Instructional Design for an Undergraduate Laboratory Course in Molecular Biophysics

ABSTRACT In this article, an approach to teaching molecular biophysics is described. The organization and course content has been carefully chosen and curated so that fundamental ideas in molecular biophysics can be taught effectively to upper classmen in higher education. Three general topic areas are introduced along with accompanying experiments that illustrate major principles related to each topic area. This article outlines an approach to organizing chosen course material and suggests multiple teaching activities within each major topic area: thermodynamics, kinetics, and structural biology. Subtopics are presented along with suggested laboratory experiments. The experiments are outlined in a way that they can be readily adopted by educators teaching a biophysical chemistry lab. The accompaniment of workshop exercises as an additional teaching modality is a component of the course intended to enhance the development of important problem-solving skills and comprehension of new content. Finally, a reflection on student feedback and course outcomes along with targeted learning goals is discussed.

“Multiplex” rheostat positions cluster around allosterically critical regions of the lactose repressor protein

Amino acid variation at “rheostat” positions provides opportunity to modulate various aspects of protein function – such as binding affinity or allosteric coupling – across a wide range. Previously a subclass of “multiplex” rheostat positions was identified at which substitutions simultaneously modulated more than one functional parameter. Using the Miller laboratory’s dataset of ∼4000 variants of lactose repressor protein (LacI), we compared the structural properties of multiplex rheostat positions with (i) “single” rheostat positions that modulate only one functional parameter, (ii) “toggle” positions that follow textbook substitution rules, and (iii) “neutral” positions that tolerate any substitution without changing function. The combined rheostat classes comprised >40% of LacI positions, more than either toggle or neutral positions. Single rheostat positions were broadly distributed over the structure. Multiplex rheostat positions structurally overlapped with positions involved in allosteric regulation. When their phenotypic outcomes were interpreted within a thermodynamic framework, functional changes at multiplex positions were uncorrelated. This suggests that substitutions lead to complex changes in the underlying molecular biophysics. Bivariable and multivariable analyses of evolutionary signals within multiple sequence alignments could not differentiate single and multiplex rheostat positions. Phylogenetic analyses – such as ConSurf – could distinguish rheostats from toggle and neutral positions. Multivariable analyses could also identify a subset of neutral positions with high probability. Taken together, these results suggest that detailed understanding of the underlying molecular biophysics, likely including protein dynamics, will be required to discriminate single and multiplex rheostat positions from each other and to predict substitution outcomes at these sites.