protein periodicity

has no periodicity

unlike DNA, does not recur at regular intervals

protein sequence dependency

dependent on sequence, unlike DNA

seq of aas determines three dimensional structure

spontaneous refolding

results in fully denatured protein regaining its enzymatic activity

in vivo, usually aided by molecular chaperones

ribonuclease

RNAse

type of nuclease that catalyzes RNA into smaller components

e.g. RNAseH removes RNA primer in DNA synthesis

DNAK

(assisted by DNAJ and GrpE)

molecular chaperones in E. Coli

aid in folding or new proteins, rescue of misfolded proteins, translocation of proteins across membranes; assembly /dissassembly of protein complexes; control of regulatory proteins' activity

require ATP

GroEl / GroES

part of molecular chaperone family

aid in folding of proteins

requires ATP

to function properly GroEL requires lid like co-chaperone GroES

zwitterion

charge of amino acids

dipolar ion

ion carying both positive and negative charge

charge of amino acids

both positive and negative

predom form at physiological pH

amino: positive--BASE

carboxyl: negative--ACID

stereoisomers

mirror images cannot be superimposed

chiral objects

stereochemistry

subdivision in chemistry involves spatial arrangement of atoms within molecules

stereochemistry of amino acids

α-C of all amino acids except glycine

is a chiral center

thus, each amino acids exists in two forms, "D" and "L"

what do all polypeptides synthesized by ribosomes have in common?

they are made exclusively of "L" amino acids

glycine stereochemistry

for glycine R group is an H

therefore it is non chiral

plane of symmetry exists

D-Ala and D-Glu amino acids found where?

rare bacterial cell walls

another use for D-amino acids

1. can be used in certain peptide antibiotics such as bacitracin

2. D-serine neurotransmitter in brain?

notation for distinguishing diff stereoisomers

four diff substituents of an aa are assigned priority according to atomic number

if progression from highest to lowest goes counterclockwise, L

if progression from highest to lowest goes clockwise, D

four categories of side chains

and how many a.a. of ea.

neutral-non polar (9)

neutral-polar (6)

acidic (2)

basic (3)

neutral-non polar amino acid side chains

how many a.a.

princ type of interaction

there are 9 of them

principle type of interaction: hydrophobic

consist of hydrocarbons

can be aliphatic or aromatic

neutral-polar amino acid side chains

three diff groups

there are six of them

those with hydroxyl group,thiol group, amide group

principle type of interaction: H bonds

acidic amino acid side chains

there are 2 of them

principle type of interaction: ionic and H bonds

basic amino acid side chains

there are 3 of them

principle type of interaction: ionic and H bonds

type of interaction that all categories of amino acid side chains make

van der Waals

aliphatic vs aromatic

aliphatic: hydrocarbon chains

aromatic: two ends are connected

Proline

Pro, P

non-polar hydrophobic

aliphatic

but differs in that hydrocarbon chain bonds to both alpha helix and nitrogen of aa

rigid structure

cannot form into alpha helix because amide H cannot act as donor  helix breaking residue

Cysteine

Cys, S

polar amino acid side chain

with thiol group

like serine but with thiol (sulfhydryl) in place of hydroxyl

thiol is much more reactive than hydroxyl

disulfide bonds

two thiol groups can form disulfide bonds

which play important role in stabilizing proteins

Phenylalanine

Phenyl ring in place of one of the Hs of Alanine

Phe, F

Tryptophan

Trp, W

like phenylalanine but with indole group instead of phenyl ring

indole group comprised of two fused rings + NH group

NH makes it a little less hydrophobic than phenylalanine

amino acids with neutral-polar (hydroxyl group) side chains

Tyrosine (Tyr, Y)

Serine (Ser, S)

Threonine (Thr, T)

Amine vs Amide

amine: doesn't have a carbonyl group attached to N--amino acids are amines

formular written NHR

if carbonyl comes btw NH and R it's an amide

amide: does have a carbonyl attached to N

e.g. when peptide bond forms btw carboxyl and amine groups of two amino acids, resulting molecule is an amide

amino acids with neutral polar (amide group) side chains

Asparagine (Asn, N)

Glutamine (Gln, Q)

uncharged derivites of Aspartate and Glutamate

they contain carboxamide instead of carboxylic acid

Amino acids with acidic side chains

negatively charged at phys pH

Aspartate (Asp, D)

Glutamate (Glu, E)

Amino acids with basic side chains

positively charged at Phys pH

Lysine (Lys, K)

Arginine (Arg, R)

Histidine (His, H)

