Biochem 2

Which of the following statements concerning protein domains is true?

They may retain their correct shape even when separated from the rest of the protein

The structural classification of proteins (based on motifs) is based primarily on their

secondary structure content and arrangement

enzyme kinetic experiments are useful for

- comparing different enzymes that catalyze the same reaction -studying the effect of amino acid changes on substrate binding and catalysis -determining if an enzyme inhibitor completes with the substrate for binding

Each of the following reagents or conditions will denature a protein: urea, polar organic solvents, detergent and low pH. For each, describe in one or two sentences what the reagent/condition does to destroy native protein structure. In this description indicate whether the mechanism of denaturation involves primarily entropy or enthalpy or both

-urea- boriz, polar molecule, H-bonds, with water, reducing order in water around hydropphebriz functional groups decreases hydrophobic forces, affects entropy -polar organic solvents: H-bond with water, reducing order of water reduces polarity of solvent decreases hydrophobic forces, affect entropy -detergent: from "coat" around hydrophobic functional groups, decreases hydrophobic forces, affects entropy -low pH: results in protonation of ionizable functional groups -COOH -NH3+ -Positive charge of repulsion loss of ionize bonds- enthalpy

Describe briefly the two major types of symmetry found in oligomeric proteins and give an example of each

1) rational: in rotational symmetry subunits are superimposable after rotation about one or more of the axes. Some examples are hemoglobin and the polivirus capsid 2) helical: on helical symmetry subunits are superimposable after helical ration. Some examples are actin filaments and the tobacco mosaic virus capsid.

two enzyme preperations have the same Km but the bacterially expressed enzyme has a 10-fold lower Vmax. the most likely explanation for this is that

90% of the enzyme expressed in the bacteria is inactive (like adding 1/10[Et])

Two molecules A and B have a Ka=10^6M^-1 for AB complex formation. When both molecules are at 10^-8M the proportion of A and B in an AB complex will be

<10%

A protein (P) has a Ka=10^7 M^-1 for a ligand (L). When [L] is at 10^-6M the proportion of P bound by L in a PL complex (the O or fraction of binding sites occupied) will be

>0.9

Two molecules A and B have a Ka=10^6M for AB complex formation. When both molecules are at 10^-5M the proportion of A and B in an AB complex will be

>90%

O2 binds the oxy or R conformation of HB . CO2 and BPG bind the deoxy or T conformation. It is false that

A HB Varient with a lower affinity for BPG binds O2 less tightly than normal Hb (both in the presence of BPG)

The following polypeptide is likely to form (non polar amino acids) Gly-Leu-Asp-Glu-Lue-Ala-Lys-Ser-Leu-Arg-His-Phe-Cys-His-Ala-Ile

An amphipathic alpha helix

A sequence of amino acids in a certain protein is found to be Ser-Gly-Pro-Gly the sequence is most probably part of of an

Beta turn

The strength and specificity ("affinity") of binding between two molecules is not dependent on

concentration of the two molecules

the Km for an enzyme catalyzed reaction

Consistently underestimates the ES binding affinity

describe three of the important features of a beta-sheet polypeptide structure

In the beta sheet structure, several exxtended polypeptides, or two regions of the same polypeptides, lie side by side and are stabalized by hydrogen bonding between adjacent chains. Adjacent chains may be either parallel (with a repeat distance of about 6.5 A) or antiparallel (7 A repeat). The R groups are often small and alternately protrude from opposite faces of the sheet

What is levathals paradox and what two models for protein folding are currently used to "solve" this paradox

Levinthal was the first to recognize that proteins could not fold through a random process of sampling are possible conformations (shapes). Consider a single protein of 100 amino acids in length. If there are just three conformations possible for each amino acid that gives you a total number of possible conformation, the now takes a few seconds to fold. the first model used to oslve this paradox is the "hierarchial folding model" swcondary structure form first and is stabalized, then tertiary, followed by quatinary structure. The socks analogy discussed in class tries to describe this idea. The monkey at a typewriter. the second model is the "hydrophobic collapse" or "molten globule model" the free energy funnel is often used to describe the process of protein folding states (medium entropy) with more structure, but still dynamic, to the fully folded state with the lowest free energy and entropy.

What do the terms native and denatured mean as applied proteins? what conditions lead to denatured of proteins?

Native protein is correctly folded into its natural conformation (its functional shape found in the cell) A denatured (unfolded) protein is not in its native conformation. It may be only slightly unfolded or it may be completely unfolded. Proteins can be denatured by heat , extreme pH or treatment with denaturants like urea. Urea is a small, polar molecule that interacts with water to disrupts the formation of H-bonded crystaline lattices. this essentially negates the hydrophobic force driving protein folding. Urea is sometimes called a chaotropic ('Chaos-forming') agent

in the lysozyme active site Glu 35 is protonated while Asp 52 is deprotonated

Since the local environmental of Glu35 is less polar than that os Asp 52

Thr and/or Leu residues tend to disrupt an alpha helix when they occur next to each other in a protein because:

Steric hinderance occurs between the bulky Thr side chains

What do the terms "structural motifs or folds" and "structural domains" (or modules) and "protein families" refer to? discuss the evolutionary implications of their definitions

Structureal domains or modules are stable, independently folding comonents of proteins. DOmains can often function alone. There are approximately 1000 unique domains identified, but about 40 of them are found in half of the known of the known proteins. proteins are often grouped by containinga common domain into "families" domains or subdominains (called motifs or protein folds) are oncoded by exons or groups of exons or groups of exons. These exons can combine with other exons to form different proteins. Evolution has selected a small subset of all the possible protein sequences and structures for those that work. These have been mixed and matched and "tweaked" to perform a wide array of functions in cell.

