Investigation of the three-dimensional solution structure of melanostatin using one- and...

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Investigation of the three-dimensional solution

structure of melanostatin using one- and

two-dimensional NMR spectroscopy and molecular

dynamics calculations

Ryan McQuillen

Department of Physics, Lafayette College, Hugel Science Center, Easton, PA 18042

Advisor: Professor Bradley C. Antanaitis

May 9, 2012


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Investigation of the three-dimensional solution structure of

melanostatin using one- and two-dimensional NMR spectroscopy

and molecular dynamics calculations

Ryan J. McQuillen

Department of Physics, Lafayette College, Hugel Science Center, Easton, PA 18042

Advisor: Professor Bradley C. Antanaitis

May 9, 2012

Abstract

Two cyclic nonapeptides derived from alpha-fetoprotein (AFP) have been synthe-sized by researchers at Albany Medical College and shown to inhibit the growth ofestrogen-receptor (ER) positive breast cancer while exhibiting minimal toxicity. How-ever, the three-dimensional structures of these nonapetides in solution are unknownand their biological receptors remain to be identified. A method has been developedto elucidate their solution structures using a combination of NMR spectroscopy andmolecular dynamics. Because of the complexity of the nonapeptides NMR spectra

and the large number of conformations presumably available to them, it was deemedprudent to first test the methodology on simpler compounds whose structures are rea-sonably well characterized in the literature. For this purpose melanostatin (Pro-Leu-Gly), a tripeptide having two amino acid residues in common with the nonapeptideswas chosen. Using a custom suite of sophisticated one- and two-dimensional NMRspectroscopic protocols all resonance peaks in melanostatin and its three constituentamino acids were identified. These assignments combined with calculations made byHyperChem, a molecular dynamics program, allowed the prediction of the dominantsolution conformations of melanostatin. In principle, similar techniques applied to thenonapeptides should give complete and unambiguous resonance assignments of theircomplex NMR spectra and identify their dominant solution structures. In turn, thatinformation will serve as input for molecular docking studies designed to identify thebiological receptors of these promising anti-breast cancer compounds and thus shedlight on their mode of action.

Keywords: melanostatin, hydrogen bond, solution structure

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Contents

1 Introduction 91.1 Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91.2 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1.3 General Protein Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111.4 Melanostatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2 Materials and Methods 132.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132.2 Nuclear Magnetic Resonance (NMR) Spectroscopy . . . . . . . . . . . . . . . 13

2.2.1 NMR Suite for Structural Determination of Polypeptides . . . . . . . 142.2.2 Solvent Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

2.3 Molecular Dynamics Calculations . . . . . . . . . . . . . . . . . . . . . . . . 162.3.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162.3.2 Understanding the AMBER Force Field . . . . . . . . . . . . . . . . 18

2.3.3 Simulated Annealing and Temperature Mimicking Scheme . . . . . . 232.4 Running a Simulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

3 Results 243.1 1H and 13C NMR Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

3.1.1 1D Carbon-13 NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . 243.1.2 1D and 2D Proton NMR . . . . . . . . . . . . . . . . . . . . . . . . . 35

3.2 Solution Conformations Predicted by Molecular Dynamics . . . . . . . . . . 60

4 Conclusion & Discussion 704.1 Future Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

A Nuclear Magnetic Resonance (NMR) Spectroscopy 74A.1 Physical Basis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74A.2 Magnetic Resonance Condition . . . . . . . . . . . . . . . . . . . . . . . . . 79A.3 Saturation and T1 Relaxation Processes . . . . . . . . . . . . . . . . . . . . 79A.4 The Bloch Equations: A Classical Description . . . . . . . . . . . . . . . . . 81

A.4.1 The Non-Rotating Reference Frame . . . . . . . . . . . . . . . . . . . 81A.4.2 The Rotating Reference Frame . . . . . . . . . . . . . . . . . . . . . 85

A.5 Introduction of the Transverse Magnetic Field . . . . . . . . . . . . . . . . . 88A.5.1 Nutation ofM by B1 . . . . . . . . . . . . . . . . . . . . . . . . . . . 91A.5.2 Pulse Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

A.6 Recording a Signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94A.7 The Chemical Shift . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

A.7.1 Contributions to Nuclear Shielding . . . . . . . . . . . . . . . . . . . 98A.7.2 The Chemical Shift Scale . . . . . . . . . . . . . . . . . . . . . . . . . 101A.7.3 The Reference Compound . . . . . . . . . . . . . . . . . . . . . . . . 101

A.8 First-Order (Weak) Spin-Spin Coupling . . . . . . . . . . . . . . . . . . . . . 103A.8.1 Singly Coupled (AX) Nuclei . . . . . . . . . . . . . . . . . . . . . . . 104

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A.8.2 Coupling to Two Inequivalent Nuclei (AMX) . . . . . . . . . . . . . . 105A.8.3 Coupling to Two Equivalent Nuclei (AX2) . . . . . . . . . . . . . . . 105A.8.4 Coupling to n Equivalent Nuclei (AXn) . . . . . . . . . . . . . . . . . 107

A.9 Pulse Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108A.9.1 The 1H and 13C Free-Induction-Decay (FID) Pulse Sequence . . . . . 108

A.9.2 The 13C Single Pulse Broadband Decoupling Pulse Sequence . . . . . 109A.9.3 The 1H Single Pulse Watergate Suppression Pulse Sequence . . . . . 110A.9.4 The 13C Attached Proton Test (APT) Pulse Sequence . . . . . . . . . 110A.9.5 The 13C Distortionless Enhancement by Polarization Transfer (DEPT)

Pulse Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111A.9.6 The Correlation Spectroscopy (COSY) Pulse Sequence . . . . . . . . 112

B The Berendsen Thermostat 113

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List of Figures

1.1 Illustrations of the cyclic AFP-derived nonapeptide. . . . . . . . . . . . . . . 101.2 The three amino acid constituents of melanostatin and their representative

structures.[12] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1.3 A ribbon model representing the secondary structure of a -bended molecule.[13] 132.1 Experimental process for a given system or model.[27] . . . . . . . . . . . . . 162.2 Mass-spring model of two atoms (i and j) separated by a single (M1) bond. . 192.3 The angle between two atoms (i and j) separated by two (M2) bonds. . . . . 192.4 Torsion angle of the bond (j,k) between two bonded pairs (M3) of atoms (i,j

and k,l). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202.5 Plot of the LJ Potential between two atoms (i and j) separated by three or

more bonds (M4) in a fictitious molecule. . . . . . . . . . . . . . . . . . . . . 212.6 Plot of the Hydrogen Bond Potential between two atoms (i and j) separated

by three or more bonds (M4) in a fictitious molecule. . . . . . . . . . . . . . 222.7 Plot of the electrostatic interaction potential as a function of atomic separation

for two atoms (i and j) separated by three or more bonds (M4) in a fictitiousmolecule. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

2.8 The free energy surface of Beta3s (a mini-protein) and assigned conformationalstates.[34] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

3.1 Glycine 100mM 90% D2O/10% H2O13C with Broadband Decoupling spectra. 26

3.2 L-4-Hydroxyproline 100mM 13C with Broadband Decoupling spectra. . . . . 283.3 Leucine 100mM 90% H2O/10% D2O

13C with Broadband Decoupling Spectra. 303.4 Melanostatin 3mM 90%H2O/10% D2O

13C with Broadband Decoupling spectra. 323.5 Hypothetical 1D proton spectrum of melanostatin resulting from superpo-

sition of L-4-hydroxyproline, leucine, and glycine assuming no inter-residue

interactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363.6 1H Single Pulse with Water Suppression Spectra of Glycine 100mM in bothsolvent mixtures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

3.7 L-4-Hydroxyproline 100mM 90% H2O/10% D2O1H Single Pulse with Water

Suppression Spectrum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393.8 Enlarged detailed regions of the L-4-Hydroxyproline 100mM 90% H2O/10%

D2O1H Single Pulse with Water Suppression Spectra. . . . . . . . . . . . . . 40

3.9 L-4-Hydroxyproline 100mM 90% D2O/10% H2O1H Single Pulse with Water

Suppression Spectrum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413.10 Enlarged detailed regions of the L-4-Hydroxyproline 100mM 90% D2O/10%

