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M Lorch

Publications and source records attributed to M Lorch.

12 recordsLinked to original sources
PubMed2005

NMR and fluorescence spectroscopy approaches to secondary and primary active multidrug efflux pumps.

Multidrug efflux pumps are found in all major transporter families. Along with a lack of three-dimensional structure information, the mechanism of drug recognition, energy coupling with drug translocation and the catalytic cycle are so far not understood. In the present study, we present first data of a fluorescence-based assay to study the pH-gradient-mediated activity of the multidrug antiporter EmrE, by co-reconstitution with the light-driven proton pump bacteriorhodopsin. In addition to biochemical approaches, the emerging technique, solid-state NMR, can be used for the investigation of these transporters. A number of experiments based on MAS (magic angle sample spinning) NMR are available to provide data on protein structure and dynamics, drug binding and protein-lipid interactions. However, these experiments dictate a number of constraints with respect to sample preparation that will be discussed for proteins from the SMR (small multidrug resistance transporter) family. In addition, 2H-NMR is used to probe protein mobility of Lactococcus lactis ABC transporter, LmrA.

ATP-Binding Cassette Transporters↗
PubMed2001

In vitro studies of membrane protein folding.

The study of membrane protein folding is a new and challenging research field. Consequently, there are few direct studies on the in vitro folding of membrane proteins. This review covers work aimed at understanding folding mechanisms and the intermolecular forces that drive the folding of integral membrane proteins. We discuss the kinetic and thermodynamic studies that have been undertaken. Our review also draws on closely related research, mainly from purification studies of functional membrane proteins, and gives an overview of some of the successful methods. A brief survey is also given of the large body of mutagenesis and fragment work on membrane proteins, as this too has relevance to the folding problem. It is noticeable that the choice of solubilizing detergents and lipids can determine the success of the method, and indeed it appears that particular lipid properties can be used to control the rate and efficiency of folding. This has important ramifications for much in vitro folding work in that it aids our understanding of how to obtain and handle folded, functional protein. With this in mind, we also cover some relevant properties of model, lipid-bilayer systems.

Bacterial Proteins↗
PubMed2000

Progressive stabilization of intermediate and transition states in protein folding reactions by introducing surface hydrophobic residues.

It can be argued from the principle of solvent exclusion that the introduction of hydrophobic residues onto the surface of a protein will not destabilize the folded state because the nonpolar side chain will be at least as exposed in the unfolded state as it is when the protein chain is folded. A comparison of the folding pathway of wild type and 11 site-directed mutants of CD2.d1 shows this to be true. In fact, owing to partial burial of nonpolar groups as folding proceeds, we find that the rapidly formed intermediate state and, to a greater extent, the transition state are generally stabilized by hydrophobic surface mutations. This effect is slightly moderated in the folded state presumably by the perturbation of van der Waals' contacts and/or local electrostatic interactions that have a greater influence in this fully compact structure. The fact that in all but one case we find that stabilization of the rapidly collapsed intermediate is accompanied by a faster acquisition of the folded state refutes the argument that I states are generally "off pathway" conformations or ensembles that lead to the inhibition of otherwise more rapid folding trajectories.

Amino Acid Substitution↗
PubMed2000

Effects of mutations on the thermodynamics of a protein folding reaction: implications for the mechanism of formation of the intermediate and transition states.

We have measured changes in heat capacity, entropy, and enthalpy for each step in the folding reaction of CD2.d1 and evaluated the effects of core mutations on these properties. All wild-type and mutant forms fold through a rapidly formed intermediate state that precedes the rate-limiting transition state. Mutations have a pronounced effect on the enthalpy of both the intermediate and folded states, but in all cases a compensatory change in entropy results in a small net free-energy change. While the enthalpy change in the folded state can be attributed to a loss of van der Waals interactions, it has already been shown that changes in the stability of the intermediate are dominated by changes in secondary structure propensity [Lorch et al. (1999) Biochemistry 38, 1377-1385]. It follows that the thermodynamic basis of beta-propensity is enthalpic in origin. The effects of mutations on the enthalpy and entropy of the transition state are smaller than on the ground states. This relative insensitivity to mutation is discussed in the light of theories concerning the nature of the rate-limiting barrier in folding reactions.

Animals↗
PubMed1999

Effects of core mutations on the folding of a beta-sheet protein: implications for backbone organization in the I-state.

A series of core mutations were introduced into beta-strand segments of an immunoglobulin fold (the isolated first domain of CD2, CD2.d1) to examine their influence on the rapidly formed intermediate state (I-state) which transiently accumulates in the folding reaction [Parker, M. J., and Clarke, A. R. (1997) Biochemistry 36, 5786-5794]. The residue changes were chemically conservative, each representing the removal of one or two methylene groups from aliphatic side chains. Predictably, the mutations destabilize the folded state with respect to the unfolded state by about 1.1 +/- 0.7 kcal mol-1 per methylene group removed. However, when the folding reaction is dissected by transient kinetic analysis into its component steps, six out of the nine mutations lead to a stabilization of the I-state. The direction and magnitude of these effects on the global stability of the transient intermediate are well correlated with changes in secondary structure propensity occasioned by the substitutions. The results show that, although side chain interactions are extremely weak in this early phase of folding, the beta-strand conformation of the polypeptide chain is established. In the next phase of the reaction, the rate-limiting transition state is attained by the formation of a tightly localized hydrophobic nucleus which includes residues V30, I18, and V78. Interestingly, in almost all immunoglobulin domains of extracellular proteins, the latter pair are cysteine residues which form a disulfide bridge.

