Biochemistry 5012: Section on Protein Biochemistry Lecturer: Chuck Sanders Lectures 1-2 Survey of proteins in life and disease. Molecular energetics. Related Reading: Voet, Chapters 1,2 Learning Objectives To gain a broad perspective of the roles of proteins in life and disease. To understand the energetic basis for molecular motion and diffusion. To understand the concepts of free energy and equilibrium and their relationship. To understand the molecular forces which determine free energy.

To gain insight into how modulation of protein function can lead to and/or alter disease states. Topic Outline Significance of proteins in life and disease Survey of human proteins Useful Observations about Protein Biochemistry Kinetic energy and Brownian motion Standard free energy vs. driving force energy Equilibrium Enthalpy and entropy Hydrophobic effect Attractive forces

“There are many more things in heaven and earth, Horatio, than are dreamt of in your philosophy.”

Hamlet, Act 1, Scene 5

E. coliRed Blood Cell and Blood Serum

“to scale” artwork by David Goodsell

From: Mathewsand Van Holde

1 Angstrom = 0.1 nm

atoms andbonds: 1-3 Angstrom

typical single domain globularprotein:35-50 Angstromsin diameter

Biomolecular Scale

Classes of Biomolecules

Fats, Oils, Lipids

Co-Factors (Metal ions, vitamin derivatives)

Carbohydrates

Metabolites

Nucleic Acids

Proteins

Minimal Ingredients for Life on Earth?

•liquid water•physical scaffold•glue•organization•semi-permeable barriers•source of energy•capacity to harvest, store, and transduce energy•information storage•information copying, and retrieval•capacity for sensing environment•logic for information use, integration, and response•capacity for regulation (switches and throttles)•catalysts•systems for quality control: error management and exploitation•capacity for reproduction

Minimal Ingredients for Life on Earth?

Proteinsplay major,often dominant,roles in…

•liquid water•physical scaffold•glue•organization•semi-permeable barriers•source of energy•capacity to harvest, store, and transduce energy•information storage•information copying, and retrieval•capacity for sensing environment•logic for information use, integration, and response•capacity for regulation (switches and throttles)•catalysts•systems for quality control: error management and exploitation•capacity for reproduction

Proteins play a central role in almost every process that is important for life.

Proteins are linear polymers Proteins are linear polymers of amino acidsof amino acids

• 20 different amino acids: many combinations

• Proteins are made in the RIBOSOME

amino acidsribosome protein

(short proteins are called petides or polypeptides)

How Significant are the Roles of Proteins in Human Disease?

To address the question, consider “simple” inherited diseases–disorders that are the result of single (heritable) genetic lesions.

There are roughly 5000 known simple disorders.

For known disorders, mutations involving about 2000 different genes have been already been identified and characterized. For some genes, there aremany known disease-linked mutations.

What do these disease-causing mutations represent in terms of biomolecular outcome?

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Heritable Mutations Linked to “Simple” Diseases

Heritable Mutations Linked to “Simple” Diseases

53,000 Total Chromosomal DNA Mutations:

10%: involve gross DNA changes

10%: affect RNA splicing

80%: fall in protein open reading frames

of these, 30,000 are missense mutations that code for a single amino acid change in a protein(missense mutation = single DNA base change)

Human Gene Mutation Database: www.hgmd.org

For “simple” disorders, the vast majority of inherited mutations perturb genes that encode proteins. In otherwords, alterations in proteins are by far the most common cause of simple disorders.

In fact, over of half of disease-causing mutations leadto a single amino change in a single protein.

Mutations lead to disease by causing loss of a certainprotein’s function, altered protein function, or byprotein “gain of function” (which includes protein toxicity).

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COO-Bold-circulated sites: a single mutation at

ANY of these sites leads to loss of V2R function, resulting in nephrogenic diabetes insipidus (NDI).

Diabetes insipidus: a disease caused by a protein’s loss of function.

Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2

S S

8-arginine vasopressin (the anti-diuretic hormone): bindsto the vasopressin V2 receptor, turning on its signaling state

Notes on protein nomenclature:

•beginning of chain is “N-terminus”•end of chain is “C-terminus”•amino acids (“residues”) are numbered from N-terminus to C-terminus

Function of vasopressin V2 receptor (V2R) in kidney epithelial cells. The hourglass-like protein is the aquaporin-2 water channel. The vasopressin hormone in the blood binds to V2R in the basolateral membrane, which then signals to the cell interior to tell the cellthat it is time to traffic aquaporin water channels from storage sites within the cellto the cell surface… allowing water to flow into the cell as part of the process of absorbing water into the body, resulting in concentrated urine.

From Morello and Bichet, Ann. Rev. Physiol 63,607-630 (2001)

Diabetes Insipidus and the Vasopressin V2 receptor

aquaporin

Aquaporins are water channels that function to allowthe flux of water across normally impermeablebiological membranes. There are about 10 differentaquaporins in humans (all of which have similar 3-Dstructures). Aquaporin-2 is the one that is involvedIn NDI.

When V2R loses its function, thenaquaporin channels do not traffic tothe membrane surface. Water is not,therefore, properly absorbed into the body and urine remains dilute.

Diabetes Insipidus

90% of cases are caused by mutations in thevasopressin V2 receptor. V2R gene is on the X chromosome, so most cases of NDI are “X-linked”.

Mostly affects males (1/250,000) becausewomen will usually have a healthy V2R gene in 50% of their cells– sufficient to maintainhealthy organ function. (Whereas, males havethe same X-chromosome in all cells).

Symptoms start soon after birth: chronicsevere thirstiness, dilute urine, high blood sodium, fever, constipation, etc.

No cure yet, but disease is manageable providedplenty of clean water is available. Also treatedby low sodium diet and certain diuretic drugs.

(diuretic = promotes urine formation and loss ofsodium)

While complex (multifactorial) diseases are much harder to characterize in termsof underlying molecular causes, it is very clear that proteins play central rolesin most of these diseases, including all 11/15 of the leading causes of death inThe US.

How many human proteins are there?

How Many Protein-Encoding Genes Are There?

M. genitalium: 500

E. coli: 4000

M. tuberculosis: 4000

Yeast: 6000

C. elegans: 18,000

Drosophilia melanogaster: 13,000

Rice: 60,000

Humans: 22,000 (best current estimate)

DNA mRNA Protein

DNA Encodes Proteins Through the Intermediation of RNA

•A protein’s identity is defined by its amino acid sequence.

•Each protein amino acid sequence is encoded by its open reading frame (ORF) within its gene in chromosomal DNA (usually 2 alleles per protein)

•For lower organisms each protein open reading frame is usually encoded bya continuous segment of chromosomal DNA.

•In humans genes are typically composed of alternating segments called exons and introns. Exons contain coding sequence for the protein, introns do not. The average human gene contains 9 introns.

•When human genes are transcribed into mRNA both exons and introns are transcribed. The mRNA must then be processed to remove the introns and to splice the exons together to create the final open reading frame which will be converted to protein. In some cases, there is more than one way splicingcan be executed (“alternate splicing”), such that different final ORFs can result, leading to related, but distinct proteins. One gene sometimes produces multipledifferent forms of a protein. It is estimated that 40-60% of all genes are expressedin multiple splice forms.

transcription 1. RNA editing/splicing

2. translation

From: Alberts

The average human genecontains 9 introns.

40-60% of all human genes are expressed in multiple splice variant forms.

There are many more proteins than the ca. 22,000 genes that encode them.

Therefore… while there are ca. 30,000 genes encoding human proteins, many of these genes will produce multiple variants of a given protein.

Post-translational modification of proteins (covalent attachment of lipids, sugars etc.) will generate even more molecular diversity in the human proteome: there could easily be > 1 million chemically-distinct proteins.

Each prong extending down from each splice variant represents a different state of post-translational modification. For example, forms of a single splice variant might be present that are: unmodified, modified only by glycosylation, modified only by lipid attachment, and modified by both lipid attachment and glycosylation.

Nature often generates diversity based on combinatorial principles.

parent gene

splicevariants

RNA splicing

post-translationmodifications

Venter, J.C. et al (2001)  Science, vol. 291, 1304 (2002).

Proteins Can Be Classified According to Molecular Function

The vast majority of drugs target proteins.

