Berg J.M., Tymoczko J.L., Stryer L. Biochemistry

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I. The Molecular Design of Life 4. Exploring Proteins 4.5. Three-Dimensional Protein Structure Can Be Determined by NMR Spectroscopy and X-Ray Crystallography

Figure 4.44. One-Dimensional NMR Spectra. (A)

H-NMR spectrum of ethanol (CH

OH) shows that the

chemical shifts for the hydrogen are clearly resolved. (B)

H-NMR spectrum from a 55 amino acid fragment of a protein

with a role in RNA splicing shows a greater degree of complexity. A large number of peaks are present and many

overlap. [(A) After C. Branden and J. Tooze, Introduction to Protein Structure (Garland, 1991), p. 280; (B) courtesy of

Barbara Amann and Wesley McDermott.]

I. The Molecular Design of Life 4. Exploring Proteins 4.5. Three-Dimensional Protein Structure Can Be Determined by NMR Spectroscopy and X-Ray Crystallography

Figure 4.45. The Nuclear Overhauser Effect. The nuclear Overhauser effect (NOE) identifies pairs of protons that are

in close proximity. (A) Schematic representation of a polypeptide chain highlighting five particular protons. Protons 2

and 5 are in close proximity (~4 Å apart), whereas other pairs are farther apart. (B) A highly simplified NOESY

spectrum. The diagonal shows five peaks corresponding to the five protons in part A. The peaks above the diagonal and

the symmetrically related one below reveal that proton 2 is close to proton 5.

I. The Molecular Design of Life 4. Exploring Proteins 4.5. Three-Dimensional Protein Structure Can Be Determined by NMR Spectroscopy and X-Ray Crystallography

Figure 4.46. Detecting Short Proton-Proton Distances. A NOESY spectrum for a 55 amino acid domain from a

protein having a role in RNA splicing. Each off-diagonal peak corresponds to a short proton-proton separation. This

spectrum reveals hundreds of such short proton-proton distances, which can be used to determine the three-dimensional

structure of this domain. [Courtesy of Barbara Amann and Wesley McDermott.]

I. The Molecular Design of Life 4. Exploring Proteins 4.5. Three-Dimensional Protein Structure Can Be Determined by NMR Spectroscopy and X-Ray Crystallography

Figure 4.47. Structures Calculated on the Basis of NMR Constraints. (A) NOESY observations show that protons

(connected by dotted red lines) are close to one another in space. (B) A three-dimensional structure calculated with these

proton pairs constrained to be close together.

I. The Molecular Design of Life 4. Exploring Proteins 4.5. Three-Dimensional Protein Structure Can Be Determined by NMR Spectroscopy and X-Ray Crystallography

Figure 4.48. A Family of Structures. A set of 25 structures for a 28 amino acid domain from a zinc-finger-DNA-

binding protein. The red line traces the average course of the protein backbone. Each of these structures is consistent

with hundreds of constraints derived from NMR experiments. The differences between the individual structures are due

to a combination of imperfections in the experimental data and the dynamic nature of proteins in solution. [Courtesy of

Barbara Amann.]

I. The Molecular Design of Life 4. Exploring Proteins 4.5. Three-Dimensional Protein Structure Can Be Determined by NMR Spectroscopy and X-Ray Crystallography

Figure 4.49. Essence of an X-Ray Crystallographic Experiment: an X-Ray Beam, a Crystal, and a Detector.

I. The Molecular Design of Life 4. Exploring Proteins 4.5. Three-Dimensional Protein Structure Can Be Determined by NMR Spectroscopy and X-Ray Crystallography

Figure 4.50. Crystallization of Myoglobin.

I. The Molecular Design of Life 4. Exploring Proteins 4.5. Three-Dimensional Protein Structure Can Be Determined by NMR Spectroscopy and X-Ray Crystallography

Figure 4.51. Myoglobin Crystal and X-Ray. (A) Crystal of myoglobin. (B) X-ray precession photograph of a

myoglobin crystal. [(A) Mel Pollinger/Fran Heyl Associates.]

I. The Molecular Design of Life 4. Exploring Proteins 4.5. Three-Dimensional Protein Structure Can Be Determined by NMR Spectroscopy and X-Ray Crystallography

Figure 4.52. Section of the Electron-Density Map of Myoglobin. This section of the electron-density map shows the

heme group. The peak of the center of this section corresponds to the position of the iron atom. [From J. C. Kendrew.

reserved.]

I. The Molecular Design of Life 4. Exploring Proteins 4.5. Three-Dimensional Protein Structure Can Be Determined by NMR Spectroscopy and X-Ray Crystallography

Figure 4.53. Section of a U.S. Geological Survey Map. Capitol Peak Quadrangle, Colorado.