H2O1H Single Pulse with Water Suppression Spectra. . . . . . . . . . . . . . 42

3.11 Leucine 100mM 90% H2O/10% D2O 1H Single Pulse with Water SuppressionSpectrum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

3.12 Enlarged detailed regions of the Leucine 100mM 90% H2O/10% D2O1H Single

Pulse with Water Suppression Spectra. . . . . . . . . . . . . . . . . . . . . . 453.13 Leucine 100mM 90% D2O/10% H2O

1H Single Pulse with Water SuppressionSpectrum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

3.14 Leucine 100mM 90% D2O/10% H2O1H Single Pulse with Water Suppression

Spectrum with detailed regions enlarged. . . . . . . . . . . . . . . . . . . . . 47

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3.15 Melanostatin 3mM 90% H2O/10% D2O1H Single Pulse with Water Suppres-

sion Spectrum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493.16 Melanostatin 3mM 90% H2O/10% D2O

1H Single Pulse with Water Suppres-sion Spectrum with detailed regions enlarged. . . . . . . . . . . . . . . . . . 50

3.17 Melanostatin 3mM 90% H2O/10% D2O1H Single Pulse with Water Suppres-

sion Spectrum with detailed regions enlarged (continued). . . . . . . . . . . . 513.18 Melanostatin 3mM 90% D2O/10% H2O

1H Single Pulse with Water Suppres-sion Spectrum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

3.19 Melanostatin 3mM 90% D2O/10% H2O1H Single Pulse with Water Suppres-

sion Spectrum with detailed regions enlarged. . . . . . . . . . . . . . . . . . 533.20 Melanostatin 3mM 90% D2O/10% H2O

1H Single Pulse with Water Suppres-sion Spectrum with detailed regions enlarged (continued). . . . . . . . . . . . 54

3.21 Glycine Correlated Spectroscopy (COSY) Spectra . . . . . . . . . . . . . . . 563.22 L-4-Hydroxyproline Correlated Spectroscopy (COSY) Spectra . . . . . . . . 573.23 Leucine Correlated Spectroscopy (COSY) Spectra . . . . . . . . . . . . . . . 583.24 Melanostatin Correlated Spectroscopy (COSY) Spectra . . . . . . . . . . . . 593.25 Two different conformations of melanostatin generated using HyperChem. . . 603.26 Diagrammatic representation of the melanostatin and its dihedral angles.[14] 613.27 Karplus plot for a dihedral angle using the empirical constants A = 7.13,

B = 1.31, and C = 1.56. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614.1 Peptide backbone of a protein with the expected correlations from COSY and

NOESY experiments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 704.2 Two advanced features of HyperChem to utilize in future studies. . . . . . . 71A.1 Splitting of spin states for a spin-12 nuclide. . . . . . . . . . . . . . . . . . . . 76A.2 The motion of a spinning top (left) with the force of gravity acting on it. The

motion of a nucleus (right) with the force of an external magnetic field acting

on it. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77A.3 A system of homogeneous nuclei (left) whose average s add up to M0 (right). 77A.4 The bulk magnetic moment vector of a system ofI = 12 nuclides.[21] . . . . . 78A.5 (a) Graphical depiction of the T1 relaxation process. (b) Exaggerated pictorial

view of each step. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80A.6 If several spins (ms) precess in the xy plane at slightly different rates, the

total spin amplitude decreases due to dephasing.[22] . . . . . . . . . . . . . . 83A.7 The path traced by the tip of the bulk magnetic moment vector M as it relaxes

according to Equations A.31A.33.[22] . . . . . . . . . . . . . . . . . . . . . 85A.8 Coordinate system defined by x, y, and z rotating about the z axis. . . . . 85A.9 Graphical depiction of the precession of the nuclear spins and M when both

the static magnetic field B0 and the oscillating magnetic field B1 are present. 88A.10 Decomposition of the linear oscillating field B1. . . . . . . . . . . . . . . . . 89A.11 The path traced by the tip of the magnetic moment vector M as it relaxes

back to M0.[22] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92A.12 Illustration of how the linearly oscillating magnetic field B1 is created in an

NMR spectrometer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92A.13 Two different illustrations of the pulsed magnetic field B1. . . . . . . . . . . 93

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A.14 Direction of the magnetic moment vector, M, in the rotating frame after a) apulse of arbitrary angle , b) after a 90 pulse, and c) after a 180 pulse.[21] 94

A.15 A dipole (m) along the x-axis generates a flux through the shaded region (coil)in the xy-plane that is equal and opposite to that through the hemisphericalcap.[22] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

A.16 Consequences of electron shielding for a given nuclide in an NMR sample. . . 97A.17 The circulation of electronic charge brought about by the mixing of electronic

wavefunctions by an external field B0. This paramagnetic current generatesa small local magnetic field that acts to deshield the nucleus at the center ofthe electron density.[19] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

A.18 Dipolar lines of flux generated by an induced magnetic moment at the centerof a cylindrically symmetric neighboring group (i.e., an aromatic ring) withtwo protons A and B nearby. Proton A is more shielded by the paramagneticeffect while proton B is deshielded. . . . . . . . . . . . . . . . . . . . . . . . 100

A.19 NMR spectra are conventionally plotted with chemical shift increasing fromright to left. Nucleus A, which is more strongly shielded than nucleus B, thusappears to the right of B, has a lower resonance frequency than B, and issometimes referred as being upfield of B.[19] . . . . . . . . . . . . . . . . . 102

A.20 Two common well-shielded NMR standards. . . . . . . . . . . . . . . . . . . 102A.21 The effect of 1H13C scalar coupling in H13CO2 on the energy levels and

spectrum of the 1H nucleus. For clarity, the energy-level shifts due to the J-coupling have been greatly exaggerated. The central pair of energy levels andthe upper spectrum are appropriate in the absence of a J-coupling interaction.Scalar coupling produces the energy levels on the left and right, and the lowerspectrum. Also, you can see here that JAX is the coupling constant measuredin Hz[19] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

A.22 NMR spectrum of nucleus A in an AMX spin system. The four components ofthe multiplet, a doublet of doublets, arise from the four combinations of M andX spins, indicated (m = + 12) and (m =

12). Drawn for JAM > JAX > 0.[19]106

A.23 NMR spectrum of nucleus A in an AX2 spin system. The triplet arises fromthe four combinations of the two X spins, as indicated. Drawn for JAX > 0.[19]106

A.24 Pascals triangle showing the binomial coefficients in the expansion of (1+ x)n.The rows give the relative intensities of the (n + 1) lines in the A multipletof an AXn spin system (n = 06), where X is a spin-

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nucleus. The columnsgive the positions of the lines, relative to the chemical shift position, in unitsof JAX.[19] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

A.25 Multiplet patterns for the A nucleus in various spin systems. M and X are spin-12 unless otherwise stated. Weak coupling is assumed throughout. Spectra aredrawn for |JAX| > |JAM|.[19] . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

A.26 Pulse sequence and signal for a free-induction-decay measurement.[22] . . . . 108A.27 Pulse sequence and signal for a heteronuclear spin decoupling measurement. 109A.28 Depiction of the fields and their associated effects on the bulk magnetic mo-

ment vectors of the carbon, MC, and the the hydrogen, MH. . . . . . . . . . 109A.29 Pulse sequence and signal for a APT measurement. . . . . . . . . . . . . . . 111A.30 Pulse sequence and signal for a DEPT measurement. . . . . . . . . . . . . . 111

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A.31 Pulse sequence and signal for a COSY measurement. . . . . . . . . . . . . . 112

List of Tables

3.1 NMR peak data for Glycine 90% D2O/10% H2O

13

C Single Pulse Experimentwith Broadband Decoupling . . . . . . . . . . . . . . . . . . . . . . . . . . . 273.2 NMR peak data for Glycine 90% D2O/10% H2O

13C Attached Proton Test(APT) Experiment with Broadband Decoupling . . . . . . . . . . . . . . . . 27

3.3 NMR peak data for Glycine 90% D2O/10% H2O13C Distortionless Enhance-

ment by Polarization Transfer (DEPT) Experiment with Broadband Decoupling 273.4 NMR peak data for L-4-Hydroxyproline 100mM 90% D2O/10% H2O