Known drugs appear to target <150 proteins… whereasit has been estimated that there are at least 600 targets based oncurrent paradigms in pharmacology and medical genetics.

Nature Reviews Drug Discovery 1; 727-730 (2002)

Targets of marketed small-molecule drugs by biochemical class.99% are proteins

Nature Reviews Drug Discovery 1; 727-730 (2002)

(G protein-coupled receptors)

This Concludes The “Biomedical Significance of Proteins”

Section

Useful Observations About Protein Biochemistry

• Water is everywhere– water insoluble molecules require special transport mechanisms in cellular milieu. Also, for any process where water must be excluded, biology must exert special effort to exclude it.

• The biochemistry of human life must work properly within a moderate and limited temperature range– a 1.5% change in absolute body temperature can be fatal.

• While covalent chemistry is important, a hallmark of biological chemistry is the use of widespread non-covalent molecular binding (think velcro). Non-covalent bonding is well suited for the constant, massive, and rapid biochemical changes which are required for life.

• The principle of functional redundancy often applies to the roles of specific biochemical systems in life: nature is interested in robustness, so often there is more than one molecule which can fulfill a certain physiological role (in case something goes wrong with one). There are proteins which seem to be absolutely essential, but others which are not.

• Just because when a protein is taken away and there seems to be no adverse affect doesn’t mean the protein is not important– maybe the right conditions were not checked. Maybe functional redundancy was at work. Maybe the normal function of the

protein is relatively unimportant, but the protein can still contribute in an importantway to disease.

Useful Observations About Biochemistry (continued)

• Biochemists tend to take reductionist approach. Reductionism represents the attempt to understand the whole by studying the parts. This is a powerful approach, but has its limitations because of the “complex” nature of physiological systems.

• The principle of pleiotropy often applies to the roles of specific biomolecules in life: biomolecules typically function as parts of networks. When the function of one molecule in a physiological system is perturbed, it may have multiple affects that impact physiology through multiple possible pathways through the network: a pleiotropic affect… the bane of reductionists.

• Biochemistry is governed by fundamental laws of thermodynamics and kinetics. Understanding these laws at an elementary level is important (really!) and, fortunately, notdifficult.

• “Nature is an endless combination and repetition of very few laws. She hums the old well-known air through innumerable variations.” -Ralph Waldo Emerson

The Energetic Governance of Biomolecular Interactions

Energy Units

Joules

Calories

1 Calorie = 4.184 Joules

Usually we use kCal or kJ

Total Energy = Kinetic Energy + Potential Energy

Quantities of Matter Used in PE

moles (biochemicals)

coulombs (electricity)

partial pressure (gas)

Chemical Potential Energy Units: kJ/mole or kCal/mole

Electrical Potential Energy Units: volts: (Joules/Coulomb)

Faraday’s constant is used to convert between electricaland chemical potential energy:

Faraday’s Constant: 96,500 coulombs/mole

(important for understanding biological oxidation/reduction reactionsin metabolism and for understanding electrophysiology)

In biochemistry, “energy” is almost always referring to potential energy. However, biological molecules do have kinetic energy and that class of energy is very important for how the world works.

Molecules in solution are not static, but experience both internal motions andwhole-molecule motions as a result of what can be called “random thermal energy”.

The average kinetic energy resulting from random thermal energy is proportionalkT, where k is Boltzmann’s constant and T is temperature. This proportionalityis important because it means that as the temperature goes up, molecules move faster, bonds vibrate faster, and conformational fluctuations increase (on the average).

The distribution of kinetic energy states around the average kinetic energy for apopulation of molecules is governed by the Boltzmann distribution.

As the temperature goes up, theaverage energy of molecules goesup and the range of well populatedenergy states broadens.

The Boltzman distribution indicates that in any given population of moleculesthere will be a whole range of energies and that molecules will be randomly jumping from one energy state to another. If a molecule can undergo a chemical reaction, the ones in the total population that will actually react are those that have jumped into energy states that exceed the activation energy barrier for the reaction in question (see above plot).

Another characteristic of “random thermal energy” is that it exerts its affects in arandom manner. Thus, molecules in solution not only spontaneously moveabout, but frequently change directions in a completely random manner: this isknown as the “random walk” or Brownian motion.

