaced 3-4 residues apart can stabilize or destabilize alpha helix -charge, size, and shape of R chains can destabilize -formation of ion pairs and hydrophobic effect can stabilize --The helixes express handedness: -right-handed alpha helixes (the R groups protruding away from the helical backbone; most common -extended left-handed (theoretically less stable; not observed in proteins) --Amino Acid Residues Near the End of the alpha Helix Segment Affect Stability: -small electric dipoles in each peptide bond align through hydrogen bonds -negatively charged amino acids often found near the NH3+ terminus -positively charged amino acids often found near the COO- terminus

Ion Exchange Chromatography (IEC)

-separates based on sign and magnitude of the net electric charge -pH and concentration of free salt ions affect protein affinity -uses bound charged groups: cation exchangers and anion exchangers

Denaturation curve

-shows how proteins can be denatured by various causes -Tm is the midpoint (for temperature curves); the higher the Tm the more stable the protein

How can proteins be separated and purified?

-size -charge -binding properties -protein solubility

Protein Tertiary and Quaternary Structure

-tertiary structure: overall three-dimensional arrangement of all the atoms in a protein (weak interactions and covalent bonds hold interacting segments in position) -quaternary structure: arrangement of 2+ separate polypeptide chains in three-dimensional complexes

collagen

-type of fibrous protein -found in connective tissue -when in a secondary structure it is very unique. It is a left-handed, repeating tripeptide unit Gly-X-Y, where X is often Pro and Y is often 4-Hyp -tertiary and quaternary structure is a right-handed twisting of 3 separate polypeptides (cross-linked triple helices) --Covalent Cross-Links in Collagen Fibrils: -cross-linked by covalent bonds involving Lys, HyLys (5-hydroxylysine), or His (links create uncommon amino acid residues; dehydrohydroxylysinonorleucine) --Scurvy, Vitamin C, and Collagen Formation: -scurvy is caused by a lack of vitamin C (characterized by general degeneration of connective tissue which is usually made up of collagen) -vitamin C is required for the hydroxylation of proline and lysine in collagen

Fibroin

-type of fibrous protein -main protein in silk -contains antiparallel beta sheets that are tightly stacked upon one another -predominantly β (beta) conformation -rich in Ala and Gly -stabilized by hydrogen bonding and van der Waals interactions

alpha Keratin in hair

-type of fibrous protein -α-keratin helix is a right-handed α (alpha) helix -two strands of α-keratin, oriented in parallel, wrap about each other to form a supertwisted coiled coil (supertwisted helical path is left-handed) --Hair Contains Many α-Keratin Filaments: -rich in hydrophobic residues: Ala, Val, Leu, Ile, Met, Phe -cross-links stabilized by disulfide bonds

Myoglobin

-type of globular protein -An oxygen-storing, pigmented protein in muscle cells. -includes: alpha helical regions, binding pocket, hydrophobic R chains -the hydrophobic side chains tend to collapse to form the interior of the globular protein -*see chp 4 slide 89 for its structural representation*

bioinformatics

-use of computer databases to organize and analyze biological data -identifies functional segments in new proteins -establishes sequence and structural relationships to known proteins Tells which proteins are more essential and which ones aren't as much essential amino acid residues are conserved over evolutionary time less important amino acid residues vary over evolutionary time

Edman degradation

-used to analyze small proteins - selectively & sequentially removes the N terminal amino acid of the protein which is analyzed by mass spectroscopy

Electrophoresis

-used to visualize and characterize purified proteins -can be used to estimate: --number of different proteins in a mixture --degree of purity --isoelectric point --approximate molecular weight Electrophoresis for Protein Analysis: -uses cross-linked polymer polyacrylamide gels -proteins migrate based on charge-to-mass ratio -visualization by coomassie blue dye which binds to proteins

oligo synthesizer

-uses Merrifield method; allows for smaller peptides to be chemically synthesized (roughly 20 amino acids long)

HPLC (High-Performance Liquid Chromatography)

-uses high-pressure pumps to move proteins down the column (vs letting column flow via gravity) -greatly improves resolution