Describe three of the important features of the alpha-helical polypeptide structure

The alpha-helical structure of a polypeptide is tightly wound a long a central axis; each turn of the right handed helix conatins 3.6 residues and stretches 5.4 A along the axis. The peptide NH is hydrogen-bonded to the carbonyl oxygen of the fourth amino acid along the sequence toward the amino terminus. The R groups of the amino acid residues protrude outward from the helical backbone

Describe the four main levels of protein folding and the types of chemical bonds and interactions involved in forming and stabilizing them

The four main levels of protein folding are primary structure (involving covalent peptide bonds between amino acids) secondary structure (stabalized by H-bonds) tertiary structure (driven by hydrophobic forces and stabalized by all four types of noncovalent bonds) and quaternary structure (stabalized by all four types of noncovalent bonds) -Primary structures consists of the amino acid sequence. secondary structure consists of alpha-helices, beta-strand, beta-sheets, and turns. Tertiary structure consists of domains that are formed from combinations of secondary structures. These combine to form the structure of the whole proteins (polypeptide) Quaternary structure refers to multi-protein (subunit) complexes forming dimers, trimers -finally, the formation of disulfide bonds does not help a protein fold properly. enzymes (disulfide isomerase) are needed to help the disulfide bonds break to allow folding to occur and then help the correct cross-linking bonds to form later. Disulfide bonds function to stabalize the correct protein structure. Howeevr, they can form in the relatively oxidizing environment outside cells and inside the endoplasmic reticulum lumen and in vesicles. Therefore, these bonds aare found most commonly in extracellular proteins like antibiotics and intestinal enzymes

An alpha helix would be destabalized most by:

The presence of the two Lys residues near the amino terminus of the alpha helix

Experiments on denaturation and renaturation after the reduction and reoxidation of the S-S- bonds in the enzyme ribonuclease (RNase) have shown that

The primary sequence of RNase is sufficient to determine its specific secondary and tertiary structure

Kendrews studies of the globular myoglobin structure demonstrated that

The structure was very compact, with virtually no internal space available for water

Why are glycine and proline often found within a beta turn

a beta turn results in a tight 180 reversal in the direction of the polypeptide chain. Glycine is the smallest and thus most flexible amino acid, and proline can readily assume that cis configuration, which facilitates a tight turn.

triosephosphate isomerase catalyzes the above reaction and its competitively inhibited by phosphoglycolate by

acting as a transition state analog

drug resistance in cancer cells

all of them are correct

explain (succintly) the theoretical and/or experimental arguments in supporrt of this statement: "the primary sequence of a protein determines its three-dimensional shape and thus its functional"

anfinsen showed that a completely denatured enzyme (ribonuclease) could fold spontaneously into its native, enzymatically active form with only the primary sequence to guide it.

Blood and metastatic cancers

are generally treated using chemotherapy

What is typically found in the interior of a water-soluble globular protein? what is found on the exterior of a globular protein?

as a general rule, aqueous soluble protein fold with the hydrophobic amino acids on the inside and the hydrophilic amino acids on the outside. - hydrophobic amino acids are on the inside away from water and hydrophilic amino acids are on the outside interacting with water. Exeptions to this usually help identify important active sites or intermembrane regions in transmembrane proteins

in general, enzymes increase the rate of reactions

by reducing the energy of activation

hemoglobin

changes conformation as O2 binds causing a higher affinity for additional O2 binding

in kinetic analyses of chemical reactions

comparing reactions is simplifies by using initial velocities

What is the rationale for many large proteins containing multiple copies of a polypeptide subunit?

each different polypeptide requires a separate gene that must be replicated and transcribed. It is therefore more efficient to have fewer gens, encoding shorter polypeptides that can be used to construct many large protein complexes.

the most important contribution to the stability of a proteins conformation appears to be the

entropy increase from the decrease in ordered water molecules forming a solvent shell around it

enzymes exhibit saturation behavoir (become insensitive to more substrate, have a Vmax) because

enzymes have a fixed number of active sites where subrstrate binds

P-glycoprotein

functions to keep toxins out of the central nervous system

The fundamental cause of sickle-cell disease is a change in the structure of

hemoglobin

In the binding of oxygen to myoglobin, the relationship between the concentration of oxygen and the fraction of binding sites occupied can best be described as

hyperbolic

a mutation in an enzymes active site that increases its affinity for the substrate 100-fold is most likely

increase the rate of the catalyzed

The catalytic mechanism of serine protease

involves all the above

concurrent acid and base catalysis

involves making reactants better electrophiles and better nucleophiles

The tertiary structure of proteins

is stabilized, but not dictated by disulfide bond formation

which of the following statements concerning the process of spontaneous folding of protein is false?

it may be an essentailly random process

an enzyme is added to a solution were its substrate and products are in equilibrium

nothing; the reaction would stay at equilibrium

when oxygen binds to a heme-containing protein, the two open coordination bonds of Fe2+ are occupied by

one O2 molecule and one amino acid atom

multidrug resistance (MDR) in cancer cells

pften arises through expression of P glycoprotein

an enzyme can not speed up a reaction by

the active site providing heat from the environment that raises the energy content of the substrate

Any given protein is characterized by a unique amino acid sequence (primary structure) and three-dimensional (tertiary) structure. How are these related?

the three-dimensional structure is determined by the amino acid sequence. This means that the amino acid sequence. This means that the amino acid sequence contains all of the information that is required for the polypeptide chain to fold up into a discrete three-dimentional shape.