PubMed Health⌕ Search

Biomedical subjects

G L Millhauser

Publications and source records attributed to G L Millhauser.

At least 19 recordsLinked to original sources
PubMed1997

Estimating the relative populations of 3(10)-helix and alpha-helix in Ala-rich peptides: a hydrogen exchange and high field NMR study.

Recent experimental and theoretical work suggests that alanine-rich peptides fold as a mixture of 3(10)-helix (i --> i + 3 hydrogen bonding) and alpha-helix (i --> i + 4 hydrogen bonding). In order to assess the relative proportions of the two conformers, NMR studies were performed on the 16 residue sequences: Ac-AAAAKAAAAKAAAAKA-NH2 (3K) and Ac-AMAAKAWAAKAAAARA-NH2 (MW). Hydrogen/deuterium-exchange kinetics measured for the first three amide protons of the 3K peptide indicate that the NH of Ala3 is partially protected from exchange. This result is consistent with the presence of an i --> i + 3 hydrogen bond between the carbonyl group of the acetyl blocking group and the NH group of Ala3. The MW peptide is a modified version of the 3K peptide, designed to increase alphaH signal dispersion. 1H NMR spectra of the MW peptide at 750 MHz reveal a series of intermediate range (NOEs) consistent with a mixture of 3(10)-helix and alpha-helix. The relative intensities of the alphaN(i,i + 3) and alphabeta(i,i + 3) (nuclear Overhauser enhancements) NOEs suggest that 3(10)-helix is present throughout the peptide, but with the greatest contribution at the termini. A model was developed to determine the relative contributions of 3(10)-helix and alpha-helix. Lower bounds for the population of 3(10)-helix are approximately 50% at the termini and 25% in the middle of the peptide. The greatest alpha-helical content is between the middle of the peptide and the N terminus.

Alanine↗
PubMed1996

Local helix content in an alanine-rich peptide as determined by the complete set of 3JHN alpha coupling constants.

Alanine-rich peptides serve as models for exploring the factors that control helix structure in peptides and proteins. Scalar C alpha H-NH couplings (3JHN alpha) are an extremely useful measure of local helix content; however, the large alanine content in these peptides leads to significant signal overlap in the C alpha H region of 1H 2D NMR spectra. Quantitative determination of all possible 3JHN alpha values is, therefore, very challenging. Szyperski and co-workers [(1992) J. Magn. Reson. 99, 552-560] have recently developed a method for determining 3JHN alpha from NOESY spectra. Because 3JHN alpha may be determined from 2D peaks outside of the C alpha H region, there is a much greater likelihood of identifying resolved resonances and measuring the associated coupling constants. It is demonstrated here that 3JHN alpha can be obtained for every residue in the helical peptide Ac-(AAAAK)3A-NH2. The resulting 3JHN alpha profile clearly identifies a helical structure in the middle of the peptide and further suggests that the respective helix termini unfold via distinct pathways.

Amino Acid Sequence↗
PubMed1995

Exploring the peptide 3(10)-helix reversible alpha-helix equilibrium with double label electron spin resonance.

Over the last several years we have used spin labeling as a means for exploring the structure of helical peptides. Two nitroxide labels are engineered into a peptide sequence and distances are ranked with electron spin resonance (ESR). We have found that there is a significant amount of 3(10)-helix in 16-residue model peptides containing only L-amino acids. This review covers several facets of the methodology including spin labeling strategy, interpretation of ESR spectra and the influence of molecular dynamics on the spectral line shapes. Also covered are recent findings of a length-dependent 3(10)-helix-->alpha-helix transition and the role of Arg+ in the stabilization of specific helix structures.

Amino Acid Sequence↗
PubMed1994

A single carboxy-terminal arginine determines the amino-terminal helix conformation of an alanine-based peptide.

