ACS Biochemistry First Semester

Buffer Order and Henderson Hasselbach

Water, Buffer, Salt, Base

pH = pKa + log(A/HA)

pKa decrease as temp increase so pH decrease

Ramachandran plots

Φ = N-R

ψ = C-R

optimal angle to prevent steric hinderance

Protein structure of a helix, B sheet, reverse turn

a: LEMARKHQ

B: VICYFTW

RT: PSDNG

how many amino acids in alpha turn helix

3.6

Super secondary structure

helix turn helix

parallel vs. antiparallel

parallel wedges face same way

antiparallel face opposite

Isoelectric point (pI)

pH at which a particular molecule carries no net electrical charge.

Protein Separation Techniques (ALL)

Homogenization

Salting Out

Affinity Chromatgraphy

Ion Exchange

Size Exclusion/Gel Filtration

H, S, A, I, SG

He said all individuals SinG

Protein Separation Techniques (H)

Homogenization

disrupt cells, sonicator, centrifuge

Protein Separation Techniques (S)

Salting out

different proteins precipitate out at different salt concentrations

Protein Separation Techniques (A)

Affinity chromatography

add a tag; histidine binds; see how well there is an affinity for the group

Protein Separation Techniques (I)

ion exchange

separation based on charge; + proteins stick to - beads

Protein Separation Techniques (SG)

Size Exclusion/Gel Filtration

separation based on size

Cuts (protein separation techniques)

cut out what dont need/isnt protein

(use 40% concen. then keep liquid; if use 60% concen. then keep pellet)

Dialysis

protein separation based on bad with pores

1. Moles in bag + moles in buffer

2. Divide moles by total volume - new concentration

Electrophoresis

gels - move by size with electric current

1. Isoelectric focusing - purely pH

2. SDS page

pH separation is by pI and charge in 2d

Protein sequencing (who cuts after what)

Cyanogen bromide = methionine

trypsin = K, and R

chymotrypsin = F, M, L, W, Y

IP

Immunoprecipitation with proteins

1. add antibodies specific to protein of interest to lysed cells

2. add antibody binding protein on beads

3. centrifuge and protein is on bead with antibodies

4. gel elect

CO-IP

observe protein protein interaction

Messelson Stalh

RATSHIT.

Western Blotting

run gel - transfer to sheet - stained with radioactive antibody - autoradiogram - exposed antibody

ELISA - indirect and sandwich

Enzyme linked immunosorbant assay

1.) indirect - antigen coated well - bind persons antibody - bind antigen with enzyme - bind substrate = rate of color

2.) Sandwich - antibody coated well - bind person antigen - bind 2nd antibody with enzyme - bind substrate = rate of color

Mass spec

identify proteins by size

MALDI

matrix assisted laser desorption ionization (BIG)

ESI

electric spray ionization - charged molecules - time of flight detector

Nucleobase

adenine (2), guanine (3), cytosine (3), thymine/Uracil (2)

nucleoside

adenosine, guanosine, cytidine, thymidine, uridine

nucleotide

ATP, ADP, AMP

DNA facts

3.4 A separates bases

10 nucleotides in one twist

A form = dehydrated (right); B form = normal (right)

z form = left handed

Base hydrolysis RNA

separating nucleosides

DNA Replication mechanism (RNA transcription)

putting two bases together with template present

pyrophosphate leaves

Transcription DNA-->RNA

initiation, elongation, termination

copied 5' --> 3'

template is 3' --> 5'

DNA polymerase vs. RNA Polymerase

High fidelity vs. low fidelity

Endonucleases

bind splice sites

Ribosomal translation

initiation: factors bind 30S/40S -- ribosome binds mRNA -- bring tRNA -- large 50S binds

elongation: peptide bonds form

Peptide bond formation

base first deprotonates the NH2

Restriction enzymes

manipulate DNA, site specific endonucleases, cleaves bases and DNA

recognition sites are a palindrome

Cut into sticky or blunt ends

agarose gels

separate DNA based on base size

more porous than polyacrilamide

southern blotting

uses cDNA from gel -- blot onto sheet -- add p32 labeled probe -- autoradiography --

northern blotting

mRNA version of southern blotting

DNA fingerprinting

tandem repeats

PCR

95. 54. 72.

amplify DNA quickly

separate parent strands

DNA sequencing

similar to PCR - but must know part of sequence

Radioactive -- 4 separate rxns; using ddNTPs because will not make any PO3 group; different fragments; different lengths = where specific nucleotide is located

fluourescence - 1 reaction; fluorescent labeled ddNTPs; separated by capillary electrophoresis; detect flourescence as ejected from capillary

ddNTP

Vectors into plasmids

phage easily infects;

recombinant DNA

Lysogenic - DNA inserts into bacterial genome

Lysic - cells lysed as more phage are made

cDNA

Site directed mutagenesis

create specific targeted changes in DNA

study changes in protein activity - DNA manipulation

need to know gene you want in PCR and non-PCR

(nonPCR) = use whole plasmid; gaps where primer replicated DNA - cant ligate - ligated into host cell = template

(PCR) = use part of plasmid - run through PCr - new sequence = new template = limit is the time of the primers

Homology

Paralog = pair of genes in species

Ortholog = genes in pair of species

identity = % exactly the same

similarity = % similar and exact same

Sequence alignments

what % is similar

Hemoglobin structure

dimer of dimers

O2 binds to Fe on top and use 2nd distal histidine to stabilize the O2 to prevent Superoxide release

concerted binding

prefer tense state when nothing is bound

prefer relaxed state when stuff if bound

sequential binding

A hypothesis that states that an ion binds to a binding site and progresses through the cell membrane by binding to sequential binding sites that develop a stronger attraction for the ion

Tense state

no O2 bound

wants to release O2

binding affinity is low

relaxed state

O2 is bound

wants to bind O2

binding affinity high

what does a shift to the right from hemoglobin binding oxygen result from?