Histadine

His, H

amino acid with basic side chain

less basic than Lys and Arg because of imidazole group

but can exchange H⁺

aromatic

ring can be pos charged depending on environ

peptide bond that links amino acids

partial covalent bond

no freedom of rotation

limits possible conformations

as a result of bond, four atoms of peptide bond are coplanar

semi rigidity

has important consquences for higher protein structure

amphipathic

molecule or structure with both a hydrophilic and hydrophobic part

α helices

type of secondary structure: rod like spiral

formed by regular pattern of H bonding btw carbonyl O of one aa to amide H of aa four residues toward c end of chain

arrangement confers polarity (H donors have same orientation)

hydrophobic or hydrophilic quality of helix conferred entirely by side chains

structural characteristics of alpha helix

right handed

pitch: 5.4Å

3.6 residues per turn

ideally positioned for intra- or intermolecular interactions (with DNA or other proteins)

Β sheets

planar alignment of 2 or more B strands--which are usually short fully extended regions of the backbone

B strands are usually 5-10 aa in length

line up along each other

can be formed from parallel B strands, antiparallel, and mixed

Most adopt a twisted shape (more compact as a result)

Turns

U shaped four residue segments stabilized by H bonds btw arms

usually at surface of protein and redirect chain into interior

allow polypeptide chains to reverse direction

frequent in antiparallel B-sheets

Motifs

combinations of secondary structures with characteristic 3D organization

Coiled Coil motif

example

2 amphipatic α helices wrap around each other

non polar side chains of one helix interact with non polar side chains of the other

e.g. Leu zipper

Leu Zipper

also called Leucine scissors

protein with coiled coil motif

Leu (hydrophobic aa is found at every 7th position)

common dimerization domain

found in some proteins regulating gene expression

DNA binding motif

leu zipper regulatory proteins interact with DNA as dimers and are called basic leu zippers

Amyloid fibrils

insoluble fibrous protein aggregates; highly resistant to proteolysis

cross-B structure (stacked B sheets)

alzheimer's disease

Protein Tertiary Structure

describes spatial arrangement of all amino acids

folding of secondary structures into fibrous or globular structures called domains

stabilized by hydrophobic interactions btw non-polar side chains and in some cases by disulfide bonds

stabilizing forces hold various secondary structure elements together into compact internal scaffold

myoglobin

70% of structure is made of alpha helices

porins

exception to the hydrophobic-in, hydrophilic-out rule

hydrophilic channel

hydrophobic exterior

protein quaternary structure

held together by what kind of bonds

interaction of several polypeptide chains to form an active multimeric molecule

held together by non covalent bonds

hemoglobin

four subunits (2 a, 2 B subunits)

each subunit structurally similar to myoglobin

secondary structure

stabilized by what bonds

localized organization (spatial arrangement) of amino acids

without stabilizing interactions, polypeptide chain assumes random coil shape

stabilized by H bonds

protein's size and shape dependent on

1. amino acid sequence

2. number size and arrangement of secondary structures

Domain

modular unit from which many larger proteins are constructed btw 40-350 aas

region of a protein with distinct tertiary structure and characteristic activity

compacy independently folded region of a protein

domains of a protein connected by flex segments of polypeptide chains--can usually be seperated by mild protease

nuclear magnetic resonance

technique that exploits magnetic properties of certain nuclei to give info about distance btw part of prot molecules

data can be recorded in solution

not good for large proteins

no crystal required

use external magnetic field to change spin of atoms (usually ¹H, but sometimes ¹³C and ¹⁵N)

record the resonance spectrum (can distinguish signals from H nuclei of diff aa residues and measure shifts when it ineracts with neighbors)

neighboring atoms will influence the signals chemical shift

ab initio method for predicting 3D protein structure

use computer to minimize free energy of a structure with a given aa seq

prob: vast number of possible configs

knowledge based methods of predicting 3D protein structure

compatibility of an aa seq of unknown structure with known structures

prob: only possible is similar structure has already been id'd

way to purify protein according to solubility

ammonium sulfate precipitation

way to purify proteins according to charge

ion exchange chromatography

way to purify proteins according to binding affinity

affinity chromatography

ion exchange chromatography

seperates proteins according to their net charge

if you want to seperate positively charged proteins, immobilize negatively charged beads on resin (Carboxymethyl or CM group)

later to get the entrapped proteins released, increase concentration of sodium chloride or other salt to buffer--will compete with pos prots for binding to CM group. prots with lower densitiy of pos charge will emerge first followed by prots with higher dens of pos charge.

