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As a first step to determine the folding pathway of a protein with an cy/P doubly wound topology, the 'H, "C, and "N backbone chemical shifts of Azotobacter vinelandii holoflavodoxin I1 (179 residues) have been determined using multidimensional NMR spectroscopy. Its secondary structure is shown to contain a five-stranded parallel P-sheet (p2-pl-p3-p4-/?5) and five a-helices. Exchange rates for the individual amide protons of holoflavodoxin were determined using the hydrogen exchange method. The amide protons of 65 residues distributed throughout the structure of holoflavodoxin exchange slowly at pH* 6.2 ( k , < s-I) and can be used as probes in future folding studies. Measured exchange rates relate to apparent local free energies for transient opening. We propose that the amide protons in the core of holoflavodoxin only exchange by global unfolding of the apo state of the protein. The results obtained are discussed with respect to their implications for flavodoxin folding and for modulation of the flavin redox potential by the apoprotein. We do not find any evidence that A. vinelandii holoflavodoxin I1 is divided into two subdomains based on its amide proton exchange rates, as opposed to what is found for the structurally but not sequentially homologous cylp doubly wound protein Che Y.Keywords: flavodoxin; hydrogenJdeuterium exchange; NMR spectroscopy; protein folding; protein stability; redox potential regulation We have chosen Azotobacter vinelandii (strain ATCC 478) flavodoxin I1 as a model protein to study protein folding. A flavodoxin was chosen because it adopts one of the nine protein domain superfolds (Orengo et al., 1994), the alp doubly wound topology, Abbreviations Amp, ampicillin; ct, constant-time; CSI, chemical shift index; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt: fid, free induction decay; PC,, free energy for transient opening; AG;;;, apparent free energy for transient opening in holoflavodoxin; PC,+ equlllbrium free energy of global unfolding; HSQC, heteronuclear single-quantum coherence; IPTG, isopropyl P-D-thiogalactopyranoside; kex, measured amide proton exchange rate; kin,, sequence-specific intrinsic amide proton exchange rate; &, dissociation constant; NOE, nuclear overhauser effect; NOESY, NOE spectroscopy: NMR, nuclear magnetic resonance; pH*, glass-electrode reading of the pH meter at room temperature, uncorrected for isotope

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A Radial Basis Boltzmann Machine (RBBM) is a specialized Boltzmann Machine architecture that combines feed-forward mapping with probability estimation in the input space, and for which very efficient learning rules exist. The hidden representation of the network displays symmetry breaking as a function of the noise in the dynamics. Thus, generalization can be studied as a function of the noise in the neuron dynamics instead of as a function of the number of hidden units. We show that the RBBM can be seen as an elegant alternative of k-nearest neighbor, leading to comparable performance without the need to store all data. We show that the RBBM has good classification performance compared to the MLP. The main advantage of the RBBM is that simultaneously with the input-output mapping, a model of the input space is obtained which can be used for learning with missing values. We derive learning rules for the case of incomplete data, and show that they perform better on incomplete data than the traditional learning rules on a 'repaired' data set. * marcel@mbfys.kun.nl 301 Int. J. Neur. Syst. 1997.08:301-315. Downloaded from www.worldscientific.com by FLINDERS UNIVERSITY LIBRARY on 02/08/15. For personal use only.