I. The Molecular Design of Life 4. Exploring Proteins 4.5. Three-Dimensional Protein Structure Can Be Determined by NMR Spectroscopy and X-Ray Crystallography

Figure 4.54. Resolution Affects the Quality of an Image. The effect of resolution on the quality of a reconstructed

image is shown by an optical analog of x-ray diffraction: (A) a photograph of the Parthenon; (B) an optical diffraction

pattern of the Parthenon; (C and D) images reconstructed from the pattern in part B. More data were used to obtain

image D than image C, which accounts for the higher quality of image D. [(A) Courtesy of Dr. Thomas Steitz. (B)

Courtesy of Dr. David DeRosier).]

I. The Molecular Design of Life 4. Exploring Proteins

Summary

The rapid progress in gene sequencing has advanced another goal of biochemistry

elucidation of the proteome. The

proteome is the complete set of proteins expressed and includes information about how they are modified, how they

function, and how they interact with other molecules.

The Purification of Proteins Is an Essential Step in Understanding Their Function

Proteins can be separated from one another and from other molecules on the basis of such characteristics as solubility,

size, charge, and binding affinity. SDS-polyacrylamide gel electrophoresis separates the polypeptide chains of proteins

under denaturing conditions largely according to mass. Proteins can also be separated electrophoretically on the basis of

net charge by isoelectric focusing in a pH gradient. Ultracentrifugation and gel-filtration chromatography resolve

proteins according to size, whereas ion-exchange chromatography separates them mainly on the basis of net charge. The

high affinity of many proteins for specific chemical groups is exploited in affinity chromatography, in which proteins

bind to columns containing beads bearing covalently linked substrates, inhibitors, or other specifically recognized

groups. The mass of a protein can be precisely determined by sedimentation equilibrium measurements or by mass

spectrometry.

Amino Acid Sequences Can Be Determined by Automated Edman Degradation

The amino acid composition of a protein can be ascertained by hydrolyzing it into its constituent amino acids in 6 N HCl

at 110°C. The amino acids can be separated by ion-exchange chromatography and quantitated by reacting them with

ninhydrin or fluorescamine. Amino acid sequences can be determined by Edman degradation, which removes one amino

acid at a time from the amino end of a peptide. Phenyl isothiocyanate reacts with the terminal amino group to form a

phenylthiocarbamoyl derivative, which cyclizes under mildly acidic conditions to give a phenylthiohydantoin-amino acid

and a peptide shortened by one residue. Automated repeated Edman degradations by a sequenator can analyze sequences

of about 50 residues. Longer polypeptide chains are broken into shorter ones for analysis by specifically cleaving them

with a reagent such as cyanogen bromide, which splits peptide bonds on the carboxyl side of methionine residues.

Enzymes such as trypsin, which cleaves on the carboxyl side of lysine and arginine residues, also are very useful in

splitting proteins. Amino acid sequences are rich in information concerning the kinship of proteins, their evolutionary

relations, and diseases produced by mutations. Knowledge of a sequence provides valuable clues to conformation and

function.

Immunology Provides Important Techniques with Which to Investigate Proteins

Proteins can be detected and quantitated by highly specific antibodies; monoclonal antibodies are especially useful

because they are homogeneous. Enzyme-linked immunosorbent assays and Western blots of SDS-polyacrylamide gels

are used extensively. Proteins can also be localized within cells by immunofluorescence microscopy and

immunoelectron microscopy.

Peptides Can Be Synthesized by Automated Solid-Phase Methods

Polypeptide chains can be synthesized by automated solid-phase methods in which the carboxyl end of the growing

chain is linked to an insoluble support. The α -carboxyl group of the incoming amino acid is activated by

dicyclohexylcarbodiimide and joined to the α -amino group of the growing chain. Synthetic peptides can serve as drugs

and as antigens to stimulate the formation of specific antibodies. They can also be sources of insight into relations

between amino acid sequence and conformation.

Three-Dimensional Protein Structure Can Be Determined by NMR Spectroscopy and

X-Ray Crystallography

Nuclear magnetic resonance spectroscopy and x-ray crystallography have greatly enriched our understanding of how

proteins fold, recognize other molecules, and catalyze chemical reactions. Nuclear magnetic resonance spectroscopy

reveals the structure and dynamics of proteins in solution. The chemical shift of nuclei depends on their local

environment. Furthermore, the spins of neighboring nuclei interact with each other in ways that provide definitive

structural information.

X-ray crystallography is possible because electrons scatter x-rays; the way in which the scattered waves recombine

depends only on the atomic arrangement. The three-dimensional structures of thousands of proteins are now known in

atomic detail.