13C Sin-gle Pulse Experiment with Broadband Decoupling . . . . . . . . . . . . . . . 29

3.5 NMR peak data for L-4-Hydroxyproline 100mM 90% H2O/10% D2O13C At-

tached Proton Test (APT) Experiment with Broadband Decoupling . . . . . 293.6 NMR peak data for L-4-Hydroxyproline 100mM 90% H2O/10% D2O

13C Dis-

tortionless Enhancement by Polarization Transfer Experiment with Broad-band Decoupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

3.7 NMR peak data for Leucine 90% H2O/10% D2O13C Single Pulse Experiment

with Broadband Decoupling. . . . . . . . . . . . . . . . . . . . . . . . . . . . 313.8 NMR peak data for Leucine 90% D2O/10% H2O

13C Attached Proton Test(APT) Experiment with Broadband Decoupling. . . . . . . . . . . . . . . . . 31

3.9 NMR peak data for Leucine 90% H2O/10% D2O13C Distortionless Enhance-

ment by Polarization Transfer (DEPT) Experiment with Broadband Decoupling. 313.10 NMR peak data for Melanostatin 90% H2O/10% D2O

13C Single Pulse Ex-periment with Broadband Decoupling. . . . . . . . . . . . . . . . . . . . . . 33

3.11 NMR peak data for Melanostatin 90% D2O/10% H2O13C Attached Proton

Test (APT) Experiment with Broadband Decoupling. . . . . . . . . . . . . . 333.12 NMR peak data for Melanostain 90% H2O/10% D2O

13C Distortionless En-hancement by Polarization Transfer (DEPT) Experiment with Broadband De-coupling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

3.13 NMR peak data for Glycine 90% H2O/10% D2O1H Single Pulse Experiment

with Water Suppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373.14 NMR peak data for Glycine 90% D2O/10% H2O

1H Single Pulse Experimentwith Water Suppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

3.15 NMR peak data for 4-L-OH-Proline 90% H2O/10% D2O1H Single Pulse Ex-

periment with Water Suppression . . . . . . . . . . . . . . . . . . . . . . . . 43

3.16 NMR peak data for 4-L-OH-Proline 90% D2O/10% H2O1

H Single Pulse Ex-periment with Water Suppression . . . . . . . . . . . . . . . . . . . . . . . . 43

3.17 NMR peak data for Leucine 90% H2O/10% D2O1H Single Pulse Experiment

with Water Suppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483.18 NMR peak data for Leucine 90% D2O/10% H2O

1H Single Pulse Experimentwith Water Suppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

3.19 NMR peak data for Melanostatin 90% H2O/10% D2O1H Single Pulse Exper-

iment with Water Suppression . . . . . . . . . . . . . . . . . . . . . . . . . . 55

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3.20 NMR peak data for Melanostain 90% D2O/10% H2O1H Single Pulse Exper-

iment with Water Suppression . . . . . . . . . . . . . . . . . . . . . . . . . . 553.21 Best fit dihedral angles and their associated parameters from MD simulations.

All energies are in kJ/mol and all angles are in degrees. . . . . . . . . . . . . 623.22 Values for the various energies and dihedral angles obtained from MD simu-

lations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63A.1 Nuclear spin quantum numbers (I) of some commonly occurring nuclides.[19] 74

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1 Introduction

1.1 Motivation

Of all the human scourges, cancer remains one of the most intractable, recalcitrant, and

deadly. In spite of large sums of money spent in research and treatment, the over-all mortality

rate for the past 40 years has changed little.[1] To this day the best outcomes for all forms

of cancer involve early detection followed by complete surgical removal of all visible tumors.

Unfortunately, by the the time symptoms appear it is often too late because the tumor has

metastasized and become inoperable. One of the few effective treatments of metastasized

cancer involves chemotherapy, the systemic administration of cytotoxins that kill or slow the

growth of rapidly dividing cells. But this also wreaks havoc with healthy tissue, especially

tissue composed of rapidly growing cells, for example, hair, blood cells and mucosa. Even

though chemotherapy may win remission in the short run, it typically fails as the cancer

cells eventually develop a resistance to chemotherapy. For these reasons, researchers are

constantly developing new chemotherapeutic agents, always with an aim of reducing toxicity

and improving long-term efficacy.

1.2 Background

Alpha-fetoprotein (AFP) is an extensively studied protein found in pregnant women, fe-

tuses, and individuals with cancer, the last observation making the protein of great interest

to the medical community. Specifically, AFP has been shown to prevent the growth of

estrogen-receptor positive breast cancer cells in vitro and thus shows promise as a poten-

tial chemotherapeutic agent in the fight against breast cancer.[2][3][4] Unfortunately, whole

proteins tend to be unwieldy, difficult to administer, inherently unstable, and expensive to

isolate. These are properties that cause pharmaceutical companies to shy away from them

as front line therapeutic agents. Recently, researchers at Albany Medical College have iden-

tified and synthesized cyclized nonapeptide derivatives, United States patent 7132400, from

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the active site of AFP that prevent the growth of carcinogen-induced mammary tumors in

rats and inhibit the growth of estrogen-receptor positive human breast cancer xenografts

in mice.[4][5][7] Furthermore, these compounds (see Figure 1.1) retain their oncostatic ef-

ficacy when taken orally, show no toxicity at levels administered to date, and evidently

have a mode of action that differs from the currently preferred chemotherapeutic agent,

tamoxifen.[2][5][6][8] Understandably, research on these and related oligopeptides, focusing

on their potential as anticancer drugs, is proceeding apace. Professor Antanaitis of the

Physics Department has recently acquired small amounts of these compounds for structural

determination. The ultimate goal of his research is the elucidation of the three-dimensional

solution structure of these compounds and identification of potential biological receptors.

However, even a cursory examination of the aqueous NMR spectra of these nonapeptides

reveals considerable complexity, making unambiguous assignment of all resonances, the first

step in solving any structure by NMR spectroscopy, very challenging. That observation

combined with the likelihood that the polypeptides conformational space is large suggest

the structural determination project be broken down into smaller projects, each with its own

specific goals.

(a) 2D Depiction of the nona-

paptides chemical structure.

(b) 1D Depiction of the nonapaptides chemical structure.

Figure 1.1: Illustrations of the cyclic AFP-derived nonapeptide.

The current research project focuses on the development and execution of a research

protocol for structural determination of melanostatin, a tripeptide that has two amino acids

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in common with the nonapeptides and, like the nonapeptides, is water soluble. In partic-

ular, the solution structure of melanostatin was determined by an approach that combines

information derived from a suite of one- and two-dimensional proton and carbon thirteen

NMR experiments with the predictive and illustrative power of computational molecular

modeling. For the most part, the structure of melanostatin is already well-defined in the

literature, except for a nagging dispute about the existence of an intramolecular hydrogen

bond. Bearing this in mind, this preliminary exercise will provide the means for elucidation

of the structure of the nonapeptides and also will help resolve the hydrogen bond dispute.

Key results of both the NMR and molecular modeling studies along with the techniques

developed are given below.

1.3 General Protein Structure

Proteins are linear polymer-like molecules consisting of a string of individual units called

amino acids. Twenty different naturally occurring amino acids have been discovered to

date; nine of which humans derive from food. Figure 1.2 gives the representative structures

of the three amino acids making up melanostatin. The three-dimensional structure and

thus the function of proteins depends on the number and sequence of the amino acids in

the chain. Because structure and function are so closely related, much research has gone

into both identifying and predicting their structures. Experimental methods, such as X-ray

crystallography and nuclear magnetic resonance (NMR) spectroscopy, have been employed

in an attempt to accurately delineate the structure of proteins.[11]

1.4 Melanostatin

To test the accuracy and feasibility of the methodology that will eventually be applied to

the nonapeptides a small, water soluble tripeptide, melanostatin, was chosen. Melanostatin,

which contains the amino acids: proline, leucine, and glycine, was thought to be appropriate

not only because it is small, but also because two of its three amino acid constituents (the

11


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Figure 1.2: The three amino acid constituents of melanostatin and their representativestructures.[12]

hydroxylated form of proline and glycine) are contained in the AFP-derived nonapeptides.

