We have expressed in E. coli a series of periodic polypeptides represented by sequence 1. Our objective has been an understanding of the role of chemical sequence in determining the chain folding behavior of periodic macromolecules. Molecular organization has been examined by infrared spectroscopy and ^1H and ^(13)C NMR methods and a preliminary model of the folded structure has been developed

Borbely, Janos

Deguchi, Yoshikuni

Fournier, Maurille J.

Krejchi, Mark T.

Mason, Thomas L.

Tirrell, David A.

Caltech Authors - Main

SYNTHESIS AND CHARACTERIZATION OF PERIODIC POLYPEPTIDES 
CONTAINING REPEATING -(AlaGly),GluGly- SEQUENCES 
Y OSHIKUNl DEGUCHI*, MARK T. KREJCHI*, JANOS BORBELY *, MAURILLE J. 
FOURNIER**, THOMAS L. MASON** and DAVID A. TIRRELL* 
*Department of Polymer Science and Engineering and **Department of Biochemistry and 
Molecular Biology, University of Massachusetts, Amherst, Massachusetts 0 1003 
ABSTRACT 
We have expressed in E. coli a series of periodic polypeptides represented by sequence 1. 
Our objective has been an understanding of the role of chemical sequence in determining the 
chain folding behavior of periodic macromolecules. Molecular organization has been 
examined by infrared spectroscopy and IH and l 3 ~  NMR methods and a preliminary model of 
the folded structure has been developed. 
INTRODUCTION 
We have been studying a series of periodic polypeptides represented by the general 
formula 2, wherein the repeating alanylglycine dyads are expected to bias the chain toward an 
anti-parallel P-sheet conformation (I 1,2]. Our objective is control of chain folding in the solid 
state, by incorporating periodic P-sheet breakers into the sequence. Our first attempt used a 
variant of sequence 2 in which x=3, Z=ProGlu, and n=54 [3]. X-ray diffraction analysis of 
this protein afforded no evidence of crystalline structure. In this paper we report recent 
progress in characterization of the similar family of polymers represented by sequence 1. 
EXPERIMENTAL SECTION 
Synthetic strutegy. The overall strategy for the production of ,the target material is shown 
in Figure 1. Detailed experimental conditions are described elsewhere [3,4]. 
Protein expression. Artificial genes encoding proteins containing sequences la-g were 
constructed and expressed in the pET3-b vector and E. coli strain BL21@E3)pLysS 
developed by Studier and coworkers [5 ] .  Expression was initially monitored by the 
incorporation of 3 ~ - g l ~ c i n e  into the target protein. A rich medium (YT) was used for sample 
preparation. P-Isopropylthiogalactoside (IPTG, final concentration 95 mg/L) was added to the 
culture to induce protein production when OD at 600 nm reached ca. 0.8. 
Purification. Cell extracts were prepared by freezing, thawing, sonication and spinning 
out of cell debris. The extract was adjusted sequentially to pH 5.0, 4.5 and 4.0 with glacial 
acetic acid. After each pH adjustment, precipitated material was removed by centrifugation. 
After enzymatic removal of contaminating nucleic acids, the target protein was precipitated 
by addition of ethanol to 40 v/v%. Crude product was washed with water repeatedly until the 
absorbance of the supernatant at 260 nm disappeared. 
Molecular Design +-- DNA Duplex 
I 
Codon Selection 
I 
Cloning and Multiplication 
Endonuclease Digestion 
Transformation 
Of :- coli \ &m 1 DNA Monomer I Self Ligation 
Multimer Screening \ --- 
Expression 
Figure 1. Overall strategy for biosynthesis of polymers of sequence 1. 
Cyanogen bromide cleavage. Fusion ends were chemically removed by selective 
cyanogen bromide cleavage at room temperature in formic acid. 
Infrared spectroscopy. A portion of cleaved protein was placed in an Eppendorf tube and 
90% formic acid was added thereto so that 1 pL of solution would contain 100 pg of solid. 
