Biochemistry week 3 and 4

protein structure

protein structure

the conformation (or shape) of a protein predicts its function

what are the different types of protein structures?

1. globular proteins

2. fibrous proteins

3. transmembrane proteins

4. DNA binding proteins

bonding in proteins

• proteins are held in their shape mostly by non-covalent bonds

electrostatic (ionic) interactions

interactions between charged groups on different amino acids

hydrogen bonds

interactions between amino acids mediated by partial charges on hydrogens

hydrophobic interactions

• grouping of non-polar amino acids

• cluster

• slight attraction with each other

levels of structures

primary: peptide bonds

secondary: hydrogen bonds

tertiary & quaternary: disulfide bonds, electrostatic interactions, hydrogen bonding, hydrophobic interactions

protein structure rules

• the three-dimensional structure must be flexible enough to function properly but stable enough that it will not convert to another conformation

• it must have amino acids with side groups that are compatible with the environment or environments (such as a transmembrane protein) the protein will function in

  • so it will have polar groups on the outer surface if it is able to function in aqueous environments, and hydrophobic groups in membrane insertion areas if it is a transmembrane protein

primary structure

• peptide bonds form the backbones of proteins

• the peptide bond is between an amino group of one amino aid and a carboxyl group of another

• because of its partial double-bond characteristics the peptide bond is rigid

• the R-groups are usually on opposite sides of the bond

secondary structure: a-helices

• regular repeating structure where the coil is maintained by hydrogen bonds between the H in the N-H bond and the O of the carbonyl group 4 amino acids away in the peptide chair

• start to see the helix shape form

• occurs between the peptide bonds and backbone

• the R-groups radiate out from the helix, this keeps them far enough apart from each other that the helix is stable

• even though the peptide bonds are somewhat rigid,, the bonds have enough ability to rotate

what is the only amino acid not present in an a-helix?

• proline because it cannot form a hydrogen bond

• it is known as a “helix breaker”

secondary structure: B-sheets

• repetitive structure

• maintained by hydrogen bonding between backbone groups- not side groups

• can be parallel or anti-parallel depending on whether the strands are in the same or opposite orientation

• more rigid structures

• can form barrels: structures that can transport hydrophobic substances or form pores in membranes

vitamin A

• fat soluble, the lining on the inside is non-polar

• outside is polar

tertiary structures

• the a-helices and B-sheets combined with irregular elements such as loops and turns, make up this structure

• the amino acids in the primary structure define which secondary structure will be adopted and which tertiary structure conformation every molecule of a protein will take

• the “native conformation” of a protein is the 3-dimensional form it should take to perform its function correctly

• this is coded for in the DNA and every molecule of this protein should assume the same conformation if folded correctly

• proteins may contain characteristic elements called “structural domains” that may be similar among quite different proteins

quaternary structure: hemoglobin

• proteins made up o more than one subunit (eg. dimers, trimers, or hemoglobin)

• the subunits can be the same (such as in a homodimer) or different (such as in heterodimer)

• in hemoglobin there are 2a-subunits and 2B-subunits

• the Fe in hemoglobin can make 6 bonds

• it has 4 bonds to heme and 1 bond to a nearby histidine

• this leaves 1 binding slot open. This can be filled with oxygen, carbon monoxide or can remain empty

• binding of oxygen causes a shift in the shape of hemoglobin, making it easier for more oxygen to bind to the other sites

quaternary structures: immunoglobulins

• antibodies or immunoglobulins perform important defense functions in our bodies

• the immunoglobulins all have this structure with 2 light and 2 heavy chains held together by disuphide bonds

• the antigens are bound at the ends of the “Y”, a variable region depending on the specific antibody, alerting the body to invasion

protein denaturation

• proteins are not stable, so they need to be folding properly to function

• if a protein is unfolded or misfolded, it may be unable to carry out its role

how can proteins be altered?