if you want to seperate negatively charged proteins, immobilize positively charged beads on resin (Diethylaminoethyl or DEAE group)

affinity chromatography

way to purify protein based on its binding affinity

involves designing a stationary phase that reversibly binds to the protein you want to purify

protein purification according to solubility

salting out

ammonium sulfate precipitation

most prots are less soluble at high salt concentrations

salt concentration at which prot precipites, varies protein to protein

Polyacrylamide Gel Electrophoresis (SDS-PAGE gel)

seperates proteins by mass at good resolution

uses SDS  which denatures proteins and applies neg charge in porportion to its mass--so that all prots have roughly same charge to mass ration and hency equal mobilities in an electric field

chain length is sole determinant of migration rate

smallest proteins move most rapidly

isoelectric point

(For a protein)

pH at which the net charge of the protein is zero

isoelectric focusing

way to seperate proteins electrophoretically based on their relative contents of basic and acidic residues

at a protein's isoelectric point (pI), it's electrophoretic mobility is zero

run proteins through a pH gradient, and they will stop at point of their pI

gradient formed by subjecting polyampholytes with various pIs to electrophoresis

polyampholytes

small multicharged polymers

2 D protein gels

protein sample seperated by charge via isoelectric focusing (horizontal direction)

and then by mass via SDS-PAGE

(vertical direction)

former drawback was that proteins were seperated but not identified. NOW you can couple 2D protein gel results with mass spectrometry to id proteins

steps of MALDI-TOF

mass spectrometry

1. laser beam ionizes protein sample

2. electric field draws ions from sample through flight tube to detector

3. smaller ions move faster

4. laser triggers a clock which records time of flight

molecular weight calculated using time of flight

western blotting

makes it possible to find a protein in a complex mixture--used in test for Hep C

after proteins are seperated by mass via SDS-PAGE

Blotting transfers proteins resolved to a polymer sheet to make them more accessible to reaction

antibody specific for antigen added to sheet

tagged second antibody specific to first antibody added; radiated antibody produces dark band on xray film (autoradiogram)

SDS Page prior to protein blot gives info about molecular weight and possible existence of protein isoforms

Hydrogen bond donors

and acceptors

for max specificity DNA prots have to bind to which groove?

major

Why? in minor groove, patterns are similar for GC and CG and for AT and TA

two common protein-DNA interactions

1. asparagine with adenine

2. arginine with guanine

because of guanine's two H bond acceptors, it can be recognized with high specificity by the side chain of arginine

both bind to major groove

Arginine and Guanine

(one of most common protein-DNA interactions)

Asparagine and Adenine

(common protein-DNA interaction)

five DNA binding motifs

commonly found where?

these are commonly found in transcriptional regulators

1.Basic Leu Zipper (bZip)

2.Helix turn Helix (HTH)

3.zinc finger

4.ribbon-helix-helix

5.basic-helix-loop-helix (bHLH)

Helix-turn-Helix motif

most common DNA binding motif

composed of two alpha helices (one a C-term and one an N term) connected by a turn

C-term helix is recognition helix

N-term is a structural positioning helix

involved in regulation of gene expression

fits into major groove

~120° angle

dyad symmetry

refers to two areas of DNA strand whose base sequences are inverted repeats (reverse complements of each other)

e.g. GAATAC and GTATTC

these areas form hairpin loops with each other

basic Leu zipper

(bZip)

2 α-helices interact to form a coiled coil

hydrophobic residues (e.g. Leus) on each helix inetract with one another

region rich in basic a.a. interacts with DNA

heterodimers with distinct DNA binding specificities can be formed

basic helix-loop-helix

two α-helices joined by extended loop

note: 1st helix is shorter

loop is flex so one helix can fold back and interact with the other

4 helix bundle

association of 2 bHTHs

basic HLH

heterodimers can be formed

basic a.a. interact with DNA

zinc finger

composed of loop of polypeptide chain held in hairpin bend held by zinc atom

Zn²⁺ coordinated by 2Cys and 2His

the four amino acids that bind the zinc hold one end of the α-helix to one end of the Β-sheet

2 antiparallel Β-strands (contains 2 Cys)

followed by an α-helix (containing 2His)

α-helix recognizes major groove of DNA

zinc finger repeats

zing finger motif often found in clusters

DNA-binding proteins can have 2-37 repeats of motif

ribbon-helix-helix motif

seq recognition by Β-sheet

peptide mass fingerprinting

unknown protein cleaved into smaller peptides

absolute mass determined by mass spectrometry

these masses compared to known databases

can be coupled with 2D gels to identify specific proteins

CAP structure

dyad symmetry, coiled coil dimerization

DNA binding induces bending

two subunits

each subunit has two domains:

Sensory domain cAMP binding

DNA binding domain 3 α-helices including HTH motif

acid carries what charge

base carries what charge

acid negative

basic positive

e value

S score is measure of similiarity of query to sequence shown

e value is measure of the reliability of the S score

The probability due to chance, that there is another alignment with a similarity greater than the given S score.

The typical threshold for a good E−value from a BLAST search is e−5=(10−5) or lower.

pfam

allows you to view the domain organization of proteins

Four levels of protein structure

primary--aa seq--polypeptide chain

secondary--folding of chain into into 3d shapes

tertiary--overall confirmation of the chain

quaternary--joining of 2 or more chains into multisubunit structures

in gel filtration chromatography, what size proteins emerge first?

the largest

the smaller proteins enter the internal volume of the beads

is it only aplha helices that recognize DNA?

no beta sheets recognize too

for example, ribbon-helix-helix motif