How does the curve of myoglobin binding oxygen compare to that of hemoglobin?

fetal binding is in between

hemoglobin binding oxygen

carbonic anhydrase o2 binding

Myoglobin

high affinity at low pO2 in blood

--> Myoglobin (tertiary structure) has a higher affinity for oxygen than hemoglobin. Tertiary structure---> can bind or release oxygen.

hemoglobin - sigmoidal curve because...?

has cooperativity - easier to bind more O2 after already bound

high pO2 in lungs - bind tightly

low pO2 in muscles - release easily

Fetal hemoglobin is special because..?

higher binding affinity - low 2,3 BPG

2,3 BPG

allosteric effector for binding hemoglobin - blocks O2 from binding - keeps in T state

Allosteric Regulation

1. Low pH = low O2 affinity

- T state is stabilized by salt bridge (proton added to Histidine)

2. 2, 3 BPG - bound to oxyanion hole

3. CO2 binds to H2O - H2CO3 forms and lowers the pH through HCO3-

4. CO2 binds to NH2 terminus to form carbamate and H+ = lower pH

Sickle Cell Anemia

Glu-->Val (polar --> nonpolar)

disrupts quaternary structure

in deoxy state

stuck in capillaries (stretched back and forth) - firmer

heterozygotes resistant to malaria because RBC are firmer

Thalassemia

a/B - not enough of alpha/beta chain produced

Michaelis Menton german

Die kinetik der invertinwirkung

Briggs-Haldane Equation

Vo = (Vmax*[S])/(Km + [S])

Briggs Equation

Vmax = max rate (usually only up to 80%)

kcat = Vmax/[E] = rate of product formation

Km = Vmax/2 rate of formation at half of the max velocity

kcat/km = 2nd order rate constant

Catalytic Efficiency

catalytically perfect - depend on rate of diffusion

Irreversible inhibition

inhibitor binds so tightly therefore inactivates AKA suicide

Reversible inhibition (3 types)

depends on dissociation constant

Competitive

Noncompetitive

Uncompetitive

Competitive Inhibition

inhibitor can bind or substrate can bind; since we have infinite substrate then it just takes longer for it to bind more than inhibitor

need increased substrate concentrations

Vmax is not changed

Km increases (Km = substrate concentration when half of active sites are filled)

Noncompetitive Inhibition

if inhibitor is bound then the substrate cannot bind

Number of substrates able to bind is decreased because there are fewer possibilities for binding location

Vmax decreases (max velocity is less because it wont take as long to fill all the available spots since inhibitor is decreasing amount)

Km is not changed

Uncompetitive Inhibition

inhibitor keeps substrate bound to enzyme

inhibitor deactivates enzyme

substrate stays bound - therefore not as many spots available

Vmax is decreased

Km is decreased (change number of substrate and ability to bind)

2 Models of substrate binding

induced fit - change

lock and key - fit like a lock and key

Enzyme rates plateau because...

have finished bind all substrate to all available enzymes

pH profile

know pKa's

Sequential binding

ordered: need to bind b/c of substrates

random: random binding

ping pong: 2 reactions - 1st reaction products modifies something in 2nd reaction

Burst/Transient Kinetics

Steady State = after product forms

Burst = initial rate of enzyme

Cysteine Protease

Serine Protease

Aspartyl Protease

Metallo Protease

Carbonic Anhydrase Mechanism

Enzyme Regulation (ALL 5)

1. Isozymes

2. Phosphorylation

3. Zymogens

4. Enzyme concentration

5. Allosteric Control

I, P, Z, E, A

Enzyme Regulation (I)

Isozymes = Multiple enzyme forms - different forms of the enzyme can regulate, altered kinetic parameters

Enzyme Regulation (P)

Phosphorylation/kinases - covalent modifications of proteins can activate/deactivate protein function

add/remove groups

Phosphorylation/dephosphorylation

Enzyme Regulation (Z)

zymogens - inactive form of enzyme - activated by cleavage

Enzyme Regulation (E)

Enzyme concentration - concentration of enzyme can control and regulate rate at which reaction is completed

Enzyme Regulation (A)

Allosteric Control - additional binding sites on enzyme that regulate activity - active sites that can inactivate or activate

Ketose and aldose

pyranose and furanose

glucose ring structure

fructose ring structure

D/L Carbon

1st achiral farthest from anomeric

anomeric carbon

determines alpha or beta

ribose and deoxyribose

glycosidic bonds

Fatty acid structure

Phospholipid/triglyceride

have no carbs for energy use - use lipids because lipids are easily stored

they serve in the cell membrane as a bi layer - highly impermeable to polar molecules