1. temperature

2. change in pH

3. protein modifications

protonation and deprotonation

• changes in pH cause structural changes due to disruption of the hydrogen and ionic bonds

• a large change in pH could cause the addition or dissociation of a proton

• loss of gain of a proton could cause the breaking of the hydrogen bonds that hold the protein in its proper conformation

• proteins without the correct tertiary conformation cannot perform their function

protein modification

• can be modified non-enzymatically

  • as hemoglobin is by glucose (increasing the HbA1c)

• the higher the concentration of glucose in the blood, the more likely “glycation” is to occur

• advanced glycation end-products or “AGEs” result from “glycation”, these pro-inflammatory molecules that are harmful to the cell

why is HbA1c a good measure of blood glucose levels?

the circulate for about 120 days

• it gives us a picture of what kind of conditions the red blood cells have been exposed to over the last few months

consequences of non-conservatives change

• because of glutamate with a negatively charged group, it has been replaced by the hydrophobic valine

• it can interact with a hydrophobic pocket on another hemoglobin

• this allow the formation of long strands or polymers of hemoglobin

• this cause the red blood cell to sickle and prevents it from doing its job of delivering oxygen to tissues

enzymes

• proteins that make chemical reactions go faster - they “catalyze” the reaction

nomenclature for enzymes

• “ase” suffix

catalysis

• enzymes increase the rate of chemical reactions

• they are each specific for one reaction (depending on the amino acids that make up the enzyme)

• they are not permanently changed in these reactions

• they bond the substances that are reacting, called “substrates” and release the “products” (bicarbonate) once the reaction is complete

steps in an enzyme-catalyzed reaction

1. binding of the substrate: E+S <--> ES

2. conversion of substrate to product: ES <--> EP

3. release of product: EP<--> E+P

what do enzymes do?

lower the amount of energy required to go from reactants to products

they do not change the reactants or products but lower the activation energy so more molecules can enter the transition state and proceed to products

the enzyme uses different strategies to lower the activation energy and so speeds up the rate of the reaction

enzyme active sites

• enzymes are specific for a particular reaction and substrates

• the shape or “conformation” of the molecule brings the substrates together so that the reaction can proceed to the active site of the molecule is dependent on the amino acids that are located there

what does zinc do?

• helps stabilize

• it is held in place by binding to 2 histidine’s in the active site

carbonic anhydrase

• the positive charge from zinc attracts electrons from water, making it easier for one of the H’s to be removed and the reaction to proceed

cofactors

• helps lower the energy required to cause the reaction

• usually minerals

• often required to bind for it to function

co-enzymes

• organic molecules

• not synthesized from vitamins

• the coenzymes contribute functional groups that help catalyze the reaction, but are not very active on their own

lock and key

• an enzyme and a substrate fit perfectly together

• this model explains the specificity of the enzyme but NOT the ability of the enzyme to stabilize the transition state

induced-fit model

• allows for flexibility of the enzyme in binding to the substrate, and so is thought to be the most accurate

• binding of the substrate induces changes in the shape or conformation of the enzyme

• this allows for changes such as closing of the actin fold of hexokinase once glucose is bound

pH dependence

• each enzyme has optimal pH at which they function most efficiently

• for many enzymes that optimal pH corresponds with physiological pH, or the pH of their environment

• some enzymes have evolved to work best in acidic conditions such as pepsin

temperature and concentration

• the rate of reaction of an enzyme increases until the temperature at which the protein (enzyme) is denatured

• normal physiological temperature is 37 C, which is the optimal temperature for function of our enzymes

• concentration is the other factor affecting enzymatic rates of reaction

• reaction rates increase as the concentration increases

competitive inhibitor

• a molecule with a similar shape to one of the substrates of an enzyme

• when the inhibitor binds instead of the substrate, the reaction cannot occur, and the product is not produced

• inhibitors can bind to the enzyme reversibly or irreversibly

• stop enzymes from working

reversible inhibitors

• only inhibit when they are bound in the active site

• many can be displaced by increasing the concentration of the substrate

irreversible inhibitors

• eg. aspirin

• do no detach and the enzyme action is permanently shut down

noncompetitive (allosteric) inhibition

• binds to a part of the enzyme away from the active site

• the binding of the inhibitor prevents the reaction from proceeding by changing the conformation of the enzyme