Further it has been observed that even though a number of papers have been published

regarding the solution structure of melanostatin, its actual three-dimensional conformation

in solution is still in dispute. Some groups claim that an intramolecular hydrogen bond exists,

and others claim it does not.[16][14] Initial X-ray crystallographic studies of melanostatin

found a -bend structure characterized by a hydrogen bond from one of the terminal amide

hydrogen atoms to the prolyl carbonyl oxygen atom (see Figure 1.3).[14][15] However, X-

ray crystallography requires that the oligopeptide be in a crystalline state; whereas we are

interested in the structure of the protein in solution. Initial molecular dynamics studies

of the tripeptide in solution claimed that the turn structure is one of the most stable

conformations of melanostatin.[16] In contrast, a later study suggested that the -bend

conformation, and thus the hydrogen bond between the N-H of the amide group and the

C=O of proline, is highly unlikely in solution.[14] Making the choice all the more interesting

is the observation that melanostatin is the tripeptide tail of the neurohormone oxytocin, a

12


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compound recently discovered to have some anticancer properties of its own.[17][18]

Figure 1.3: A ribbon model representing the secondary structure of a -bended molecule.[13]

2 Materials and Methods

2.1 Materials

Reagent grade L-4-hydroxyproline, L-leucine (pH = 5.19), glycine, and melanostatin (pH

= 8.97) were obtained from the Sigma-Aldrich Chemical Company and were used without

further purification. Two 10mM solutions of the amino acids were prepared, one in 1:9 and

one in 9:1, H2O:D2O solvent volume ratios. Similarly, 3mM solutions of melanostatin were

prepared in both 1:9 and 9:1 H2O:D2O solvent volume ratios. Because both L-leucine andmelanostatin have low solubilities, they were gently heated above room temperature using a

hot plate to bring them into solution. Samples were then placed in NMR tubes, evacuated

using a water aspirator, pressurized with ultra-pure argon gas, and stored at 4 C. We chose

4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) as an internal standard because of its low

chemical shift, sharp signal and low reactivity. The DSS standard was added in a 10:1 volume

ratio.

2.2 Nuclear Magnetic Resonance (NMR) Spectroscopy

NMR spectra were recorded with an JEOL Eclipse+ 400MHz spectrometer and analyzed

using both JEOL Delta (v. 4.36 or 5.01) and Mnova (v. 7.1.2) software. All spectra are

13


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reported as intensity vs. chemical shift. Chemical shifts are reported in parts per million

(ppm) relative to DSS. The ensuing discussion is intended to briefly describe the ideas behind

NMR spectroscopy. For a more detailed explanation of the theory behind NMR spectroscopy,

refer to Appendix A.

2.2.1 NMR Suite for Structural Determination of Polypeptides

A main goal of this study is to devise a method for the unambiguous assignment of all

resonances in the 1D 1H and 13C spectra of a given polypeptide. To complete this task,

a number of NMR pulse sequences (experiments) were used. An outline of the approach

follows:

Step 1: Initial 13C Peak Assignments and Carbon Counting Before looking at the

1D proton spectrum of a given amino acid or polypeptide, the 1D 13C single pulse Free

Induction Decay (FID) spectrum was analyzed. Peaks were counted to confirm that the

number of resonances corresponded to the number of carbons in the compound. Then with

the aid of amino acid data tables in reference [24], preliminary peak assignments were made

based on the relative shielding of a given carbon atom in the molecule from well-shielded

carbons with low chemical shifts to less shielded atoms with higher chemical shifts.

Step 2: Confirmation of 13C Peak Assignments Preliminary peak assignments in the

1D 13C single pulse FID spectrum were confirmed using both Attached Proton Test (APT)

and Distortionless Enhancement by Polarization Transfer (DEPT) spectra which identify

carbon resonances via their phase angle with respect to the chemical shift axis. Information

gleaned from the APT and DEPT spectra allow us to more confidently assign resonances to

each of the carbons. Specifically, in an APT experiment, C and CH2 groups are displayed

+90 with respect to the chemical shift axis whereas CH3 and CH groups are displayed 90

with respect to the chemical shift axis. In a DEPT experiment, CH and CH3 groups are

displayed +90 with respect to the chemical shift axis whereas CH2 groups are displayed

14


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90 with respect to the chemical shift axis. In DEPT carbon spectra, carbons with no

hydrogen coordination are not present.

Step 3: Initial 1H Peak Assignments After assigning carbon resonance peaks, the 1D

1H Single Pulse FID spectrum of the amino acid or polypeptide is analyzed. Preliminary

resonance assignments are made based on chemical shifts, multiplet structure, relative peak

intensities, and when appropriate spectral linewidths.

Step 4: Confirmation of1H Resonances To confirm the initial peak assignments made

on the 1D proton spectrum, a combination of 2D experiments were carried out, specifically,

COSY (Correlated Spectroscopy), NOESY (Nuclear Overhauser Spectroscopy), and TOCSY

(Total Correlated Spectroscopy). Specifically, proton-proton COSY spectra were used to

identify sets of protons coupled via two (geminal coupling) or three (vicinal coupling) covalent

bonds. TOCSY is similar to COSY but identifies not only those sets of protons coupled

through two or three covalent bonds but those connected by coupling relays. Thus, TOCSY

allows one to map out the entire spin tree of a given amino acid residue.

Step 5: HETCOR (Heteronuclear Spin Coupling) HETCOR establishes links be-

tween specific carbon resonances in the 13C spectrum and proton multiplets in the 1D 1H

spectrum.

Step 6: 1D Proton Spectrum Simulation and Peak Deconvolution HyperChem

v. 8.1 has two very useful modules for interpreting proton NMR spectra: a proton spectral

simulation program called NMR and a deconvolution program for resolving overlapping

peaks. The programs can be used to clear up any remaining uncertainties in a spectral

assignment. An example of each is shown in Figures 4.2a and 4.2b respectively.

15


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2.2.2 Solvent Solutions

As mentioned in Section 2.1, two different solvent solutions (90% H2O/10% D2O and 90%D2O/10%

H2O) were used in the NMR experiments. These solvents allow one to readily identify labile

protons, i.e., those that are exchanged with the solvent. In 90:10 H2O/D2O such resonances

are usually present, but disappear in 10:90 H2O/D2O.

2.3 Molecular Dynamics Calculations

All simulations were carried out using HyperChem (v. 8.1.). Custom Excel macros were

written to run repeated simulations and record data.

2.3.1 Background

Molecular Dynamics (MD) simulations attempt to mimic the behavior of a real system by

manipulating observables and making calculations based on a simplified model of the system

(see Figure 2.1).[27] All MD simulations involve making approximations of physical theories

with the goal of producing the most accurate results within a reasonable computational time

frame. There are many different forms of molecular dynamics, and choosing the form thatis best suited for a given study depends on a number of factors including: type of system,

degrees of freedom, number of particles, how they interact, phenomena being observed, and

computing power available. Basically, the available methods fall into two broad categories:

quantum mechanical (QM) simulations and classical simulations.

Manipulate & control Measure or compute

certain observables SYSTEM other observables(inputs) (outputs)

Figure 2.1: Experimental process for a given system or model.[27]

Two of the most common QM methods are ab-initio and hybrid Classical/QM (semi-

classical) simulations. Both are very useful and the decision to use one method or the

16


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other often comes down to the complexity of the system being studied and the available

computing power. Given the complexity of melanostatin and the limitations of computers

readily available, it is not practical to use QM-based calculations.[28]

The simulations used in the present study fall into the fully classical mechanical domain.

Classical simulations are well suited for modeling the behavior of large biomolecules as they

can efficiently handle a large number of particles because their computations scale as O(N2)

compared to the scaling ofO(N7) for QM methods. The behavior of melanostatin, consisting

of 43 atoms, can easily be simulated using an empirical force field. The semi-empirical

molecular simulations used involve a two-part model: one for intramolecular interactions

and one for system-environment interactions.[27] In these simulations the molecule is treated

classically and is coupled to an external bath to maintain a constant temperature. This

treatment causes the system to reside in the Canonical regime with each atom obeying

Newtons laws. As is customary in such treatments, the system is assumed to obey the

ergodic hypothesis which states that the time averages for quantities of interest are, to a good

approximation, equal to their associated ensemble averages. Although the present treatment

of the tripeptide does not exactly meet the qualifications of the Canonical ensemble, one may

treat it as such because no analytical partition function is calculated.