From: Krell Institute

Shape Isn’t Everything Amino acid side chains are more than just pieces of a 3-D jigsaw puzzle- we must consider attractive and repulsive forces that give each side chain its distinctive properties… but first, it is important to understand the concept of equilibrium…

Experimental Electron Density Maps for the 20 Amino Acid Side ChainsFrom: Web site at UCLA: mbi.ucla.edu/~sawaya/m230d/Modelbuilding/aadensity.png

What does it mean when something is at equilibrium?

Consider binding of a protein “R” with a ligand “L”

R + L RL

At equilibrium, there is no net flux between states and the free and boundreceptor and ligand populations are constant.

Another way of saying this is that at equilibrium:

forward rate = reverse rate

kforward.[R]..[L] = kreverse

. [RL]

Indeed, one way to define the equilibrium constant is:

Keq = kforward/kreverse where k are the rate constants.

However, the more common (and useful) way of describing the equilibrium constant is as a ratio of the concentrations of the species in the two states. This is seen by rearranging the equilibrium rate condition:

kforward.[R].[L] = kreverse

. [RL]

[RL]

kforward/kreverse= = Keq

[R] . [L]

The “position” of the equilibrium is defined by the equilibrium constant. If Keq is large then it means that under equilibrium conditions the concentration of the complex [RL] will be large relative to the free ligand and receptor concentrations.

By definition: Go = -RTlnKeq

Goreactants

productsEnergy

Go is known as the “standard Gibbs free energy”. This should not be confusedwith G, which is the energy potential (“driving energy”) for a system not at equilibrium.

For the case of a receptor and ligand forming a complex, complex formation is favorable (Keq >1 and Go is negative) because the free energy of the complex is lower than the free energy of the free ligand/receptor state.

Note the logarithmic relationship between energy and Keq: only a small changein ΔGo results in a large change in the equilibrium constant. This is very important.

While the position of an equilibrium process is described by its equilibrium constant,it is determined by the free energy difference between the two states.

Consider also a case where a protein “R” has two binding sites for a ligand “L”.In this specific example, the second site can filled only when the first site is filled.

In this case, two equilibria are possible and are said to be coupled.

R + L RL

Keq,1

RL + L RLL

Keq,2

For cases such as this where two equilibrium processes are coupled, the overallequilibrium constant:

R + 2L RLL

Keq,overall

is equal to the product of the individual equilibrium constants:

Keq,1 x Keq,2 = Keq

and the overall free energy is equal to the sum of the energies for each step:

Go1 + Go

2 = Gooverall

Coupled Equilibria

Important Classes of Equilibria in Biological Systems

Ligand binding to a “receptor”carrier-ligandenzyme-substrateantibody-antigenprotein-nucleic acidprotein-protein

Membrane partitioning (ligand in solution vs. ligand in membrane)Keq = [Membrane-Bound Ligand]/[Ligand in Solution]

Biochemical Reactions (reactants vs. products)[C] . [D]

For: A + B C + D Keq = [A] . [B]

Protein Folding (unfolded form versus folded form)Keq = [Folded]/[Unfolded]

Acid/Base Buffering (the “ligand” is the proton)

C

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hormone(i.e., vasopressin)

K >>> 1activationK << 1basal

In the absence of hormone, only the first equilibrium is operative, and so the receptor is mostly in its off signalingstate. When the hormone becomes abundant, the overall equilbrium shifts towards the “on” signaling statebecause Kactivation is >> K basal. In other words, under normal physiological conditions, the receptor significantlypopulates the signaling-active state only when the hormone is present.

In “loss of function” disorders such as diabetes insipidus, some of the causitive mutations can lower the free energy of the“off” state to the exent that it becomes the predominate state of the receptor, even under conditions where the hormonewould normally drive the equilibrium completely towards the “on” state. In other words, the basal equilibrium constant isreduced to be even smaller than usual.

Alternately, mutations can induce “loss of function” by mutating the hormone binding site so that the receptor does notsignificantly bind the hormone at normal activating concentrations. In other words, some mutations lower the activationequilibrium constant dramatically, making the receptor unresponsive to hormone at normally-stimulating concentrations.