• this can either alter the affinity of the enzyme or the structure of the enzyme

cytochrome P450 Enzymes

• larger family of enzymes

• important in drug metabolism, particularly in the liver, but also the intestine

• they oxidize hydrophobic compounds making them more soluble so they can be excreted (rather than building up in the adipose tissue)

  also in intestinal cells, this is where grapefruit juice interferes with statins are they are not metabolized and can build up to toxic levels in the blood

bacterial cells

• prokaryotic and do not contain a nucleus or other membrane-bound organelles

eukaryotic cells

• contain many compartments bounded by membranes

• in human cells

• these structures allow cells to concentrate molecules and proteins to control when and how reactions take place

plasma membrane

• all cells are enclosed by this

• lipid bilayer

• the membrane serves to restrict the entry of substances into the cell

• there are proteins both embedded in the membrane and anchored to it

• non-polar

• the lipids in the membrane consist of molecule with polar head groups and hydrophobic tails (amphipathic molecules)

• different head groups appear more often on one side or the other side of the cell membrane

• phosphatidylserine (negative charge) makes up more of the inner membrane, along with phosphatidylethanolamine

integral proteins

• embedded in the membrane with hydrophobic regions in the membrane and hydrophilic regions on either side

• they connect to the outside and inside of the cell

• functions include channels or transporters, as well as receptors for hormones or neurotransmitters

peripheral proteins

• attach to integral membrane proteins or to the edges of the lipid bilayer temporarily and carry out specific functions while there

cell transport

• transport across the lipid bilayer establishes an electrochemical gradient

what are the 2 components to an electrochemical gradient?

• concentration gradient

• the charge on the membrane

electrochemical gradient

• the interior of the cell is more negatively charged, while the exterior is more positively charged

• this means that positive ions will be more likely to diffuse through while negative ions may require energy to transport the

• the concentration gradient is more important for uncharged molecules

• molecules more to equalize the concentration gradient

  • high to low

what are the modes of transport?

1. simple diffusion

2. facilitative diffusion

3. active transport

4. endocytosis

simple diffusion

• small nonpolar molecules can diffuse through the plasma membrane directly quickly and easily

• small polar molecules, like water, can also move across but slower

facilitated diffusion

• movement from an area of high concentration to an area of low concentration

• think larger polar molecules

• the molecule must bind to its transported, but it is passive, no energy is required

• if there is a high concentration outside the cell and all transported are occupied, transport is faster than it would be by simple diffusion

active transport

• energy is required to transport substances against their electrochemical gradient

• this occurs with the sodium-potassium pump

• the maintenance of high Na outside the cell can be used to drive secondary active transport or to allow membrane depolarization in action potential

secondary active transport

• sodium is pumped out of the intestinal cells so that it is low within the cell compared to the lumen

• sodium can bind to a transported that will move sodium into the cells along its electrochemical gradient, but this is combined with the movement of glucose against its concentration gradient

• glucose can then go into the extracellular fluid, where its concentration is lower, by passive transport

• this mechanism moves sodium and glucose from the digestive tract into the body

nucleus

• the library and command center

• largest organelle in the cell

• most of the DNA is in the nucleus, with a small amount located in the mitochondria

• DNA replication and transcription of the DNA into RNA both occur in the nucleus

• once the mRNA is transcribed and processed it travels out of the nucleus for translation into proteins

nuclear transport

• proteins involved in processes in the nucleus, such as DNA replication are targeted to re-enter the nucleus after translation

• these proteins have a nuclear localization signal (NLS) attached that allows them to traverse the nuclear pores and re-enter the nucleus

endoplasmic reticulum

• protein factory

• made up of smooth and rough (ribosomes attached) areas

• the rough ER is the site of translation of proteins bound for outside the cell or within the membrane or organelles (where posttranslational modifications occur)