To describe the interactions between various atoms in melanostatin an empirical force

field consisting of a sum of various potential energies is used. A standard (and complete)

potential function may be written as follows:

V(r) = Vstretch + Vbend + Voop + Vtors + Vcross + VvdW + Ve (2.1)

with Vstretch describing individual bond lengths, Vbend describing angles between two consec-

utive bonds, Voop describing the out of plane bending of rings, Vtors describing the various

torsional angles within the molecule, Vcross describing the cross terms that come from the

potentials previously mentioned, VvdW describing the long-range Van der Waals interactions

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of non-bonded atoms, and Ve which describes the long-range electrostatic interactions of

non-bonded atoms.[25]

The Assisted Model Building and Energy Refinement (AMBER) force field package in

the latest version of HyperChem (v8.1) was chosen for this study because it was designed

specifically for use with biomolecules. The AMBER potential function is similar to the one

described by Equation 2.1 except it neglects the Voop and Vcross potential function components

as previous studies suggest that these interactions are unimportant for most amino acid

residues.[29]

2.3.2 Understanding the AMBER Force Field

The AMBER potential consists of five terms, each describing a different interaction within

the system. For a more detailed discussion on the evaluation schemes of these potentials,

see references [30] and[31]. Following is an abridged version of Equation 2.1 containing only

those potential components used in the AMBER force field.

V(r) =

(i,j)M1fr +

(i,j)M2f +

(i,j)M3f +

(i,j)M4fHB +

(i,j)M4fvdW +

(i,j)M4fe (2.2)

It is important to note that in Equation 2.2, M1, M2, M3, and M4 in the indices of the

summations above refer to atoms separated by one, two, three, or more than three cova-

lent bonds respectively and the fs denote different components of the complete potential

function.

Bond Length Potential Atoms separated by one bond can be visualized as connected

by a spring with an associated spring constant Krij specific to the bond type (Figure 2.2

below). The bond length potential can then be written as a linear approximation in the

form of Hookes law:

fr =

(i,j)M1

Krij(rij r0ij)

2, (2.3)

18


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where rij = |rj ri| is the bond length of interest, r0ij is the preferred bond length as if the

diatomic system were alone in vacuo, and Kij is the associated spring constant associated

with each bond. The effective spring constants for each bond type have been predetermined

by the creators of AMBER and are based on a large body of experimental data.[30] Though

not exact, a linear approximation is sufficient for present purposes since one is predicting

near-equilibrium states of the tripeptide.

Figure 2.2: Mass-spring model of two atoms (i and j) separated by a single (M1) bond.

Bond Angle Potential The bond angle portion of the potential function is the angular

analog of the bond length potential as shown below:

f =

(i,k)M2

Kik(ik 0ik)

2 (2.4)

Note the definition of the indices here: ik refers to two atoms (i and k) bound to a third

atom (j) as indicated in Figure 2.3. In this potential, 0ik is the equilibrium bond angle and

Kik is the angular spring constant associated with the bonded pairs.

Figure 2.3: The angle between two atoms (i and j) separated by two (M2) bonds.

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Torsion Angle Potential The torsion potential (sometimes referred to as the dihedral

angle potential) as depicted in Figure 2.4 is modeled by the periodic potential function:

f =

(i,l)M3

1

2Vn[1 + cos(nilil il)] (2.5)

where Vn is the equilibrium potential of the three-bond component, il is the phase angle

between bonds ij and kl (see Figure 2.4), nil is the number of bonds separating atoms i

and l, and il is the dihedral angle optimized on the simplest possible molecule.[30][31] This

potential is usually applicable only for molecules with C symmetry; however, with a simple

phase shift resulting in an alteration to Vn, the potential can be transformed to work in

asymmetric molecules such as the tripeptide being examined as displayed in Equation 2.4

above.

Figure 2.4: Torsion angle of the bond (j,k) between two bonded pairs (M3) of atoms (i,j

and k,l).

Lennard-Jones (LJ) Potential Atoms more than three bonds away are considered to

interact through long-range interactions, and can be described by the Lennard-Jones (LJ)

potential shown in Figure 2.5:

fLJ =

(i,j)M4

AijR12ij

Bij

R6ij

(2.6)

where Rij is the distance between atoms i and j, and Aij and Bij are interaction terms for

these two atoms derived from fitting to experimental data.[30] The LJ potential accurately

models legitimate physical interactions and provides for ease of calculation. In particular,

20


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23/124

Figure 2.6: Plot of the Hydrogen Bond Potential between two atoms (i and j) separated bythree or more bonds (M4) in a fictitious molecule.

Electrostatic Potential The electrostatic portion the potential function, shown in Figure

2.7, describes the long-range charge-charge interactions of non-bonded atoms and is modeled

by

fe =

(i,j)M4

qiqjrij

, (2.8)

which is simply Coulombs Law for point charges. Here, is the dielectric constant of the

baths medium defined by the user.

Figure 2.7: Plot of the electrostatic interaction potential as a function of atomic separationfor two atoms (i and j) separated by three or more bonds (M4) in a fictitious molecule.

22


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25/124

of the tripeptide during the simulation period.[35] Values for various energies and torsion

angles were collected after each simulation.

Figure 2.8: The free energy surface of Beta3s (a mini-protein) and assigned conformationalstates.[34]

3 Results

This section contains 1D and 2D proton and carbon NMR spectra with key information sum-

marized in tables. There is also a subsection devoted to predictions made by MD calculations

and another joining the two.

3.1 1H and 13C NMR Spectra

3.1.1 1D Carbon-13 NMR

The 13C-NMR spectra yielded no surprises with the number and type of carbons in each

amino acid and the tripeptide easily identifiable. Specifically, glycine depicted in Figure 3.1a

showed two sharp, well-separated peaks at 172.58 ppm and 41.74 ppm with the lower one

representing the methylene alpha carbon and the upper one the carbon of an ionized carboxyl

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group. For this simple amino acid containing only one type of protonated carbon, APT

and DEPT experiments, shown in Figure 3.1b and 3.1c, respectively, provide no additional

information (both spectra are consistent with this result).

L-4-hydroxyproline yields to a similar analysis with the 13C spectrum, Figure 3.2a, show-

ing five sharp peaks easily identified on the basis of their relative chemical shift. Both APT

in Figure 3.2b and the DEPT spectra in Figure 3.2c confirm these assignments, with the

metheylene carbons having positive phase and the methines having opposite phase. As usual

the carboxyl carbon has the same phase as the methylene carbons and appears far upfield.

These results are summarized in Tables 3.43.6.

The leucine spectrum in Figure 3.3a, though more complicated, still yields to this type

of analysis, with six sharp peaks each corresponding to a specific carbon environment. Here

APT and DEPT spectra in Figures 3.3b and 3.3c respectively substantiate the tentative

assignments based on relative chemical shift. In particular the APT spectrum has four

sharp peaks below the chemical shift axis and one sharp peak above, with the CH2 carbon

having a positive phase and the methyl and methine carbons having a negative phase. The

DEPT spectrum in Figure 3.18c confirms these assignments. These results are summarized

in Tables 3.73.9.

Even the crowded 13C spectrum for melanostatin in Figure 3.4a allows tentative assign-

ments of all resonances aided by the fact that it is a virtual superposition of peaks found in

the spectra of its three constituent amino acids. Any ambiguity about resonance assignments

is clarified by both the APT spectrum in Figure 3.4b and the DEPT spectrum 3.4c. Again,

these results are summarized in Tables 3.103.12.

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-20

-10

0

1

0

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

210

220

ChemicalShift(ppm)

-200000

-100000

0100000

200000

300000

400000

500000

600000

700000

800000

900000

Intensity

41.74[4]

172.58[1]

4

1

C1O2

N

H23

CH2

4

OH

5

Glycine100mM

90%D

2O/10%H

2O13CSinglePulseExperimentwithBroadbandDecoupling

(a)SinglePulseSpectrum.