Arginine is a stabilizing element in both thermophilic and low molecular weight proteins. Similarly Lys+-->Arg+ substitutions increase the helix content of designed helical peptides. Here we explore this 'arginine effect' by examining how Lys+-->Arg+ substitutions influence the 3(10)-helix-->alpha-helix equilibrium in the helical peptide Ac-(AAAAK)3A-NH2. The unsubstituted sequence contains a significant amount of 3(10)-helix, however, single Lys+-->Arg+ substitutions shift the peptide conformation toward alpha-helix in a position-dependent fashion. The single substitution closest to the carboxy terminus induces the largest conformational change at the helix amino terminus. These findings suggest that a single strategically-placed arginine can exert long range control on helix structure.

Amino Acid Sequence↗
PubMed1993

Increasing sequence length favors alpha-helix over 3(10)-helix in alanine-based peptides: evidence for a length-dependent structural transition.

Ala-based peptides form marginally stable helices at low temperature and are conventionally considered as mixtures of alpha-helix and random coil. However, recent work with doubly spin-labeled peptides suggests that short 16-residue sequences contain a significant fraction of 3(10)-helix near the N-terminus (positions 4-8). Using the same double-label strategy, we report on the helix geometry of the peptides Ac-(AAAAK)nA-NH2 with n = 3 and n = 4. The 16-mer (n = 3) is now examined at a region near the C-terminus, and there is evidence for 3(10)-helix here as well. The 21-mer (n = 4) is examined in three regions of the sequence. In dramatic contrast to the 16-mer, the 21-mer exhibits the signature of alpha-helix at the N-terminus and on through the middle of the peptide. The 21-mer C-terminus, however, adopts the 3(10)-helix geometry as is often found for C-termini in protein alpha-helices. These data indicate that the proportion of alpha-helix and 3(10)-helix in Ala-based peptides depends upon the sequence length.

Alanine↗
PubMed1993

Experimental molecular dynamics of an alanine-based helical peptide determined by spin label electron spin resonance.

The alanine-based 3K(I) peptide is reported to be very helical in aqueous solution. We have prepared a series of six nitroxide spin labeled analogs of the 3K(I) sequence and measured the variable-temperature ESR spectra for each in order to reveal the position-dependent peptide dynamics. From analysis of these local dynamics under helix-forming conditions at 1 degree C, we find that the helix termini show greater local dynamics than the peptide cancer. Further, the C-terminus is more mobile than the N-terminus. Even in the helix-promoting solvent trifluoroethanol, the results indicate that there is still substantially greater dynamics at the helix termini than at the peptide center. The unfolded state is also investigated, and we find that the peptide unfolded by guanidine hydrochloride is somewhat different than that found for high-temperature aqueous solution. Recently it was suggested that short 16-mer peptides may adopt a 3(10)-helix structure instead of the expected alpha-helix. The data presented here at 1 degree C show that there is sufficient disorder within the peptide to accommodate the 3(10) structure. Also calculated are the backbone torsional fluctuations, and the results compare well to those from computer molecular dynamics studies. A proposal is outlined that explains how the enhanced dynamics found at the C-terminus results from the exposure of the helix hydrogen bonds to aqueous solvent in this region of the peptide.

Alanine↗
PubMed1993

Side-chain mobility of the beta-lactamase A state probed by electron spin resonance spectroscopy.

beta-Lactamase from Bacillus licheniformis forms a stable compact intermediate state at low pH and moderate salt concentration (the A state), with properties consistent with a molten globule. A single cysteine residue was introduced into this class A beta-lactamase by site-directed mutagenesis at position 166. A spin label was attached to the thiol of this cysteine residue via a disulfide bond as a probe of the side-chain mobility. The mutant protein and the spin-labeled derivative exhibited similar conformational properties to the wild-type enzyme at acidic pH. The A state induced by chloride or trichloroacetate (TCA) anions was characterized by circular dichroism and esr. The A state at pH 0.5 (0.32 M HCl), or at pH 2 in the presence of 8 mM TCA or 0.4 M Cl-, had comparable amounts of secondary structure to the native state but lacked significant tertiary structure, as judged by the lack of near-UV circular dichroism. Analysis of the esr spectral line widths showed that the mobility of the spin label in the A state was similar to that in the native state and much less mobile than in the unfolded state, indicating significant constraints on the side-chain mobility in this region of the molecule in the A state. The implications of this finding to the structure of the A state are discussed.