Equilibria Involved in SignalingBy G Protein-Coupled Receptors(such as the Vasopressin V2 Receptor)

“off” signaling state “on” s ignaling state “on” signaling state

hormone(i .e., vasopressin)

K >>> 1activationK << 1basal

Above: Normal Conditions for Signaling

For the vasopressin V2 receptor (and a number of other related receptors), there is another class of mutations that canchange the difference in free energy between the basal “on” and “off” states, such that the “on” state structure becomesthe low energy state (meaning becomes the energetically favored state even in the absence of hormone). This is illustratedbelow.

“off” signaling state “on” signaling state “on” signaling state

K >>> 1activationK > 1basal

In this case, the signaling active form of the receptor predominates even in the absence of its cognate hormone. This isinappropriate. The unstimulated signaling from such “constitutively active” mutant receptors can cause disease.

Equilibria Involved in Signaling By G Protein-Coupled Receptors

How mutations can can cause constitutive “gain of function”.

Nephrogenic Syndrome of Inappropriate Anti-Diuresis (NSIAD)

A recently discovered disorder (May 5 2005 NEJM, 2 baby boys).

Very similar to the Syndrome of Inappropriate Anti-Diuretic HormoneSecretion (SIADH). This disorder is caused by higher-than-usual levels ofvasopressin, the hormone that triggers vasopressin V2 receptor signaling.

Phenotype is opposite of diabetes insipidus: inappropriate retention of water,resulting in hypoosmolar blood (specifically, low blood sodium) and overlyconcentrated urine. One patient suffered seizures.

Patients are babies, so can’t be treated by restricting liquid intake. Instead,urea was administered orally– an agent that increases osmolarity and therebytriggers the formation of higher volumes of urine and fluid reduction. Babies now OK (so far).

Vasopressin levels normal in these babies. Cause of NSIAD is mutationsof Arg137 of the vasopressin V2 receptor that make it constitutively active.

Recall that turning on the V2R turns on water absorption into the body, leading to concentrated urine.

Human Vasopressin V2 Receptor and NSIAD

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Change of arginine-137 toeither cysteine or leucinecauses NSIAD: NeprhogenicSyndrome of InappropriateAntidiuresis. It has been shown that these mutations result in constitutive activation ofthe receptor, such that signaling occurs at all times.

When are systems not at equilibrium?

When the actual concentration ratio for the “reactants” and “products” does not match the equilibrium constant the system is not at equilibrium. In this case, the energy potential driving the system towards equilibrium is described by G (sometimes referred to as the “driving force” energy; note there is no superscripted o).

G describes how far the system is from equilibrium. When the potential is such that the process will spontaneously proceed in the forward direction, then ΔG will be negative. A process that with a positive ΔG will spontaneously proceed towards equilibrium in the reverse direction. When equilibrium is reached G = 0. The driving force energy will be described later in the course, in preparation for metabolism.

Examples of non-equilibrium conditions:

A receptor plus ligand solution right after mixing a receptor solution with a ligand solution (before there has been time for equilibrium to be establish).

A metabolic pathway (a series of coupled biomolecular reactions) under conditions where there is a net flux of materials through the pathway.

A set of newly-synthesized proteins that have not had a chance to fold.

Systems where a population of molecules (misfolded protein, for example) is trapped in a state for which the rate barrier to unfolding is very high.

An enzyme reaction right after mixing an enzyme with substrates in the absenceof product.

What Are the Molecular Determinants of Go?

First, recall that: Go = Ho - TSo

where T is the temperature, H is the enthalpy and S is the entropy. S is a measure of randomness, or disorder. H is a measure of both the heat generated (or absorbed) by a process and the amount of work involved.

Contributions to energy which involve direct attraction or repulsion are generally enthalpic contributions. A negative enthalpic is energetically favorable. Entropy is a measure of the disorder or randomness: a positive entropy is energetically favorable.

Organizing anything involves a loss of entropy:

When there is a concentration gradient, this is a form of organization. Entropy would prefer a uniform concentration throughout space. This is why when there is a higher concentration of salt, sugar, or some other solute on one side of a membrane than on the other, there will be an osmotic force pushing on the membrane.