-20

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

210

220

ChemicalShift(ppm)

-150000

-100000

-50000

050000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

Intensity

41.754

C1O2

N

H23

CH2

4

OH

5

Glycine100mM

90%D

2O/10%H

2O13CAttachedProtonTest(APT)

(b)AttachedProtonTest(APT)Spectrum.

-20

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

210

220

ChemicalShift(ppm)

-150000

-100000

-50000

050000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

Intensity

41.754

C1O2

N

H23

CH2

4

OH

5

Glycine100mM90%D

2O/10%H

2O13C

AttachedProtonTest(APT)

(c)

Distortionless

Enhancement

by

Polarization

Transfer

(DEPT)Spectrum

.

26


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Carbon Group Number center

C COOH 1 172.52C CH2 4 41.73

Table 3.1: NMR peak data for Glycine 90% D2O/10% H2O13

C Single Pulse Experimentwith Broadband Decoupling


C CH2 4 41.75

Table 3.2: NMR peak data for Glycine 90% D2O/10% H2O13C Attached Proton Test (APT)

Experiment with Broadband Decoupling


C CH2 4 41.75

Table 3.3: NMR peak data for Glycine 90% D2O/10% H2O13C Distortionless Enhancement

by Polarization Transfer (DEPT) Experiment with Broadband Decoupling

27


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-20

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

210

220

ChemicalShift(ppm)

-200000

-100000

0100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

1700000

1800000

1900000

Intensity

37.53

53.05

59.93

70.17

174.38

7

6

3

8

9

O1

OH

2

CH2

3

NH

4

OH

5

CH

6

CH2

7

CH

8

C9

4-OH-Proline100mM

90%D

2O/10%H


(a)90%

D2

O/10%

H2

O

SinglePulseSpectrum.

-20

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

130

14

0

150

160

170

180

190

200

210

220

ChemicalShift(ppm)

-700000

-600000

-500000

-400000

-300000

-200000

-100000

0100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

Intensity

37.60

53.24

60.06

70.29

174.44

3

7

9

6

8

O1

OH

2

CH2

3

NH

4

OH

5

CH

6

CH2

7

CH

8

C9

4-OH-Proline100mM

90%H

2O/10%D

2O

(b)90%

H2

O/10%D

2

O

AttachedProtonTest(APT)Sp

ectrum.

-20

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

1

50

160

170

180

190

200

210

220

ChemicalShift(ppm)

-4500000

-4000000

-3500000

-3000000

-2500000

-2000000

-1500000

-1000000

-500000

0500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

Intensity

37.59[7]

53.23[3]

60.04[8]

70.28[6]6

8

3

7

L-4-Hydroxyproline90%H2O/10%D2O

13CDistortionlessEnhancementbyPolarizationTransfer(DEPT)

O1

OH2

CH2

3

NH4

OH5

CH6

CH2

7

CH8

C9

(c)90%

H2

O/10%

D2

O

DistortionlessEnhancement

byPolar-

izationTransfer(D

EPT)Spectrum.

28


30/124


31/124

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

ChemicalShift(ppm)

-100000

0100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

Intensity

21.0321.9624.02

40.08

51.99[2]

173.25

86

7

4

2

1

C1

CH

2

O3

CH2

4

NH2

5

CH

6

CH37

CH3

8

OH

9

Noise

Leucine100mM

90%H

2O/10%D2O13CSinglePulsewithBroadbandDecoupling

(a)SinglePulseSpectrum.

-20

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

210

220

ChemicalShift(ppm)

-300000

-250000

-200000

-150000

-100000

-50000

050000

100000

150000

200000

250000

300000

350000

400000

450000

500000

Intensity

21.04[7]21.97[8]24.03[6]

40.09[4]

52.00[2]

173.25[1]

4

1

2

786

C1

CH

2

O3

CH2

4

NH2

5

CH

6

C

H37

CH3

8

OH

9

Leucine100mM

90%H

2O/10%D

2O13CAttac

hedProtonTest(APT)

(b)AttachedProtonTest(APT)Spectrum.

-20

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

210

220

ChemicalShift(ppm)

-800000

-700000

-600000

-500000

-400000

-300000

-200000

-100000

0100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

Intensity

21.01[7,8]24.02[6]

40.09[4]

51.97[2]

6

2

7,8

4

C1

CH

2

O3

CH

2

4

NH2

5

CH

6

C

H37

CH3

8

OH

9

Leucine100mM

90%H

2O/10%D

2O13

CDistortionlessEnhancementbyPolarizationTransfer(DEPT)

(c)

Distortionless

Enhancement

by

Polarization

Transfer

(DEPT)Spectrum

30


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C COOH 1 173.25C CH 2 51.99C CH2 4 40.08

C CH 6 24.02C1 CH3 7 21.03C2 CH3 8 21.96

Table 3.7: NMR peak data for Leucine 90% H2O/10% D2O13C Single Pulse Experiment

with Broadband Decoupling.


C CH 2 52.00C CH2 4 40.09C CH 6 24.03C1 CH3 7 21.04C2 CH3 8 21.97

Table 3.8: NMR peak data for Leucine 90% D2O/10% H2O13C Attached Proton Test (APT)

Experiment with Broadband Decoupling.


C CH 2 51.97C CH2 4 40.09C CH 6 24.02C1 CH3 7 21.01C2 CH3 8 21.01

Table 3.9: NMR peak data for Leucine 90% H2O/10% D2O13C Distortionless Enhancement

by Polarization Transfer (DEPT) Experiment with Broadband Decoupling.

31


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-20

-10

0

1

0

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

210

220

ChemicalShift(ppm)

-150000

-100000

-50000

050000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

700000

750000

Intensity

20.79[18]22.21[20]24.46[19]25.30[4]30.54[17]39.70[5]42.21[13]46.59[2]

52.70[9]59.95[3]

174.22[10]175.59[6]

177.30[14]

18

19

3

20

4

17

6

9

2135

10

14

NH

1

C

H22

CH

3

C

H24

CH2

5

C6O7N

H8

CH

9

C10

O11

NH

12

CH2

13

C14O15N

H2

16

CH2

17

CH3

18

CH

19

CH3

20

Melanostatin3mM90%H

2O/10%D


(a)SinglePulseSpectrum

-20

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

210

220

ChemicalShift(ppm)

-500000

-450000

-400000

-350000

-300000

-250000

-200000

-150000

-100000

-50000

050000

100000

150000

200000

250000

300000

350000

400000

450000

500000

Intensity

20.79[18,20]24.47[19]25.30[4]30.55[17]39.71[5]42.21[13]46.59[2]52.70[9]

59.95[3]

17

213 5

4

3

9

191

8,2

0

NH

1

CH22

CH

3

CH24

CH2

5

C6O7N

H8

CH

9

C10

O11

NH

12

CH2

13

C14O15N

H2

16

CH2

17

CH3

18

CH

19

CH3

20

Melanostatin3mM

90%H

2O/10%D

2O13CAttachedProtonTest(APT)

(b)Attach

edProtonTest(APT)Spectrum

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

ChemicalShift(ppm)

-1000000

-900000

-800000

-700000

-600000

-500000

-400000

-300000

-200000

-100000

0100000

200000

300000

400000

500000

600000

700000

800000

900000

Intensity

22.17[18,20]24.45[19]24.93[4]

30.39[5]

39.67[17]

42.19[13]

46.59[2]

52.81[9]

59.86[3]3

9

419

18,2

0

13

2

17

5

Me

lanos

tatin

3m

M

90%

H2

O/10%

D2O

13CDistort

ion

less

Enc

hancemen

tby

Po

lariza

tion

Trans

fer

NH

1

CH2

2

CH

3

CH2

4

CH2

5

C6O7N

H8

CH

9

C10

O11

NH

12

CH2

13

C14O15N

H2

16

CH2

17

CH3

18

CH

19

CH3

20

(c)

Distortionless

Enhancement

by

Polarization

Transfer

(DEPT)Spectrum

32


34/124


C (G) COOH 14 177.30C (P) COOH 6 175.59C (L) COOH 10 174.22

C (P) CH 3 59.95C (L) CH 9 52.70C (G) CH2 13 42.21C (P) CH2 5 39.70C (L) CH2 17 30.54C (L) CH 19 24.46C (P) CH2 4 25.30C (P) CH2 2 46.59C1 (L) CH3 20 22.21C2 (L) CH3 18 20.79

Table 3.10: NMR peak data for Melanostatin 90% H2O/10% D2O 13C Single Pulse Experi-ment with Broadband Decoupling.