The solution was kept at room temperature until use. After an appropriate time period, a 
portion of the solution was spread on a NaCl plate and air dried. 
Computer graphics. Molecular modeling was performed using Biograf version 2.2 
(BioDesign, Inc.) running on a Silicon Graphics Iris work station. 
NMR spectroscopy. NMR analyses were conducted at room temperature on Varian XL- 
300, Varian 500 MHz Unity, and Bruker 200 AF, 300 MSL and 500 AMX spectrometers. 
Formic acid formyl-dl was used as solvent. 
RESULTS and DISCUSSION 
Protein expression. The expression strain, BL21@E3)pLysS, carries a chromosomal 
gene encoding bacteriophage T7 RNA polymerase under lacUVS control [5]. The pLysS 
plasmid produces a low level of T7 lysozyme, which suppresses leaky T7 RNA polymerase 
activity prior to induction with IPTG. Each of the proteins la-g was expressed in E. coli 
successfully. None of these proteins stained well with the conventional dye Coomassie Blue, 
so in vivo labelling techniques were used to monitor accumulation. 
Purification. Contaminating proteins were removed readily from the product by stepwise 
acidification of whole cell lysates, owing to the fact that the target proteins remain soluble in 
mildly acidic solutions that precipitate native E. coli proteins. Purification leads to products of 
general sequence 3, in which the repetitive oligopeptide building blocks are flanked by leader 
and trailer sequences derived from the cloning and expression vectors. Typical yields of 
purified protein were 20-70 mg/L, decreasing slightly with increased spacing between 
glutamic acid residues. The leader and trailer sequences were removed by cyanogen bromide 
digestion prior to structural analysis by infrared and NMR methods. 
Infrared spectroscopy. Figure 2 shows IR absorption spectra of films of polymer l c  cast 
from formic acid after aging of the casting solution for various times. The broad band in the 
amide I region is apparently composed of two main peaks, one centered near 1660 cm-I and 
the other around 1625 cm-l. The former band decreased in intensity as the solution aged, 
while the intensity of the latter band increased. The rate of change in the relative intensities of 
these two bands was found to depend on the sample concentration, and was reduced in more 
dilute solutions. Gelation of each solution was observed concurrently with the rise in intensity 
at 1625 cm-1- We attribute the 1625 cm-1 band to an antiparallel P-sheet structure and the 
higher frequency absorption to a disordered conformation. 
1800 1 700 1 600 1 500 1400 
Wavenumber 
Figure 2. Infrared spectra of cast films. Cleaved lc  protein was dissolved in formic acid and films were cast on 
NaCl. From the top, curves correspond to 5 min, 2 hr, 8 hr, and 26 hr of solution aging prior to film casting. 
NMR spectroscopy. The infrared resdlts discussed above, coupled with the observed 
gelation of the formic acid solutions upon aging, suggest that an ordered p-structure develops 
with time in these polymer solutions. This structure was probed more fully in preliminary 
high-field NMR experiments. Of special interest were questions of: i). whether or not the 
chain folds in regular fashion in solution, and ii). if regular folding does occur, what the local 
structure of the fold may be. 
The ' 3 ~  NMR spectrum of polymer lc  dissolved in formic acid-dl is shown in Figure 3. 
Chemical shifts relative to fonnic acid (6166.31 ppm) and peak assignments are listed in 
Table I. Assignments were made on the basis of repeating unit sequence 4, in which the 
designation of 3 distinct glycyl units and 2 distinct alanyl units is suggested by the appearance 
of multiple carbonyl resonances for each of these residues. 
172 PPM 