Amino Acid Substitution↗
PubMed1998

Thermodynamic properties of transient intermediates and transition states in the folding of two contrasting protein structures.

The N-terminal domain of phosphoglycerate kinase (N-PGK) and domain 1 of the T-cell adhesion protein CD2 (CD2.d1) fold through rapidly formed and transiently populated intermediate states in reactions which have no kinetic complications arising from proline isomerization or disulfide bonding. We have evaluated the thermodynamic parameters (DeltaCp, change in heat capacity; DeltaS, entropy change; DeltaH, enthalpy change) for each experimentally accessible step in these folding reactions. Despite their different topologies and amino acid compositions, the individual steps [U-I (unfolded to intermediate state), I-t (intermediate to major transition state), and t-F (transition state to the fully folded state)] have closely similar qualitative properties in the two proteins. For both, the heat capacity changes are proportional to m-value changes (Deltam) for every step in the reaction, but the ratio DeltaCp/Deltam is lower for N-PGK, presumably owing to a much larger compliment of aromatic amino acids in the core. According to measurements of DeltaCp and Deltam, the I-states are highly condensed (65-70% for N-PGK and 40-45% dehydrated for CD2.d1), yet the changes in entropy in the U-to-I transition are small, showing that the entropy gained from desolvation must be balanced by that lost in ordering the chain. The high degree of conformational order in the I-state, implied by these measurements, is mirrored by the extensive, native secondary structure revealed by amide exchange measurements [Hosszu, L. L. P., et al. (1997) Nat. Struct. Biol. 4, 801-804; Parker, M. J., et al. (1997) Biochemistry 36, 13396-13405]. At 25 degreesC the transition state barrier has an entirely enthalpic origin, the entropic contribution being favorable. The latter observation implies that, during the consolidation of structure occurring in the I-to-F step, further dehydration (positive DeltaS) precedes side-chain locking (negative DeltaS). Only after the transition state is surmounted do we see a net entropic penalty arising from the widespread ordering of side chains.

Animals↗
PubMed1998

Meta-analysis of botulinum toxin treatment of spasmodic dysphonia: a review of 22 studies.

This meta-analysis focuses on the treatment effects of the use of botulinum toxin in laryngeal dystonia. The vocal symptoms are characterized by spasms of the laryngeal muscles thus the use of the term spasmodic dysphonia (SD). The objective of the intervention, i.e. botulinum toxin treatment, is to reduce or eliminate the vocal spasms and thus improve the acoustic regularity of the voice. The purpose of the present study was to assess the efficacy of botulinum toxin in the treatment of SD by engaging in a 'best synthesis' systematic summary of existing research. The average treated SD patient in the 22 studies subjected to meta-analysis obtained 97% improvement as a result of treatment with botulinum toxin.

Analysis of Variance↗
PubMed1997

Acquisition of native beta-strand topology during the rapid collapse phase of protein folding.

The 98 residue C-terminal domain of the cell-surface receptor protein CD2 (CD2.D1) has a beta-sandwich fold belonging to the immunoglobulin superfamily but lacking the usual disulfide bridges. Kinetic studies on the folding/unfolding of CD2.D1 reveal that folding proceeds through a rapidly formed intermediate state [Parker, M. J., & Clarke, A. R. (1997) Biochemistry 36, 5786-5794]. To characterize the structural properties of this intermediate we have performed a series of amide hydrogen exchange studies using the pH competition method, in which folding and exchange are initiated simultaneously. The complex beta-sheet topology of this molecule makes it an ideal object for examining the acquisition of backbone hydrogen bonds made between sequence-local and sequence-distant segments of the chain during folding. The pattern of protected amides in the intermediate reveal that the essential features of the beta-sheet topology of CD2.D1 are defined early in the folding pathway, before the development of intimate side chain interactions characteristic of the native state. The results are discussed in light of current issues concerning the mechanistic relevance of kinetic protein folding intermediates.

Amides↗
PubMed1993

The use of botulinum toxin in the treatment of adductor spasmodic dysphonia.

Botulinum toxin injections have been used to treat 31 patients with adductor spasmodic dysphonia. Injections of 3.00-3.75 units of botulinum toxin were performed bilaterally into the thyroarytenoid muscle. This treatment significantly decreased the standard deviation of the fundamental frequency of the speech sample, indicating a reduction in the variability of pitch amongst patients. A total of 96% of patients' subjective diary reports showed an improvement with a median of 7 days to peak effect and a 5 week duration of peak effect.

Adult↗
PubMed1992

Treatment of cervical dystonia hand spasms and laryngeal dystonia with botulinum toxin.

One hundred and twenty-six patients with different forms of focal dystonia (89 with cervical dystonia, 12 with hand cramps and 25 with laryngeal dystonia) were treated with localised injections of botulinum toxin. Mean doses per muscle were 200 mouse units (m.u.) for treating cervical dystonia, 40-120 m.u. for forearm muscles in writers' cramp and 3.7 m.u. for the thyroarytenoid muscle in laryngeal dystonia. Responder rates have been above 80% in all patient groups and beneficial effects could be reproduced over follow-up periods of up to 4 years. The commonest side-effects were dysphagia after treatment of spasmodic torticollis, weakness of neighbouring muscles after injections for hand cramps and breathiness and hypophonia following laryngeal injections. All these were transient and generally well tolerated. It is concluded that botulinum toxin injections are a safe and effective treatment in all three types of focal dystonia.

Botulinum Toxins↗