A 2-DIMENSIONAL NMR-STUDY OF EXCHANGE BEHAVIOR OF AMIDE HYDROGENS IN A LYSOZYME DERIVATIVE WITH AN EXTRA CROSS-LINK BETWEEN GLU35 AND TRP108 - QUENCHING OF COOPERATIVE FLUCTUATIONS AND EFFECTS ON THE PROTEIN STABILITY

Two-dimensional nmr spectra [correlated spectroscopy (COSY), homonucle ar Hartmann-Hahn (HOHAHA), nuclear Overhauser effect spectroscopy (NOE SY)] have been observed for cross-linked lysozyme, a chemically, modif ied lysozyme derivative with an extra ester cross-link between residue s E35 and W108. Eight shifted cross-peaks were found in the fingerprin t region of COSY spectra. By searching COSY, HOHAHA and NOESY spectra, they have been assigned to A32, E35, S36, I58, A107, W108, V109, and A110. The NOE connectivities (d(NN) and d(dN)) found for the cross-lin ked lysozyme are quite similar to those for the intact lysozyme.Exchan ge behavior of amide hydrogens has been studied for both intact and cr oss-linked lysozymes by observing the fingerprint region of COSY spect ra. Hydrogen exchange reactions were carried out at pH 7.0 and at seve ral temperatures. There exist 41 amide hydrogens whose exchange reacti ons are detectable under this experimental condition. Not only exchang e rates but also their activation enthalpies were determined for indiv idual amide hydrogens. They are classified into two groups, which are called categories III and IV. Category III hydrogens are distributed i n relatively flexible peripheral parts of protein, and category IV hyd rogens are deeply buried in the core region of protein. Category III h ydrogens are exchanged through localized unfolding around their sites with a low activation enthalpy ranging from 10 to 25 kcal/ mol. The fo rmation of an extra cross-link affects neither the exchange rate nor t he activation enthalpy of category III hydrogens. However, amide hydro gens of residues 34-39 in the vicinity of the hinge are exceptions. Th ey are easily exchanged in the intact lysozyme but their exchange rate s are drastically retarded by cross-linking. In the intact lysozyme, s tructural fluctuations mediating the exchange of category IV hydrogens are highly cooperative with a large activation enthalpy. These large- scale structural fluctuations are the global unfolding of the overall structure and also concerted motions within a domain. Especially near 38 degrees C, it was found that the dominant fluctuation occurring in the alpha-domain is different from that in the beta-domain. However, t hese concerted motions are strongly quenched by the formation of the c ross-link because of the cooperativity of such a large-scale fluctuati on. The stabilization of a localized area of protein by cross-linking results in the great suppression of large-scale and concerted motions. The exchange rules of category IV hydrogens are extremely retarded in the cross-linked lysozyme, so that they are exchanged through the so- called penetration mechanism characterized by a low activation enthalp y. These experimental results are discussed with regard to the contrib ution of cross-linking to the stabilization of the folded structure of protein. (C) 1997 John Wiley & Sons, Inc.