Figure 3. 13c NMR spectra of cleaved lc. Upper: carbonyl carbons; lower: aliphatic carbons. 
Table I. Assignment of 13C NMR Spectrum of Polymer l c  
6 in ppm Assignment 
17.03 p-CH3 of all Ala residues 
26.63 P-CH2 of Glu 
30.21 y-CH2 of Glu 
43.28 a-CH2 of Gly 
43.33 a-CH2 of Gly* or Gly** 
43.37 a-CH2 of Gly* or Gly** 
50.5 1 a-CH of Ala and Ala* 
53.76 a-CH of Glu 
171.88 Carbonyl of Gly* 
172.00 Carbonyl of Gly 
172.31 Carbonyl of Gly ** 
174.26 Carbonyl of Glu 
175.94 Carbonyl of Ala 
175.98 Carbonyl of Ala* 
177.83 Side chain carboxyl group of Glu 
The relative intensities of the carbonyl signals are consistent with this assignment, and the 
presence of sharp resonances suggests that the chain adopts a limited set of conformations in 
formic acid solution. In contrast, the l3C NMR spectrum obtained in HFIP-d2 solution shows 
very broad carbonyl peaks, suggesting that in HFIP the polypeptide chain exists as a random 
coil. 
Figure 4 shows a partial NOESY spectrum of l c  taken in formic acid solution. The 
observation of strong sequential a-proton-NH NOEs and absence of amide-amide NOEs 
suggest that pleated p-sheets are present. This is supported by the appearance of &-Ha and 
I&-Hp NOEs which are to be expected in a regular antiparallel p-pleated sheet, in which the 
interstrand Ha-Ha distance is approximately 2.4 A. Furthermore, the NH peak of Gly** 
indicates a short interresidue interaction with the a-CH and a weak interaction with the P-CH2 
of Glu. This observation is consistent with the occurrence of a p-turn. The very strong 
correlation of Glyi**NH ->Glui-laCH suggests a turn of type 11 rather than type I or 1'. 
a-protons 
Figure 4. Partial NOESY spectrum of cleaved lc. 
CONCLUSIONS 
Although this is a preliminary analysis, the 1H and 13C NMR data obtained in formic acid 
are consistent with a chain conformation characterized by a predominance of P-s trands 
interrupted by reverse turns. In contrast, the polymer in HFIP appears to be a statistical coil, 
giving rise to broad resonances in the 13C NMR spectrum. Given these differences, it is 
intriguing that polymer films cast from formic acid exhibit infrared spectra characteristic of P- 
sheet structure, while those from HFIP are disordered. We are pursuing refinement of the 
solution structure of l c  and related polymers, with the objective of understanding the 
mechanism of structure formation during solidification. 
ACKNOWLEDGMENT 
This work was supported by a grant from the Polymers and Genetics Programs of the 
National Science Foundation (DMR-8914359) and by the Materials Research Laboratory at 
the University of Massachusetts. J. BorMly thanks the Fulbright Foundation for financial 
support. Dr. BorMly is on leave from the Department of Applied Chemistry, Kossuth Lajos 
University, H-40 10 Debrecen, Hungary. Yoshikuni Deguchi was on leave from Central 
Research Laboratories, Kaneka Corporation, 1-2-80, Yoshida-cho, Hyogo-ku, Kobe 652, 
JAPAN. 
REFERENCES 
1. D.A. Tinell, M.J. Fournier and T.L. Mason, MRS Bulletin 16(7), 23 (199 1). 
2. R.D.B. Fraser, T.P. MacRae, F.H.C. Stewart and E. Suzuki, J. Mol. Biol. 11,706 (1965). 
3. K.P. McGrath, M.J. Fournier, T.L. Mason and D.A. Tirrell, J. Am. Chem. Soc. 114,727 
(1992). 
4. Y. Deguchi, M J. Fournier, T.L. Mason and D.A. Tirrell, in preparation. 
5. F.W. Studier, A.H. Rosenberg and J.J. Dunn, Methods Enzymol. 185,60 (1990). 
6. P.Y. Chou and G.D. Fasman, J. Mol. Biol. 115,135 (1977). 


English

Synthesis and Characterization of Periodic Polypeptides Containing Repeating —(AlaGly)_xGluGly— Sequences

https://authors.library.caltech.edu/54569/1/112-Deguchi.pdf

Synthesis and Characterization of Periodic Polypeptides Containing Repeating —(AlaGly)_xGluGly— Sequences

Abstract

Similar works

Full text

Available Versions

Caltech Authors - Main