Ramachandran plots

-visualizes all φ (phi) and ψ (psi) angles that can be made -tests quality of three-dimensional protein structures -in a plot there will be dots that represent each amino acid and their corresponding phi and psi angles; where the dots occur represent the region of the angles; note: glycine frequently falls outside the expected ranges

protein conformation

-while there is basically a limitless amount of conformations in which a protein can fold, there are a limited number of conformations that predominate under biological conditions of cells -we only refer to the most thermodynamically most stable conformations which are the conformations with the lowest free energy (G) -proteins in their functional form are called the "native" version of that protein --A Protein's Conformation Is Stabilized Largely by Weak Interactions: -stability is the tendency of a protein to maintain a native conformation; some proteins might only last an hour or two while the more stable ones can last several days -unfolded proteins have high conformational entropy cause they move around randomly, unhindered; because of this chemical interactions are needed to stabilize native conformations... these include strong disulfide (covalent) bonds which are uncommon (and only occur b/w a met and cys) and weak (noncovalent; nonpolar) interactions and forces which are numerous (hydrogen bonds, hydrophobic effect, ionic interactions) --hydrophobic effect is the predominating weak interaction that keeps proteins from unfolding: -solvation layer is a highly structured shell of H2O around a hydrophobic molecule: *decreases when nonpolar groups cluster together *decrease causes a favorable increase in net entropy -surrounding molecules that force hydrophobic R groups on proteins towards inner area -hydrophobic R chains form a hydrophobic protein core --Polar Groups Contribute Hydrogen Bonds and Ion Pairs to Protein Folding: -these are often found in repeating secondary structures (alpha helices and beta sheets) where hydrogen bonding is fully optimized --interaction of oppositely charged groups = ion pair = salt bridge -strength increases in an environment of lower dielectric constant, ε: polar aqueous solvent (ε ~ 80) and nonpolar protein interior (ε ~ 4)... she didn't really talk about this :/ --Individual van der Waals Interactions Are Weak but Combine to Promote Folding -van der Waals interactions are dipole-dipole interactions over short distances -individual interactions contribute little to overall protein stability, however there are a high number of interactions that are actually substantial

X-ray Diffraction Produces Electron Density Maps from Protein Crystals

-x-ray crystallography = pattern of diffracted x-rays is collected directly and an image is reconstructed by mathematical techniques -limited to molecules that can be crystallized -usually doesn't give much information on how the observed protein works -information obtained depends on the degree of structural order in the sample Fourier transform- constructs an electron-density map from the overall diffraction pattern of spots. Problem with x-ray crystallography: the physical environment in a crystal is not identical to the physical environment in solution or in a living cell; imposes a space and time average on the structure; provides little information about molecular movement of protein; crystal-derived structure usually only represents a functional conformation

Protein Crystallography Steps

1) X-ray diffraction patterns from protein crystals 2) three-dimensional electron-density map using a Fourier transform 3) localized atomic nuclei 4) completed protein structure

Protein structure

4 levels: --primary structure- covalent bonds linking amino acid residues in a polypeptide chain --secondary structure- recurring structural pattern --tertiary structure- 3D folding of polypeptide --quaternary structure- 2+ polypeptide subunits -amino acid sequence confers 3D structure -3D structure confers function -most human proteins are polymorphic (have amino acid sequence variants) Edman degradation: classic method of sequencing amino acids

Methods for Purifying Proteins

First step: break open tissue or microbial cells which releases the crude extract, that is all the proteins contained in solution Second step: fractionation (separate proteins into fractions based on size or charge). One method is called "salting out" where you lower solubility of proteins in salt to selectively precipitate proteins Third step: dialysis (use semipermeable membrane to separate proteins from small solutes) Most common method for purifying proteins is Column Chromatography: First step: buffered solution (mobile phase) migrates through porous solid material (solid phase) Second step: buffered solution containing protein migrates through solid phase; protein properties affect migration rates Types of column chromatography: -Ion-Exchange -Size-Exclusion -Affinity Chromatography -HPLC (High-Performance Liquid Chromatography) *see chp 3 slides 44-48* Protein purification can use multiple of the above steps to isolate the desired protein. Purification results: The ratio of the final specific activity (in units/mg) to the starting specific activity (in units/mg) gives the purification factor. The percentage of the total activity at the last step (units) relative to the total activity in the starting material (units) gives the yield from the purification procedure

Classifying Proteins

Four major types of protein groups based on polypeptide chains: -fibrous proteins: arranged in long strands (fibers) or sheets -globular proteins: folded into a spherical or globular shape -membrane proteins: embedded in hydrophobic lipid membranes; allow them to be embedded in lipid bilayer -intrinsically disordered proteins: lacking stable tertiary structures; mostly unfolding and often times partly form secondary structure until they are activated by a different binding protein to serve a new function

Levinthal's Paradox

It is mathematically impossible for protein folding to occur by randomly trying every conformation until the lowest energy one is found

Sodium Dodecyl Sulfate (SDS)

Negatively-charged detergent used to denature and confer a negative charge to proteins before separation by electrophoresis -binds and partially unfolds proteins -gives all proteins a similar charge-to-mass ratio -electrophoresis in the presence of SDS separates proteins by molecular weight; smaller proteins migrate more rapidly