C (P) CH 3 59.95C (L) CH 9 52.70C (G) CH2 13 42.21C (P) CH2 5 39.71C (L) CH2 17 30.55C (L) CH 19 24.47C (P) CH2 4 25.30C (P) CH2 2 46.59C1 (L) CH3 20 22.79C2 (L) CH3 18 20.79

Table 3.11: NMR peak data for Melanostatin 90% D2O/10% H2O13C Attached Proton Test

(APT) Experiment with Broadband Decoupling.

33


35/124


C (P) CH 3 59.95C (L) CH 9 52.81C (G) CH2 13 42.19C (P) CH2 5 39.39C (L) CH2 17 36.67C (L) CH 19 24.45C (P) CH2 2 46.59C1 (L) CH3 20 22.17C2 (L) CH3 18 22.17

Table 3.12: NMR peak data for Melanostain 90% H2O/10% D2O13C Distortionless En-

hancement by Polarization Transfer (DEPT) Experiment with Broadband Decoupling.

34


36/124


37/124

upfield attributable to rapid exchange with the aqueous solvent. The identification of these

peaks as labile NH peaks is confirmed by the fact that they do not appear in the 90% D2O

spectra. These spectral features will be further investigated in the future by pH dependent

studies. Furthermore, the interesting stereochemistry of leucine suggests that NOESY may

be be used to investigate interactions between methyl protons and NH2 protons at a later

date.

Figure 3.5: Hypothetical 1D proton spectrum of melanostatin resulting from superpositionof L-4-hydroxyproline, leucine, and glycine assuming no inter-residue interactions.

Even a cursory glance at the 1D proton spectrum of melanostatin reveals its complexity,

but as was noted in the case of its complicated 13C spectrum, its proton spectrum is, to a first

approximation, a superposition of the spectra of its three constituent amino acids as shown

in Figure 3.15. Of course a detailed comparison of the hypothetical superposition spectrum

with the actual spectrum of melanostatin reveals a number of differences highlighted by fea-

36


38/124


39/124

-2-10123456789101112Chemical Shift (ppm)

-500000

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

Intensity

3.

55

4

DSS

C1

O2

NH23 CH2

4OH5

Glycine 100mM 90% H2O/10% D2O1H Single Pulse with Water Suppression

(a) Glycine 100mM 90% H2O/10% D2O1H Single Pulse with Water Suppression

Spectrum.

-2-10123456789101112Chemical Shift (ppm)

-200000

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

3000000

3200000

3400000

3600000

3800000

4000000

4200000

4400000

Intensity

3.

55

4

DSS

C1

O2

NH23

CH24

OH5

(b) Glycine 100mM 90% D2O/10% H2O1H Single Pulse with Water Spectrum.

Figure 3.6: 1H Single Pulse with Water Suppression Spectra of Glycine 100mM in bothsolvent mixtures.

38


40/124


41/124

1.96

1.98

2.00

2.02

2.04

2.06

2.08

2.10

2.12

2.14

2.16

2.18

2.20

2.22

2.24

2.26

2.28

2.30

2.32

2.34

2.36

.38

ChemicalShift(ppm)

-100000

0100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

1700000

1800000

1900000

2000000

2100000

Intensity

8(t)

2.14

J(4.09)

7(dd)

2.14

J(4.16,25.13)

4.094.09

25.13

4.16

87

O1

OH2

CH23

NH4

OH5

CH6

CH2

7

CH8

C9

4-L-Hydroxyproline100mM90%

H2O/10%D2O1HSinglePulsewithWaterSuppression

(a)

2.24

2.26

2.28

2.30

2.32

2.34

2.36

2.38

2.40

2.42

2.44

2.46

2.48

2.50

2.52

2.54

2.56

2.58

2.60

2.62

2.64

2.66

ChemicalShift(ppm)

-100000

0100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

Intensity

7(dd)

2.42

J(8.13,16.17)

16.17 8

.13

7

O1

OH2

CH2

3

NH4

OH5

CH6

CH2

7

CH8

C9

4-L-Hydroxyproline100mM90%H2O/10%D2O

1HSinglePulsewithWaterSuppression

(b)

3.26

3.28

3.30

3.

32

3.34

3.36

3.38

3.40

3.42

3.44

3.46

3.48

3.50

3.52

3.54

3.56

3.58

3.60

3.62

3.64

3.66

3.68

ChemicalShift(ppm)

-100000

0100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

1700000

Intensity

3(dd)

3.35

J(3.61,12.65)

3(dd)

3.47

J(3.67,12.53)

12.65

3.

61

12.53

3.67

3

3

O1

OH2

CH23

NH4

OH5

CH6

CH2

7

CH8

C9

4-L-Hydroxyproline100mM90%

H2O/10%D2O1HSinglePulsewithWaterSuppression

(c)

4.1

8

4.2

0

4.2

2

4.2

4

4.2

6

4.2

8

4.3

0

4.3

2

4.3

4

4.3

6

4.3

8

4.4

0

4.4

2

4.4

4

4.4

6

4.4

8

4.5

0

4.5

2

4.5

4

4.5

6

4.5

8

4.6

0

ChemicalShift(ppm)

010000

20000

30000

40000

50000

60000

70000

80000

90000

100000

110000

120000

130000

140000

Intensity

6(d)

4.3

2

J(10.2

2)

6(d)

4.3

5

J(7.0

8)

10.2

2

7.0

8 6

6

O1

OH2

CH2

3

NH4

OH5

CH6

CH2

7

CH8

C9

4-L-Hydroxyproline100mM90%H2O/10%D2O


(d)

40


42/124

-2

-1

0

1

2

3

4

5

6

7

8

9

10

11

12

ChemicalShift(ppm)

-200

000

0200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

Intensity

6,8(d)

4.35

J(6.94)

6(d)

4.32

J(9.98)

7(dd)

2.14

J(4.13,25.10)

7(dd)

2.42

J(8.35,14.53)

8(t)

2.14

J(4.17)

3(dd)

3.35

J(2.02,12.65)

3(dd)

3.47

J(3.77,12.56)

33

87

7

6

O1

OH2

CH2

3

NH

4

OH5

CH

6

CH2

7

CH

8

C9

L-4-Hydroxyproline100mM90%D2O/10%H2O

1HSinglePulseExperimentwithWaterSuppressio

n

DSS

Noise

Figure3.9:L-4

-Hydroxyproline100mM

90%

D2O/10%H2O1HSinglePulsewithWaterSuppressionSp

ectrum.

41


43/124

2.1

0

2.1

5

2.2

0

2.2

5

2.3

0

2.3

5

2.40

2.4

5

.50

ChemicalShift(ppm)

-200000

0200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

Intensity

7(dd)

2.1

4

J(4.1

3,

25.1

0)

7(dd)

2.4

2

J(8.3

5,

14.5

3)

8(t)

2.1

4

J(4.1

7)

25.1

0

4.1

3

14.5

3 8.3

5

4.1

74.1

7

87

7

O1

OH

2

CH

2

3

NH

4

OH

5

CH

6

CH

2

7

CH

8

C9

L-4-Hydroxypro

line

100m

M90%

D2

O/10%H2

O1HSing

lePu

lse

Experimen

tw

ithWa

ter

Suppress

ion

(a)

3.30

3.3

5

3.4

0

3.4

5

3.5

0

3.5

5

3.6

0

3.6

5

3.7

0

ChemicalShift(ppm)

-200000

0200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

Intensity

3(dd)

3.3

5

J(2.0

2,

12.6

5)

3(dd)

3.4

7

J(3.7

7,

12.5

6)

12.6

5

2.0

2

12.5

6

3.7

7

3

3

O1

OH

2

CH

2

3

NH

4

OH

5

CH

6

CH

2

7

CH

8

C9

L-4-Hydroxypro

line

100m

M90%D2

O/10%H2O

1HSing

lePu

lse

Experimen

tw

ithWa

ter

Suppress

ion

(b)

4.20

4.25

4.30

4.35

4.40

4.45

4

.50

4.55

4.60

ChemicalShift(ppm)