Connecting molecules together chemically or by forming a non-covalent complexresults in a loss of translational/rotational entropy.

Folding a protein results in a loss of conformational entropy

Binding of a flexible drug molecule to a rigid protein site results in a loss of conformational entropy.

Solvation leads loss of translational entropy when solvent organizes around solute.

The hydrophobic effect.

From: Mathews and Van Holde

above: water forms “cage” or “clathrate” around hydrocarbons: this is entropically unfavorable

When water-hydrocarboninteractions are replaced withwater-water and hydrocarbon-hydrocarbon interactions, water has much more freedom:entropy therefore prefershydrocarbons to aggregate

aggregation

free alkylchains in water

genesis of oil droplet

Van der Waals interactions

Interactions That Are Primarily Electrostatic

From Mathews and Van Holde

All atoms undergo VDW interactions.“Polar” atoms aren’t better at thisthan non-polar. What counts isintimate molecular packing– part of why proteins fold snugly andprotein binding sites are highlycomplementary to ligand shape.

VDW energy also is the basis for “steric hindrance” or “steric clashes”.

Energy Between Two Ions in Solution = q1 x q2

k x x d

+-d

q: charges of the ions

: dielectric constant of solution between the ions78 for water2 for hydrocarbons

k: constant (get from a physics book)

Ionic Interactions

C=O

H-O

H-N

C-O

partial negative charge

partial positive charge

Bond Dipoles

N-terminus

C-terminus

partial negative charge

partial positive charge

Helix Dipole

H Hpartial positive charge

partial negative charge

net dipole(dipolemoment)of water

when salt dissolves, salt bridgesbeween Na+ and Cl- are replaced byenergetically favorable ion-waterelectrostatic interactions

Hydrogen Bonding

Hydrogen bonds have only partial covalent character, but do allow the interacting atoms to get a little closer than normally would be allowed by van der Waals forces. H-bonds form between atoms that have a lone pair of electrons (“hydrogen bond acceptor”) and a (polarized) H that is attached to O, N, or S (the “H-bond donor”). Water is especially good at forming H-bonds-- a single molecule can participate in 4 H-bonds at once. C-H do not form good hydrogen bonds because C almost never has a lone pair of electrons and because the electrons in the C-H bond are evenly shared between C and H.

Generally, it is energetically unfavorable for polar molecule to be in a hydrophobic environment. Conversely, hydrophobic molecules prefer to be in hydrophobic environment. Water is a polar solvent. The interior of a membrane bilayer is hydrophobic and represents an alternative solvent in biology.

Hydrophobic (“hates water”) = Nonpolar = Apolar: Used to describe a substance that doesnot undergo many electrostaticinteractions except for van der Waals.

Some Terminology

Hydrophilic (“loves water”) =Polar: Used to describe a molecule or molecular fragment that can readily undergo electrostatic interactions beyond simple van der Waals forces.

How much energy are these various interactions usually worth in aqueous solution?

H-Bond and salt bridges: -1 to -5 kCal/mol

Loss of 1 degree of conformational entropy when rotation arounda single bond is frozen out: +1 kCal/mol

Hydrophobic Effect: transfer of 1 hydrocarbon –CH2- or –CH3 fromwater to hydrocarbon phase: -0.9 to -2.1 kCal/mol

These are good numbers to keep in mind. In many cases it is perturbations in protein structure and function that may involve energy shifts of only 1-5 kCal/mol that result in disease. This is partlybecause modest changes in energy lead to much larger changes inthe associated equilibrium constants.

For example, a single glutamate (negatively charged) to valine mutation on the surface of hemoglobin results in hemoglobin forming ordered aggregates that lead to sick cell anemia. The negative charge in wild type glutamate helps to solubilize the protein and to prevent aggregation. By making this site hydrophobic, the mutation creates at sticky patch on the protein surface.

polarhead group

apolar tail(acyl chains)

How Different Contributions to Molecular Energetics Can Create Order: Formation of Lipid Bilayers, the Critical Structural Unit of All Biological Membranes

A bilayered lipid vesicle-- also called a liposome. By formingsealed vesicles, the remaining hydrocarbon tails are shielded from water.