CHP 4 (part 1) Principles

Principle 1: ---Protein structures are stabilized by noncovalent interactions and forces: -Formation of a thermodynamically favorable structure depends on the influences of the hydrophobic effect, hydrogen bonds, ionic interactions, and van der Waals forces -Natural protein structures are constrained by peptide bonds, whose configurations can be described by the dihedral angles φ and ψ. Principle 2: --Protein segments can adopt regular secondary structures such as the α helix and the βconformation: -These structures are defined by particular values of φ and ψ and their formation is impacted by the amino acid composition of the segment. -All of the φ and ψ values for a given protein structure can be visualized using a Ramachandran plot. Principle 3: --Tertiary structure describes the well-defined, three-dimensional fold adopted by a protein: -Protein structures are often built by combinatorial use of common protein folds or motifs. -Quaternary structure describes the interactions between components of a multisubunit assembly Principle 4: --Tertiary structure is determined by amino acid sequence: -Even though protein folding is complex, some denatured proteins can spontaneously refold into their active conformation based only on the chemical properties of their constituent amino acids. -Cellular proteostasis involves numerous pathways that regulate the folding, unfolding, and degradation of proteins. -Many human diseases arise from protein misfolding and defects in proteostasis. Principle 5: --The three-dimensional structures of proteins can be defined. -Structural biologists use a variety of instruments and computational methods to solve biomolecular structures. -The choice of method may depend on factors such as the size of the protein being studied, its properties, or the desired resolution of the final structure.

CHP 3 principles

Principle 1: In every living organism, proteins are constructed from a common set of 20 amino acids .•Each amino acid has a side chain with distinctive chemical properties. •Amino acids may be regarded as the alphabet in which the language of protein structure is written Principle 2: In proteins, amino acids are joined in characteristic linear sequences through a common amide linkage, the peptide bond .•The amino acid sequence of a protein constitutes its primary structure, a first level we will introduce within the broader complexities of protein structure. Principle 3: For study, individual proteins can be separated from the thousands of other proteins present in a cell, based on differences in their chemical and functional properties arising from their distinct amino acid sequences. •As proteins are central to biochemistry, the purification of individual proteins for study is a quintessential biochemical endeavor. Principle 4: Shaped by evolution, amino acid sequences are a key resource for understanding the function of individual proteins and for tracing broader functional and evolutionary relationships.

Conjugated Proteins

Proteins that contain chemical groups other than amino acids; non-amino acid part is the prosthetic group Types: Lipoproteins- contains lipids (i.e. β1-Lipoprotein of blood) Glycoproteins- contains carbohydrates/sugar (i.e. immunoglobulin G) Phosphoproteins- contains phosphate groups (i.e. Glycogen phosphorylase) Hemoproteins- contains heme (iron porphyrin) (i.e. Hemoglobin) Flavoproteins- contains flavin nucleotides (i.e. Succinate dehydrogenase) Metalloproteins- contain specific metals; Iron, Zinc, Calcium, Molybdenum, Copper (i.e. Ferritin, Alcohol dehrogenase, Calmodulin, Dinitrogenase, and Complex IV)

intrinsically disordered prote

Proteins, or segments of proteins, that lack a definable three-dimensional structure in solution.

isoelectric point (pI)

The point in the titration of an amino acid with a strong base where all of the carboxylic acid functional groups have been deprotonated - molecule has no net charge between the (R-COO)- and the (R-NH3)+. Maximum number of zwitterions. Dictated by the side group of the amino acid. The more acidic the side group, the lower the pI. Ex: only amino acids with an acidic isoelectric point (all except histidine, arginine, and lysine) will be negatively charged at a basic pH (8) - for electrophoresis where the negatively charged amino acids will run towards the anode. For amino acids without ionizable side chains, the isoelectric point (pI) is: pI = (pk1 + pk2) / 2 If: -pH = pI = net charge is zero (amino acid least soluble in water, does not migrate in electric field) -pH > pI = net negative change -pH < pI = net positive charge Ionizable side chains: -have a pKa value -act as buffers -influence the pI of the amino acid -can be titrated (titration curve has 3 ionization steps)

proteases

catalyze the hydrolytic cleavage of peptide bonds

Titration of amino acids

pk1- pka of carboxylic group; when molecule is a cation; usually around pH of 2 pI- isoelectric point. Point where molecule is a zwitterion pkr- pka of the side chain (residue) pk2- pka of amino group; when molecule is an anion; usually around pH of 9

topology diagram

represent elements of secondary structure and the relationships among segments of secondary structure in a protein