Amino Acid Sequence↗
PubMed1992

Short alanine-based peptides may form 3(10)-helices and not alpha-helices in aqueous solution.

Short alanine peptides, containing 16 or 17 residues, appear to form alpha-helices in aqueous solution. But the main spectroscopic analyses used on helical peptides (circular dichroism and nuclear magnetic resonance) cannot distinguish between an alpha-helix (in which the ith residue is hydrogen-bonded to residue i + 4; ref. 9) and the next most common peptide helix, the 3(10)-helix10 (i-->i + 3 hydrogen-bonding). To address this problem we have designed single and doubly spin-labelled analogues of alanine-based peptides in which the nitroxide spin label forms an unbranched side chain extending from the sulphur atom of a cysteine residue. Here we report the circular dichroism, Fourier-transform infrared and electron-spin resonance spectra of these peptides under helix-forming conditions. The infrared absorbance gives an amide I' band with a frequency that is substantially different from that observed for alpha-helices. The electron-spin resonance spectra of doubly labelled helices show that the ranking of distances between side chains, around a single turn (residues 4-8), is inconsistent with an alpha-helical structure. Our experiments suggest that the more likely peptide geometry is a 3(10)-helix.

Alanine↗
PubMed1992

Rotational diffusion and intermolecular collisions of a spin labeled alpha-helical peptide determined by electron spin echo spectroscopy.

Short peptides that are composed mainly of alanine have recently been shown to form alpha-helices in aqueous solution at low temperature (Marqusee, S., and R. L. Baldwin. 1987. Proc. Natl. Acad. Sci. 84:8898-8902; Marqusee, S., V. H. Robbins, and R. L. Baldwin. 1989. Proc. Natl. Acad. Sci. USA. 86:5286-5290). These peptides are excellent models for probing structure and dynamics in isolated helical domains. In previous work we have designed and synthesized spin labeled analogs of these helix-forming peptides and we have shown that these analogs retain the folding characteristics of the parent peptide (Todd, A. P., and G. L. Millhauser. 1991. Biochemistry. 30:5515-5523). Using conventional continuous wave electron spin resonance (CW ESR) we have further shown that local motion is more pronounced near the helix amino terminus than in the central region as the peptide is thermally unfolded (Miick, S. M., A. P. Todd, and G. L. Millhauser. 1991. Biochemistry. 30:9498-9503). In this present work we use electron spin echo (ESE) spectroscopy to further refine our understanding of the solution dynamics of the 3K-8 peptide, which is a 16-mer with a nitroxide spin label attached at position 8. We find that the spin echo decays are well described by a single exponential function and that the determined correlation times are close to those previously derived from CW experiments. Variable concentration ESE experiments have directly revealed Heisenberg spin exchange (HSE) interactions and we find that the interpeptide collision rate is near to that expected for a free species in solution. This provides strong evidence that the helical conformation of these peptides is not stabilized by intermolecular interactions.

Alanine↗
PubMed1991

Position-dependent local motions in spin-labeled analogues of a short alpha-helical peptide determined by electron spin resonance.

We have used electron spin resonance and circular dichroism to examine and compare the dynamics in two analogues of the Ala-based 3K(I) peptide [Marqusee, S., Robbins, V.H., & Baldwin, R. L. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5286-5290], labeled at positions 4 and 8, throughout the alpha-helix----coil transition. In the middle of the thermal unfolding transition, our results demonstrate that the local mobility near the N-terminus is greater than at the center of the peptide. This provides evidence, from the perspective of dynamics, that the ends of Ala-based alpha-helices are frayed. We further find that the position dependence of the mobility for the thermally unfolded state differs from that of the denaturant unfolded state. Only the latter state exhibits the local dynamics expected for a genuine random coil.

Alanine↗
PubMed1991

ESR spectra reflect local and global mobility in a short spin-labeled peptide throughout the alpha-helix----coil transition.