-10000

010000

20000

30000

40000

50000

60000

70000

80000

90000

100000

110000

120000

130000

140000

150000

Intensity

6,8(d)

4.35

J(6.94)

6(d)

4.32

J(9.98)

6.94

9.98

6

O1

OH

2

CH

2

3

NH4

OH

5

CH

6

CH

2

7

CH8

C9

L-4-Hydroxypro

line

100m

M90%D2

O/1

0%H2

O1HSing

lePu

lse

Experimen

tw

ithWa

ter

Suppress

ion

(c)

42


44/124

Proton Group Number center J Intensity

H CH 8 4.32, 4.354 7.08, 10.22 -H CH2 7 2.14 4.16, 25.13 -H CH 6 2.14 4.09t -

H CH2 3 3.47, 3.35 12.53, 12.65 -

Table 3.15: NMR peak data for 4-L-OH-Proline 90% H2O/10% D2O1H Single Pulse Exper-

iment with Water Suppression

Proton Group Number center J Intensity

H CH 8 2.14 4.17 -H CH2 7 2.42, 2.14 14.53, 25.10 -

H CH 6 4.33 - -H CH2 3 4.34 6.94, 9.98 -

Table 3.16: NMR peak data for 4-L-OH-Proline 90% D2O/10% H2O1H Single Pulse Exper-

iment with Water Suppression

43


45/124


46/124

0.8

0

0.8

2

0.8

4

0

.86

0.8

8

0.9

0

0.9

2

0.9

4

0.9

6

0.9

8

1.0

0

1.0

2

1.0

4

1.0

6

1.0

8

1.1

0

1.1

2

1.1

4

1.1

6

1.1

8

1.2

0

1.2

2

1.2

4

1.2

6

ChemicalShift(ppm)

-500000

0500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

5500000

6000000

6500000

Intensity

8(d)

0.9

7

J(4.3

4)7

(d)

0.9

6

J(5.3

6)

4.3

4

5.3

60.95

0.97

0.97

0.97

0.98

7

8

C1

CH

2

O3

C

H2

4

NH2

5

CH

6

CH37

CH3

8

OH

9

Leucine100mM90%H2O/10%

D2O1HSinglePulseExperimentwithWaterSuppression

(a)

1.62

1.64

1.66

1.68

1.70

1.72

1.74

1.76

1.78

1.80

1.82

1.84

1.86

1.88

1.90

1.92

1.94

1.96

ChemicalShift(ppm)

0100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

1700000

1800000

1900000

Intensity

4(q)

1.74

J(7.79,9.57)

6(d)

1.68

J(7.83)

9.57

7.797.79

7.83

4

6

C1

CH

2

O3

CH2

4

NH2

5

CH

6

CH37

CH3

8

OH

9

Leucine100mM90%H2O/10%D2O1HSinglePulseExperimentwithWaterSuppression

(b)

3.91

3.93

3.95

3.97

3.99

4.01

4.03

4.05

4.07

4.09

4.11

4.13

4.15

4.17

4.19

4.21

4.23

4.25

ChemicalShift(ppm)

-20000

-10000

010000

20000

30000

40000

50000

60000

70000

80000

90000

100000

110000

120000

130000

140000

150000

160000

170000

180000

190000

200000

210000

220000

230000

240000

250000

Intensity

0.20

0.24

2(td)

4.03

J(4.15,7.73) 7

.74

7.73 4

.15

2

C1

CH

2

O3

C

H2

4

NH2

5

CH

6

CH37

CH3

8

OH

9

Leucine100mM90%H2O/10%

D2O1HSinglePulseExperimentwithWaterSuppression

(c)

7.2

7.3

7.4

7.5

7.6

7.7

7.8

7.9

8.0

8

.1

8.2

8.3

8.4

8.5

ChemicalShift(ppm)

-50000

050000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

700000

750000

Intensity

1.00

0.76

7.33

7.92

5

C1

CH

2

O3

CH2

4

NH2

5

CH

6

CH37

CH3

8

OH

9

NH3

Leucine100mM90%H2O/10%D2O1HSinglePulseExperimentwithWaterSuppression

(d)

45


47/124

-2

-1

0

1

2

3

4

5

6

7

8

9

10

11

12

ChemicalShift(ppm)

-200

000

0200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

3000000

3200000

3400000

Intensity

8(d)

0.98

J(4.68)

4,6(dt)

1.75

J(4.51,7.19)

2(d)

4.03

J(4.05)

7(d)

0.96

J(4.23)

78

4,6

2

C1

CH

2

O3

CH

2

4

NH

2

5

CH

6

CH

37

CH

3

8

OH

9

NH3andNH2

disappear

DSS

Leucine100m

M90%D2O/10%H2O


Figure3.13:Leucine100mM

90%D2O/10%H2

O1HSinglePulsewithWaterSuppressionSpectru

m.

46


48/124

0.8

8

0.9

0

0.9

2

0.9

4

0.9

6

0.9

8

1.0

0

1.0

2

1.0

4

1.0

6

1.0

8

1.1

0

1.1

2

1.1

4

1.16

1.1

8

1.2

0

1.2

2

1.2

4

ChemicalShift(ppm)

-200000

0200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

3000000

3200000

3400000

Intensity

8(d)

0.9

8

J(4.6

8)

7(d)

0.9

6

J(4.2

3)

4.6

8

4.2

3

7

8

C1

CH2

O3

CH2

4

NH2

5

C

H6

CH37

C

H3

8

OH9

Leucine100mM90%D2O/10%

H2O1HSinglePulsewithWaterSuppression

(a)

1

.64

1.6

6

1.6

8

1.7

0

1.7

2

1.7

4

1.7

6

1.7

8

1.8

0

1.8

2

1.8

4

1.8

6

1.8

8

1.9

0

1.9

2

1.9

4

1.9

6

1.9

8

2.0

0

ChemicalShift(ppm)

0100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

Intensity

4,6

(dt)

1.7

5

J(4.5

1,

7.1

9)

7.1

9 4.5

1 4.5

1

4,6

C1

CH2

O3

CH2

4

NH2

5

CH6

CH37

CH3

8

OH9

Leucine100mM90%D2O/10%H2O1HSinglePulsewithWaterSuppression

(b)

3.9

4

3.9

6

3.9

8

4.0

0

4.0

2

4.0

4

4.0

6

4.0

8

4.1

0

4.1

2

4.1

4

4.1

6

4.1

8

4

.20

4.2

2

4.2

4

4.2

6

4.2

8

4.3

0

Chem

ica

lShift(ppm

)

-10000

010000

20000

30000

40000

50000

60000

70000

80000

90000

100000

110000

120000

130000

140000

150000

160000

170000

180000

190000

200000

210000

220000

230000

Intensity

2(d)

4.0

3

J(4

.05) 4

.05

2

C1

CH2

O3

C

H2

4

NH2

5

CH6

CH37

CH3

8

OH9

Leucine100mM90%D2O/10%H2O1H

SinglePulsewithWaterSuppression

(c)

47


49/124


50/124


51/124


52/124

3.73

3.74

3.75

3.76

3.77

3.78

3.79

3.80

3.81

3.82

3.83

3.84

3.85

3.86

3.87

3.88

3.89

3.90

3.91

3.92

3.93

3.94

3.95

3.96

3.97

3.98

3.99

4.00

4.01

4.02

03

ChemicalShift(ppm)

-100000

0100000

200000

300000

400000

500000

600000

700000

800000

900000

Intensity

2(d)

3.76

J

(5.98)

2(d)

3.80

J(2.02)

13(dd)

3.90

J(3.24,10.25)

5.98

2.02

10.253.24

13

2

NH

1CH

2

2

CH

3

CH2

4CH2

5

C6

O7

NH

8CH

9

C10

O11

NH

12

CH2

13C14

O15

NH216

CH2

17

CH318

C

H

19

CH3

20

Melanostatin3mM90%H2O/10%D2O1HSinglePulseExperimentwithWaterSuppression

(a)

4.2

2

4.2

3

4.2

4

4.2

5

4.2

6

4.2

7

Date post:	05-Apr-2018
Category:	Documents
Upload:	ryan-joseph
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Investigation of the three-dimensional solution structure of melanostatin using one- and...

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