Key Terms

proteome

assay

homogenate

salting out

dialysis

gel-filtration chromatography

ion-exchange chromatography

affinity chromatography

high-pressure liquid chromatography (HPLC)

gel electrophoresis

isoelectric point

isoelectric focusing

two-dimensional electrophoresis

sedimentation coefficient (Svedberg units, S)

matrix-assisted laser desorption- ionization-time of flight spectrometry (MALDI-TOF)

dabsyl chloride

dansyl chloride

Edman degradation

phenyl isothiocyanate

cyanogen bromide (CNBr)

overlap peptides

diagonal electrophoresis

antibody

antigen

antigenic determinant (epitope)

monoclonal antibodies

enzyme-linked immunosorbent assay (ELISA)

Western blotting

fluorescence microscopy

green fluorescent protein (GFP)

solid-phase method

nuclear magnetic resonance (NMR) spectroscopy

x-ray crystallography

I. The Molecular Design of Life 4. Exploring Proteins

Problems

Valuable reagents. The following reagents are often used in protein chemistry:

Which one is the best suited for accomplishing each of the following tasks?

(a) Determination of the amino acid sequence of a small peptide.

(b) Identification of the amino-terminal residue of a peptide (of which you have less than 0.1 µg).

disulfide bonds were present?

(d) Hydrolysis of peptide bonds on the carboxyl side of aromatic residues.

(e) Cleavage of peptide bonds on the carboxyl side of methionines.

(f) Hydrolysis of peptide bonds on the carboxyl side of lysine and arginine residues.

See answer

Finding an end. Anhydrous hydrazine (H

N NH

) has been used to cleave peptide bonds in proteins. What are the

reaction products? How might this technique be used to identify the carboxyl-terminal amino acid?

See answer

Crafting a new breakpoint. Ethyleneimine reacts with cysteine side chains in proteins to form S-aminoethyl

derivatives. The peptide bonds on the carboxyl side of these modified cysteine residues are susceptible to hydrolysis

by trypsin. Why?

See answer

Spectrometry. The absorbance A of a solution is defined as

in which I

is the incident light intensity and I is the transmitted light intensity. The absorbance is related to the

molar absorption coefficient (extinction coefficient) ε (in M

-1

), concentration c (in M), and path length l (in

cm) by

The absorption coefficient of myoglobin at 580 nm is 15,000 M

-1

. What is the absorbance of a 1 mg ml

-1

solution across a 1-cm path? What percentage of the incident light is transmitted by this solution?

See answer

A slow mover. Tropomyosin, a 93-kd muscle protein, sediments more slowly than does hemoglobin (65 kd). Their

sedimentation coefficients are 2.6S and 4.31S, respectively. Which structural feature of tropomyosin accounts for its

slow sedimentation?

See answer

Sedimenting spheres. What is the dependence of the sedimentation coefficient S of a spherical protein on its mass?

How much more rapidly does an 80-kd protein sediment than does a 40-kd protein?

See answer

Size estimate. The relative electrophoretic mobilities of a 30-kd protein and a 92-kd protein used as standards on an

SDS-polyacrylamide gel are 0.80 and 0.41, respectively. What is the apparent mass of a protein having a mobility of

0.62 on this gel?

See answer

A new partnership? The gene encoding a protein with a single disulfide bond undergoes a mutation that changes a

serine residue into a cysteine residue. You want to find out whether the disulfide pairing in this mutant is the same as

in the original protein. Propose an experiment to directly answer this question.

See answer

Sorting cells. Fluorescence-activated cell sorting (FACS) is a powerful technique for separating cells according to

their content of particular molecules. For example, a fluorescence-labeled antibody specific for a cell-surface protein

can be used to detect cells containing such a molecule. Suppose that you want to isolate cells that possess a receptor

enabling them to detect bacterial degradation products. However, you do not yet have an antibody directed against

this receptor. Which fluorescencelabeled molecule would you prepare to identify such cells?

See answer

10.

Column choice. (a) The octapeptide AVGWRVKS was digested with the enzyme trypsin. Would ion exchange or

molecular exclusion be most appropriate for separating the products? Explain. (b) Suppose that the peptide was

digested with chymotrypsin. What would be the optimal separation technique? Explain.

See answer

11.

Making more enzyme? In the course of purifying an enzyme, a researcher performs a purification step that results in

an increase in the total activity to a value greater than that present in the original crude extract. Explain how the

amount of total activity might increase.

See answer

12.

Protein purification problem. Complete the table below.

See answer

Chapter Integration Problems

13.

Quaternary structure. A protein was purified to homogeneity. Determination of the molecular weight by molecular

exclusion chromatography yields 60 kd. Chromatography in the presence of 6 M urea yields a 30-kd species. When

the chromatography is repeated in the presence of 6 M urea and 10 mM β-mercaptoethanol, a single molecular

species of 15 kd results. Describe the structure of the molecule.

See answer