A series of short alanine-based synthetic peptides (16 or 17 residues) have previously been shown to exhibit an anomalously high degree of alpha-helicity [Marqusee, S., et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5286-5290; Marqusee, S., & Baldwin, R.L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8898-8902]. These peptides are ideal models for extracting position-dependent structural and dynamic information. Using the methanethiosulfonate nitroxide spin label (MTSSL), we labeled an analogue of the salt-bridge-stabilized "i+4" peptide, called the "i+4c", which has a specific attachment site created by replacing the central alanine with a cysteine. Circular dichroism (CD) spectra demonstrate that the i+4c-MTSSL peptide retains nearly the same helicity as the original i+4 peptide. The ESR spectra of the labeled peptide indicate no significant aggregation. ESR spectra were acquired throughout the helix-coil transition by temperature variation. From the motionally narrowed spectra, we extracted the rotational correlation times of the nitroxide label. Parallel measurements with circular dichroism enabled us to relate these parameters directly to the fractional helicity. For comparison, we followed a similar procedure with MTSSL-labeled glutathione (GS-MTSSL), a tripeptide that does not form an alpha-helix. Our results are interpreted in terms of a local tumbling volume, V(L), which reflects the portion of the peptide that reorients with the nitroxide label. At high fractional helicity, V(L) is similar to the volume expected for a 17-residue helix.

Amino Acid Sequence↗
PubMed1991

Diffusion model in ion channel gating. Extension to agonist-activated ion channels.

Previously, we described a model which treats ion channel gating as a discrete diffusion problem. In the case of agonist-activated channels at high agonist concentration, the model predicts that the closed lifetime probability density function from single channel recording approximates a power law with an exponent of -3/2 (Millhauser, G. L., E. E. Salpeter, and R. E. Oswald. 1988a. Proc. Natl. Acad. Sci. USA. 85: 1503-1507). This prediction is consistent with distributions derived from a number of ligand-gated channels at high agonist concentration (Millhauser, G. L., E. E. Salpeter, and R. E. Oswald. 1988b. Biophys. J. 54: 1165-1168.) but does not describe the behavior of ion channels at low activator concentrations. We examine here an extension of this model to include an agonist binding step. This extended model is consistent with the closed time distributions generated from the BC3H-1 nicotinic acetylcholine receptor for agonist concentrations varying over three orders of magnitude.

Animals↗
PubMed1990

Reptation theory of ion channel gating.

Reptation theory is a highly successful approach for describing polymer dynamics in entangled systems. In turn, this molecular process is the basis of viscoelasticity. We apply a modified version of reptation dynamics to develop an actual physical model of ion channel gating. We show that at times longer than microseconds these dynamics predict an alpha-helix-screw motion for the amphipathic protein segment that partially lines the channel pore. Such motion has been implicated in several molecular mechanics studies of both voltage-gated and transmitter-gated channels. The experimental probability density function (pdf) for this process follows t-3/2 which has been observed in several experimental systems. Reptation theory predicts that channel gating will occur on the millisecond time scale and this is consistent with experimental results from single-channel recording. We examine the consequences of reptation over random barriers and we show that, to first order, the pdf remains unchanged. In the case of a charged helix undergoing reptation in the presence of a transmembrane potential we show that the tail of the pdf will be exponential. We provide a list of practical experimental predictions to test the validity of this physical theory.

Elasticity↗
PubMed1988

A reevaluation of the mathematical models for simulating single-channel and whole-cell ionic currents.

We have developed a technique that allows for the simulation of both single-channel and whole-cell ionic currents given any arbitrary first-order kinetic scheme for the conformational states of an ion channel. The procedure is based on the solution of the master equation, which, in turn, is a general expression for a Markov process. The solution is expressed in terms of the eigenvalues and eigenvectors of the kinetic system and the system's deviation from equilibrium. Our derived expression provides a general recipe for the calculation of whole-cell currents. By further manipulation of this expression, we show how conditional probabilities are derived that can be used for the simulation of single-channel currents. We discuss computer implementation of the results so that complicated kinetic schemes can be solved numerically. Finally, we demonstrate the procedure by providing a worked example of a simple model of activation followed by inactivation.

Computer Simulation↗