# The Experimental Energy Landscape and Protein Function

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2021 · $424,974

## Abstract

Project Summary:
The goal of this project is to understand how enzymes utilize conformational fluctuations in particular, local
unfolding, to facilitate catalysis. Over the past several decades it has become increasingly clear that rather
existing as static structures, proteins are actually ensembles of sometimes very different conformational states,
and the fluctuations are critical to function. It is of great import to know how this is done. Are there unifying
principles that connect proteins with different functions? Here we take advantage of several key discoveries
discoveries by our group during the previous funding cycles, which demonstrates that the enzyme adenylate
kinase (AK) from E. coli, uses local unfolding to modulate its enzymatic activity – in effect, the energy landscape
has unfolding within its functionally important repertoire. We found unfolding to occur in both the LID and the
AMPbd domains and that unfolding in the different regions selectively modulated different key enzymatic
parameters, with changes in one lid modulating Km , and changes in the other modulating kcat. Unexpectedly we
found that local unfolding actually controlled cold adaptation in the enzyme, thus directly demonstrating the
functional importance. Our discovery stands in stark contrast to the current accepted model (which posits a
rigid-body opening and closing reaction facilitated by a hinge that is believed to facilitate catalytic turnover). The
fact that AK is representative of more than 3,000 high-resolution structures in the Protein Data Bank )PDB) that
have been hypothesized (but never actually demonstrated) to utilize the rigid-body open/closing motions to
facilitate catalysis, suggests that order/disorder fluctuations may be more prevalent than previously believed.
How general is unfolding and how does its presence impact the more than 40 years of structural biology-based
functional studies? Our approach is two-fold. First, as our results directly undermine the existing models of AK
(and thus require a new model) we will determine how the disordered states discovered by us are responsible
for the function of the enzyme. Second, we must interrogate the database of enzymes to determine how general
local unfolding and disorder are. Do all enzymes utilize unfolding? Can we develop a quantitative,
experimentally-derived model of AK and other enzymes? We will perform binding and stability measurements
using isothermal titration calorimetry (ITC), circular dichroism (CD) monitored thermal unfolding and hydrogen
exchange (HX), and we will monitor the kinetics of the conformational and enzymatic processes using NMR
CEST (conformational exchange saturation transfer) and steady state enzymatic analysis.

## Key facts

- **NIH application ID:** 10264158
- **Project number:** 5R01GM063747-20
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** VINCENT J. HILSER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $424,974
- **Award type:** 5
- **Project period:** 2001-08-01 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10264158

## Citation

> US National Institutes of Health, RePORTER application 10264158, The Experimental Energy Landscape and Protein Function (5R01GM063747-20). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10264158. Licensed CC0.

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