• the smooth ER contains enzymes for lipid synthesis and the cytochrome P450 oxidative enzymes of drug metabolism

golgi complex

• the amazon warehouse

• the golgi complex modifies, sorts, and distributes proteins around the cell

• proteins translated in the rough ER move the golgi where they receive important modifications and are transported to where they need to go

mitochondria

• powerhouse of the cell

• site of energy production

• consist of an outer and inner membrane

• the inner membrane is the site of enzymes involved in cellular respiration, while the TCA cycle and together oxidative pathways occur in the matrix

lysosomes

• the recycling depot

• membrane bound organelles that contain enzymes that breakdown molecules

• anything the cell doesnt need or foreign invaders can be broken down in the lysosome

• their pH is maintained at about 5.5 so that the enzymes enclosed in the organelle can function

• the pH is maintained by transporters that use energy to pump H+ into the lysosome

cellular signaling

• cells need to communicate with each other to maintain homeostasis

• this means sharing information across different cells and cell types

endocrine hormones

• secreted into the blood and travel to act on target cells

paracrine messengers

act on cells that are close by

autocrine messengers

bind on the same cell from which they are released

juxtacrine messengers

direct cell to cell interactions

signal receptors

• the type of receptor and its location is determined by the messenger molecule

• if the molecule can cross the plasma membrane, the receptor will likely be inside the cell

• if the molecule cannot cross the plasma membrane, the receptor will need to be on the cell surface

cortisol signaling

The signaling molecule ACTH is released by the pituitary gland in the brain and released into the bloodstream. When it reaches the adrenal cortex, it stimulates the production of cortisol.

insulin signaling

• 1.1 Insulin biosynthesis and transcription.

• 1.2 Insulin secretion.

• 1.3 Fatty acids and insulin secretion.

• 1.4 Hormonal regulation of insulin secretion.

• 1.5 Action on the cell.

• 1.6 Regulation of the insulin receptor signal.

B2-adrenergic receptor

• a protein made up of 7 transmembrane helices

central dogma of molecular biology

• DNA transcription to RNA and its translation into protein

• refers to the information flow for biological systems which goes in the direction from DNA to protein

DNA

• chains of deoxyribonucleotides (dATP, dTTP, DGTP, dCTP)

• resides in nucleus

RNA

• chains of ribonucleotides (ATP, CTP, UTP, GTP)

• transcribed in nucleus, translated in cytosol or rough ER

proteins

• chains of amino acids

• involved in all different areas of the cell

DNA and RNA

• deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are the source of genetic information for humans, animals and pants, as well as bacteria and viruses

• eukaryotic species have nuclei within their cells where the DNA is found, prokaryotes (bacteria) do not have their DNA separated from the rest of the cell contents (no organelles)

• a small amount of DNA (less than 0.1% of cell total) is located within the mitochondria - this DNA is not housed in a separate compartment, it is more like prokaryotic

viruses

• may use DNA or RNA for their genetic information but cant replicate on their own viruses require other host cells whose replication machinery they “hijack” in order to multiply

• can infect eukaryotic organisms and prokaryotes

• viruses that infect bacteria are called bacteriophage

plasmids

• circular DNA molecules that can enter bacterial cells and replicate independently on the genomic DNA

• they are not infectious but they are important since they can confer antibiotic resistance to the bacteria they enter

• used extensively in molecular biology and genetic engineering

nucleotide structure

• consist of a base, sugar, and phosphate group

nucleoside vs nucleotide

• the sugar bonded to a base

• phosphates are added and use energy

DNA sugars

• deoxyribose

• have and -H position

RNA sugars

• ribose

• carbon position at 2 has and -OH group

DNA bases

Purine: adenine and guanine

pyrimidines: thymine and cytosine

• purines are big, double ring structures

• guanine and adenine are stronger because they have double bonds

RNA base pairs

Uracil is the complementary base to adenine

• it had an H on C5 rather than a methyl group

DNA/RNA bond formation

• the bonds between nucleotides are an ester bond

• 2 phosphoester bonds are formed so this is a phosphodiester bond

• strong covalent bonds

ester bonds

formed by an acid and an alcohol and is a condensation reaction that releases water

