Tyrosine

ABSTRACT Tyrosine ring dynamics of the gastrointestinal hormone motilin was studied using two independent physical methods: fluorescence polarization anisotropy decay and NMR relaxation. Motilin, a 22-residue peptide, was selectively '3C labeled in the ring E-carbons of the single tyrosine residue. To eliminate effects of differences in peptide concentration, the same motilin sample was used in both experiments. NMR relaxation rates of the tyrosine ring C^sup epsilon^-H^sup epsilon^ vectors, measured at four magnetic field strengths (9.4,11.7,14.1, and 18.8 Tesla) were used to map the spectral density function. When the data were analyzed using dynamic models with the same number of components, the dynamic parameters from NMR and fluorescence are in excellent agreement. However, the estimated rotational correlation times depend on the choice of dynamic model. The correlation times estimated from the two-component model-free approach and the three-component models were significantly different (1.7 ns and 2.2 ns, respectively). Various earlier studies of protein dynamics by NMR and fluorescence were compared. The rotational correlation times estimated by NMR for samples with high protein concentration were on average 18% longer for folded monomeric proteins than the corresponding times estimated by fluorescence polarization anisotropy decay, after correction for differences in viscosity due to temperature and D^sub 2^O/H^sub 2^O ratio.

INTRODUCTION

NMR relaxation and fluorescence polarization anisotropy decay (FAD) are two important experimental methods to study the dynamics of biomolecules. The results from the two methods on protein dynamics have been compared for a number of proteins listed in Table 1. In several cases the global rotational correlation time deviates significantly between the two methods. In most cases the correlation time observed by fluorescence is shorter than the correlation time observed by NMR. The details of this table will be discussed later. A fraction of the observed discrepancies between the NMR data and the fluorescence data can be explained by the typical difference in concentration between NMR and fluorescence studies.

Because FAD and NMR relaxation are about the only two experimental approaches to detailed studies of molecular dynamics, it is important to try to reconcile the results and find out where the results from the two methods deviate from each other and become less reliable.

Motilin is a gastrointestinal peptide hormone with 22 amino acids, among them the single Tyr^sup 7^ fluorophore. In our previous study of motilin dynamics (Allard et al., 1995; Jarvet et al., 1996) we found that the overall rotational correlation time of motilin is ~5 ns at 20 deg C and 3 ns at 35 deg C in 30% hexafluoro-2-propanol (HFP), evaluated by spectral density mapping and with Leuo a-carbon as the probe for the relaxation measurements. These results were significantly different from the value (2.2 ns at 20 deg C in 30% HFP) measured by FAD on the single Tyr residue of the peptide.

We thank Britt-Marie Olsson for peptide synthesis and Kalle Kaljuste for help with attaching the t-Boc protecting group on the labeled tyrosine. We acknowledge the Swedish NMR center for the use of their 500- and 800-MHz NMR spectrometers.

This work was supported by a grant from the Swedish Research Council.

Submitted May 19, 2002, and accepted for publication June 5, 2002.

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Peter Damberg,* Juri Jarvet,* Peter Allard,^ Ulo Mets,^^ Rudolf Rigler,^^ and Astrid Graslund*

*Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, S-106 91 Stockholm, Sweden; ^Structural Biochemistry, Department of Biotechnology, Stockholm Center for Physics, Astronomy, and Biotechnology, The Royal Institute of Technology, S-106 91 Stockholm, Sweden; and ^^Department of Medical Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden

Address reprint requests to Dr. Astrid Graslund, Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, S-106 91 Stockholm, Sweden. Tel.: 46-8-162450; Fax: 46-8-155597; E-mail: astrid@dbb.su.se.

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