DNA structure

• the phosphates and sugars form the backbone of the molecule

• two of the oxygen molecules of the phosphate group form the phophodiester bond

• the free OH loses its H at physiological pH, so DNA carries a negative charge

• this allows binding or association of proteins or other molecules in the major and minor grooves

anti-parallel nature of DNA strands

• strands run in opposite directions and a pyrimidine is always H-bonded to the same purine (complementarity), each strand is the same but in the opposite direction

• each strand can serve as a template for the other strand

• bases in the strands are always listed in the 5’→ 3’ direction and called either the sense (coding) or antisense (template) strand

DNA packaging

• the DNA is each cell is very large

• all this DNA must be packed into each cell, inside the nucleus, so there needs to be a way of condensing it to fit

• eukaryotic DNA binds to an equal weight of histone proteins, giving it an appearance in the microscope as “beads on a sting”

nucleosomes

• the histones are small proteins that contain many arginine’s and lysines

• 2 of each of 4 different histones make up the nucleosome core, while H1 attaches to the linker DNA between each nucleosome (DNA and core)

• the coiled pattern of would nucleosomes that further compacts the DNA is called the solenoid structure

mRNA

• messenger RNA

• provides instructions for protein synthesis

• transcribed from the template strand of DNA

• undergoes processing withing the nucleus to remove “introns” that do not code for the protein and to add stabilizing structures such as the guanosine cap at the 5’ end and the poly A tail at the 3’ end

• it is then reported into the cytoplasm where it can direct the synthesis of a protein

rRNA

• ribosomal RNA

• combines with proteins to form the ribosomes on which the proteins are synthesized

• in eukaryotes there is an 80S ribosome that conducts the synthesis of proteins; it consists of 60S and 40S subunits

tRNA

• transfer RNA

• the molecules that carry the individual amino acids to the ribosomes, according to the instructions on the mRNA, to be incorporated into the growing pattern

• cloverleaf structure

• the anticodon is complementary to the mRNA sequence so the code can be ”read” and the correct amino acid added to the polypeptide chain

viral replication

• viruses dont have all the proteins or molecules needed to replicate; they need a host to hijack

• some viruses use RNA as their genetic material

• if they are a retrovirus they use an enzyme called reverse transcriptase to convert their RNA into DNA and insert that into a hosts chromosome the virus has “hijacked” the transcription and translation machinery of the host

cell cycle

interphase: G1, S, and G2

Short mitosis phase: M

• during the S-phase of the cell cycle DNA is replicated and histone and other protein synthesis is greatly increased

• once replicated and divided, cells can further divide or can enter a resting phase (G0) where they can stay or be induced back into the cycle

mitosis

process of creating two diploid cells

bacterial replication

• circular DNA with an origin

• the origin is the place that DNA replication begins in both directions

• the circular nature of bacterial DNA and its single origin are a couple of the difference between prokaryotic and eukaryotic DNA replication

eukaryotic replication

• multiple origins of replication in DNA in eukaryotes

• these replication bubbles arise at points along the DNA and synthesis proceeds along a form in each direction from these points

prokaryotic replication

• the new strand is formed from the template of the old one

• this is semi-conservative replication

semi-conservative replication

• because each strand of DNA is used a the template to create a copy, it is semi-conservative each daughter cell received 1 strand of the original DNA and 1 strand of the new copy

steps of DNA replication

1. helicase unwinds the strands

2. SSB proteins keep it from coming back together

3. topoisomerase keeps the DNA from supercoiling

4. primase starts the process of connecting the bases- creating a primer

5. DNA polymerase binds to the primer and makes the new strand of DNA in the 5’ to 3’ direction

6. leading strand is made continuously and the lagging strand cannot be so it can only stay in small chunks called okazaki fragments

7. exonuclease removes all the RNA primes

8. DNA ligase seals up the fragments of DNA to form a continuous double strand

topoisomerase

• an enzyme that can break and rejoin the strands to relieve tension from twisting it wraps around the strand and cuts the phosphodiester bond to allow the helix to spin freely and relax